Rod A Wing

Interests

Research

The use of genome biology to discover and implement sustainable solutions to help solve world hunger and to feed the planet. My lab's current focus is to understand the genome biology of the wild relatives of rice which possess many important biotic and abiotic stress traits that have been lost in present day cultivars are a consequence of domestication. My work at the International Rice Research Institute is to translate these discoveries in to practical solutions to help solve the 10 billion people question.

Teaching

Genomics, Genetics, Bioinformatics, Evolutionary Biology

Courses

Scholarly Contributions

Chapters

• McCouch, S., Wing, R., Semon, M., Venuprasad, R., Atlin, G., Sorrells, M., & Jannink, J. (2013). Realizing Africa's Rice Promise. In Making Rice Genomics Work For Africa. CABI.
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  Editor(s): Horie, T | Jalloh, A | Johnson, D | Tollens, E | Wopereis, MCS
• Sanchez, P., Wing, R., & Brar, D. (2013). The wild relatives of rice: Genomes and Genomics. In Genetics and Genomics of Rice. Springer Science Press.
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  Editor(s): Zhang, Q | Wing, R
• Wing, R., & Zhang, Q. (2013). Rice 2020 Revised: An Urgent Call to Mobilize and Coordinate Rice Functional Genomics Research Worldwide. In Genetics and Genomics of Rice. Springer Science Press.
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  Editor(s): Zhang, Q | Wing, R
• Yu, Y., Wing, R., & Li, J. (2013). Understanding the molecular genetic basis of rice grain quality. In Genetics and Genomics of Rice. Springer Science Press.
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  Editor(s): Zhang, Q | Wing, R

Journals/Publications

• Fornasiero, A., Wing, R. A., & Ronald, P. (2022). Rice domestication. Current biology : CB, 32(1), R20-R24.
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  Rice is a staple food crop for more than one-third of the global population (http://www.sustainablerice.org/), of which 90% live at or near the poverty line. Thus, rice genetic improvement is important for global food security and is critical for enhancing socioeconomic benefits and reducing the environmental impacts of agriculture. In continued efforts to address the long-standing problem of food security and sustainable agriculture, scientists are utilizing genes from diverse varieties of rice to improve the resilience of rice to pests, diseases and environmental stress. This Primer describes the history of rice domestication, the importance of wild relatives of rice for crop improvement, and the domestication of wild species of rice not previously planted by farmers - a new approach called neodomestication.
• Yi, X., Liu, J., Chen, S., Wu, H., Liu, M., Xu, Q., Lei, L., Lee, S., Zhang, B., Kudrna, D., Fan, W., Wing, R. A., Wang, X., Zhang, M., Zhang, J., Yang, C., & Chen, N. (2022). Genome assembly of the JD17 soybean provides a new reference genome for Comparative genomics. G3 (Bethesda, Md.).
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  Cultivated soybean (Glycine max) is an important source for protein and oil. Many elite cultivars with different traits have been developed for different conditions. Each soybean strain has its own genetic diversity, and the availability of more high-quality soybean genomes can enhance comparative genomic analysis for identifying genetic underpinnings for its unique traits. In this study, we constructed a high-quality de novo assembly of an elite soybean cultivar Jidou 17 (JD17) with chromsome contiguity and high accuracy. We annotated 52,840 gene models and reconstructed 74,054 high-quality full-length transcripts. We performed a genome-wide comparative analysis based on the reference genome of JD17 with three published soybeans (WM82, ZH13 and W05), which identified five large inversions and two large translocations specific to JD17, 20,984-46,912 PAVs spanning 13.1-46.9 Mb in size. 1,695,741-3,664,629 SNPs and 446,689-800,489 Indels were identified and annotated between JD17 and them. Symbiotic nitrogen fixation (SNF) genes were identified and the effects from these variants were further evaluated. It was found that the coding sequences of 9 nitrogen fixation-related genes were greatly affected. The high-quality genome assembly of JD17 can serve as a valuable reference for soybean functional genomics research.
• Lovell, J. T., MacQueen, A. H., Mamidi, S., Bonnette, J., Jenkins, J., Napier, J. D., Sreedasyam, A., Healey, A., Session, A., Shu, S., Barry, K., Bonos, S., Boston, L., Daum, C., Deshpande, S., Ewing, A., Grabowski, P. P., Haque, T., Harrison, M., , Jiang, J., et al. (2021). Genomic mechanisms of climate adaptation in polyploid bioenergy switchgrass. Nature, 590(7846), 438-444.
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  Long-term climate change and periodic environmental extremes threaten food and fuel security and global crop productivity. Although molecular and adaptive breeding strategies can buffer the effects of climatic stress and improve crop resilience, these approaches require sufficient knowledge of the genes that underlie productivity and adaptation-knowledge that has been limited to a small number of well-studied model systems. Here we present the assembly and annotation of the large and complex genome of the polyploid bioenergy crop switchgrass (Panicum virgatum). Analysis of biomass and survival among 732 resequenced genotypes, which were grown across 10 common gardens that span 1,800 km of latitude, jointly revealed extensive genomic evidence of climate adaptation. Climate-gene-biomass associations were abundant but varied considerably among deeply diverged gene pools. Furthermore, we found that gene flow accelerated climate adaptation during the postglacial colonization of northern habitats through introgression of alleles from a pre-adapted northern gene pool. The polyploid nature of switchgrass also enhanced adaptive potential through the fractionation of gene function, as there was an increased level of heritable genetic diversity on the nondominant subgenome. In addition to investigating patterns of climate adaptation, the genome resources and gene-trait associations developed here provide breeders with the necessary tools to increase switchgrass yield for the sustainable production of bioenergy.
• Paritosh, K., Yadava, S. K., Singh, P., Bhayana, L., Mukhopadhyay, A., Gupta, V., Bisht, N. C., Zhang, J., Kudrna, D. A., Copetti, D., Wing, R. A., Reddy Lachagari, V. B., Pradhan, A. K., & Pental, D. (2021). A chromosome-scale assembly of allotetraploid Brassica juncea (AABB) elucidates comparative architecture of the A and B genomes. Plant biotechnology journal, 19(3), 602-614.
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  Brassica juncea (AABB), commonly referred to as mustard, is a natural allopolyploid of two diploid species-B. rapa (AA) and B. nigra (BB). We report a highly contiguous genome assembly of an oleiferous type of B. juncea variety Varuna, an archetypical Indian gene pool line of mustard, with ~100× PacBio single-molecule real-time (SMRT) long reads providing contigs with an N50 value of >5 Mb. Contigs were corrected for the misassemblies and scaffolded with BioNano optical mapping. We also assembled a draft genome of B. nigra (BB) variety Sangam using Illumina short-read sequencing and Oxford Nanopore long reads and used it to validate the assembly of the B genome of B. juncea. Two different linkage maps of B. juncea, containing a large number of genotyping-by-sequencing markers, were developed and used to anchor scaffolds/contigs to the 18 linkage groups of the species. The resulting chromosome-scale assembly of B. juncea Varuna is a significant improvement over the previous draft assembly of B. juncea Tumida, a vegetable type of mustard. The assembled genome was characterized for transposons, centromeric repeats, gene content and gene block associations. In comparison to the A genome, the B genome contains a significantly higher content of LTR/Gypsy retrotransposons, distinct centromeric repeats and a large number of B. nigra specific gene clusters that break the gene collinearity between the A and the B genomes. The B. juncea Varuna assembly will be of major value to the breeding work on oleiferous types of mustard that are grown extensively in south Asia and elsewhere.
• Park, H. S., Jayakodi, M., Lee, S. H., Jeon, J. H., Lee, H. O., Park, J. Y., Moon, B. C., Kim, C. K., Wing, R. A., Newmaster, S. G., Kim, J. Y., & Yang, T. J. (2021). Author Correction: Mitochondrial plastid DNA can cause DNA barcoding paradox in plants. Scientific reports, 11(1), 9599.
• Song, J. M., Xie, W. Z., Wang, S., Guo, Y. X., Koo, D. H., Kudrna, D., Gong, C., Huang, Y., Feng, J. W., Zhang, W., Zhou, Y., Zuccolo, A., Long, E., Lee, S., Talag, J., Zhou, R., Zhu, X. T., Yuan, D., Udall, J., , Xie, W., et al. (2021). Two gap-free reference genomes and a global view of the centromere architecture in rice. Molecular plant, 14(10), 1757-1767.
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  Rice (Oryza sativa), a major staple throughout the world and a model system for plant genomics and breeding, was the first crop genome sequenced almost two decades ago. However, reference genomes for all higher organisms to date contain gaps and missing sequences. Here, we report the assembly and analysis of gap-free reference genome sequences for two elite O. sativa xian/indica rice varieties, Zhenshan 97 and Minghui 63, which are being used as a model system for studying heterosis and yield. Gap-free reference genomes provide the opportunity for a global view of the structure and function of centromeres. We show that all rice centromeric regions share conserved centromere-specific satellite motifs with different copy numbers and structures. In addition, the similarity of CentO repeats in the same chromosome is higher than across chromosomes, supporting a model of local expansion and homogenization. Both genomes have over 395 non-TE genes located in centromere regions, of which ∼41% are actively transcribed. Two large structural variants at the end of chromosome 11 affect the copy number of resistance genes between the two genomes. The availability of the two gap-free genomes lays a solid foundation for further understanding genome structure and function in plants and breeding climate-resilient varieties.
• Tian, P., Zhang, X., Xia, R., Liu, Y., Wang, M., Li, B., Liu, T., Shi, J., Wing, R. A., Meyers, B. C., & Chen, M. (2021). Evolution and diversification of reproductive phased small interfering RNAs in Oryza species. The New phytologist, 229(5), 2970-2983.
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  In grasses, two types of phased, small interfering RNAs (phasiRNAs) are expressed largely in young, developing anthers. They are 21 or 24 nucleotides (nt) in length and are triggered by miR2118 or miR2275, respectively. However, most of their functions and activities are not fully understood. We performed comparative genomic analysis of their source loci (PHAS) in five Oryza genomes and combined this with analysis of high-throughput sRNA and degradome datasets. In total, we identified 8216 21-PHAS and 626 24-PHAS loci. Local tandem and segmental duplications mainly contributed to the expansion and supercluster distribution of the 21-PHAS loci. Despite their relatively conserved genomic positions, PHAS sequences diverged rapidly, except for the miR2118/2275 target sites, which were under strong selection for conservation. We found that 21-nt phasiRNAs with a 5'-terminal uridine (U) demonstrated cis-cleavage at PHAS precursors, and these cis-acting sites were also variable among close species. miR2118 could trigger phasiRNA production from its own antisense transcript and the derived phasiRNAs might reversibly regulate miR2118 precursors. We hypothesised that successful initiation of phasiRNA biogenesis is conservatively maintained, while phasiRNA products diverged quickly and are not individually conserved. In particular, phasiRNA production is under the control of multiple reciprocal regulation mechanisms.
• Wu, Z., Liu, H., Zhan, W., Yu, Z., Qin, E., Liu, S., Yang, T., Xiang, N., Kudrna, D., Chen, Y., Lee, S., Li, G., Wing, R. A., Liu, J., Xiong, H., Xia, C., Xing, Y., Zhang, J., & Qin, R. (2021). The chromosome-scale reference genome of safflower (Carthamus tinctorius) provides insights into linoleic acid and flavonoid biosynthesis. Plant biotechnology journal, 19(9), 1725-1742.
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  Safflower (Carthamus tinctorius L.), a member of the Asteraceae, is a popular crop due to its high linoleic acid (LA) and flavonoid (such as hydroxysafflor yellow A) contents. Here, we report the first high-quality genome assembly (contig N50 of 21.23 Mb) for the 12 pseudochromosomes of safflower using single-molecule real-time sequencing, Hi-C mapping technologies and a genetic linkage map. Phyloge nomic analysis showed that safflower diverged from artichoke (Cynara cardunculus) and sunflower (Helianthus annuus) approximately 30.7 and 60.5 million years ago, respectively. Comparative genomic analyses revealed that uniquely expanded gene families in safflower were enriched for those predicted to be involved in lipid metabolism and transport and abscisic acid signalling. Notably, the fatty acid desaturase 2 (FAD2) and chalcone synthase (CHS) families, which function in the LA and flavonoid biosynthesis pathways, respectively, were expanded via tandem duplications in safflower. CarFAD2-12 was specifically expressed in seeds and was vital for high-LA content in seeds, while tandemly duplicated CarFAD2 genes were up-regulated in ovaries compared to CarFAD2-12, which indicates regulatory divergence of FAD2 in seeds and ovaries. CarCHS1, CarCHS4 and tandem-duplicated CarCHS5˜CarCHS6, which were up-regulated compared to other CarCHS members at early stages, contribute to the accumulation of major flavonoids in flowers. In addition, our data reveal multiple alternative splicing events in gene families related to fatty acid and flavonoid biosynthesis. Together, these results provide a high-quality reference genome and evolutionary insights into the molecular basis of fatty acid and flavonoid biosynthesis in safflower.
• Yu, H., Lin, T., Meng, X., Du, H., Zhang, J., Liu, G., Chen, M., Jing, Y., Kou, L., Li, X., Gao, Q., Liang, Y., Liu, X., Fan, Z., Liang, Y., Cheng, Z., Chen, M., Tian, Z., Wang, Y., , Chu, C., et al. (2021). A route to de novo domestication of wild allotetraploid rice. Cell, 184(5), 1156-1170.e14.
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  Cultivated rice varieties are all diploid, and polyploidization of rice has long been desired because of its advantages in genome buffering, vigorousness, and environmental robustness. However, a workable route remains elusive. Here, we describe a practical strategy, namely de novo domestication of wild allotetraploid rice. By screening allotetraploid wild rice inventory, we identified one genotype of Oryza alta (CCDD), polyploid rice 1 (PPR1), and established two important resources for its de novo domestication: (1) an efficient tissue culture, transformation, and genome editing system and (2) a high-quality genome assembly discriminated into two subgenomes of 12 chromosomes apiece. With these resources, we show that six agronomically important traits could be rapidly improved by editing O. alta homologs of the genes controlling these traits in diploid rice. Our results demonstrate the possibility that de novo domesticated allotetraploid rice can be developed into a new staple cereal to strengthen world food security.
• Flynn, J. M., Long, M., Wing, R. A., & Clark, A. G. (2020). Evolutionary Dynamics of Abundant 7-bp Satellites in the Genome of Drosophila virilis. Molecular biology and evolution, 37(5), 1362-1375.
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  The factors that drive the rapid changes in abundance of tandem arrays of highly repetitive sequences, known as satellite DNA, are not well understood. Drosophila virilis has one of the highest relative amounts of simple satellites of any organism that has been studied, with an estimated >40% of its genome composed of a few related 7-bp satellites. Here, we use D. virilis as a model to understand technical biases affecting satellite sequencing and the evolutionary processes that drive satellite composition. By analyzing sequencing data from Illumina, PacBio, and Nanopore platforms, we identify platform-specific biases and suggest best practices for accurate characterization of satellites by sequencing. We use comparative genomics and cytogenetics to demonstrate that the highly abundant AAACTAC satellite family arose from a related satellite in the branch leading to the virilis phylad 4.5-11 Ma before exploding in abundance in some species of the clade. The most abundant satellite is conserved in sequence and location in the pericentromeric region but has diverged widely in abundance among species, whereas the satellites nearest the centromere are rapidly turning over in sequence composition. By analyzing multiple strains of D. virilis, we saw that the abundances of two centromere-proximal satellites are anticorrelated along a geographical gradient, which we suggest could be caused by ongoing conflicts at the centromere. In conclusion, we illuminate several key attributes of satellite evolutionary dynamics that we hypothesize to be driven by processes including selection, meiotic drive, and constraints on satellite sequence and abundance.
• Hofmeister, B. T., Denkena, J., Colomé-Tatché, M., Shahryary, Y., Hazarika, R., Grimwood, J., Mamidi, S., Jenkins, J., Grabowski, P. P., Sreedasyam, A., Shu, S., Barry, K., Lail, K., Adam, C., Lipzen, A., Sorek, R., Kudrna, D., Talag, J., Wing, R., , Hall, D. W., et al. (2020). A genome assembly and the somatic genetic and epigenetic mutation rate in a wild long-lived perennial Populus trichocarpa. Genome biology, 21(1), 259.
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  Plants can transmit somatic mutations and epimutations to offspring, which in turn can affect fitness. Knowledge of the rate at which these variations arise is necessary to understand how plant development contributes to local adaption in an ecoevolutionary context, particularly in long-lived perennials.
• Mussurova, S., Al-Bader, N., Zuccolo, A., & Wing, R. A. (2020). Potential of Platinum Standard Reference Genomes to Exploit Natural Variation in the Wild Relatives of Rice. Frontiers in plant science, 11, 579980.
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  As the world's population expands from 7.6 billion to 10 billion over the next 30 years, scientists and farmers across the globe must explore every angle necessary to provide a safe, stable and sustainable food supply for generations to come. Rice, and its wild relatives in the genus , will play a significant role in helping to solve this 10 billion people question due to its place as a staple food for billions. The genus is composed of 27 species that span 15 million years of evolutionary diversification and have been shown to contain a plethora of untapped adaptive traits, e.g., biotic and abiotic resistances, which can be used to improve cultivated rice. Such traits can be introduced into cultivated rice, in some cases by conventional crossing, and others genetic transformation and gene editing methods. In cases where traits are too complex to easily transfer to cultivated rice [e.g., quantitative trait loci (QTL)], an alternative strategy is to domesticate the wild relative that already contains the desired adaptive traits - i.e., "neodomestication". To utilize the genus for crop improvement and neodomestication, we first need a set of genomic resources that can be used to efficiently identify, capture, and guide molecular crop improvement. Here, we introduce the concept of platinum standard reference genome sequences (PSRefSeq) - a new standard by which contiguous near-gap free reference genomes can now be produced. By having a set of PSRefSeqs for every species we set a new bar for how crop wild relatives can be integrated into crop improvement programs.
• Park, H. S., Jayakodi, M., Lee, S. H., Jeon, J. H., Lee, H. O., Park, J. Y., Moon, B. C., Kim, C. K., Wing, R. A., Newmaster, S. G., Kim, J. Y., & Yang, T. J. (2020). Mitochondrial plastid DNA can cause DNA barcoding paradox in plants. Scientific reports, 10(1), 6112.
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  The transfer of ancestral plastid genomes into mitochondrial genomes to generate mitochondrial plastid DNA (MTPT) is known to occur in plants, but its impacts on mitochondrial genome complexity and the potential for causing a false-positive DNA barcoding paradox have been underestimated. Here, we assembled the organelle genomes of Cynanchum wilfordii and C. auriculatum, which are indigenous medicinal herbs in Korea and China, respectively. In both species, it is estimated that 35% of the ancestral plastid genomes were transferred to mitochondrial genomes over the past 10 million years and remain conserved in these genomes. Some plastid barcoding markers co-amplified the conserved MTPTs and caused a barcoding paradox, resulting in mis-authentication of botanical ingredients and/or taxonomic mis-positioning. We identified dynamic and lineage-specific MTPTs that have contributed to mitochondrial genome complexity and might cause a putative barcoding paradox across 81 plant species. We suggest that a DNA barcoding guidelines should be developed involving the use of multiple markers to help regulate economically motivated adulteration.
• Shenton, M., Kobayashi, M., Terashima, S., Ohyanagi, H., Copetti, D., Hernández-Hernández, T., Zhang, J., Ohmido, N., Fujita, M., Toyoda, A., Ikawa, H., Fujiyama, A., Furuumi, H., Miyabayashi, T., Kubo, T., Kudrna, D., Wing, R., Yano, K., Nonomura, K. I., , Sato, Y., et al. (2020). Evolution and Diversity of the Wild Rice Oryza officinalis Complex, across Continents, Genome Types, and Ploidy Levels. Genome biology and evolution, 12(4), 413-428.
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  The Oryza officinalis complex is the largest species group in Oryza, with more than nine species from four continents, and is a tertiary gene pool that can be exploited in breeding programs for the improvement of cultivated rice. Most diploid and tetraploid members of this group have a C genome. Using a new reference C genome for the diploid species O. officinalis, and draft genomes for two other C genome diploid species Oryza eichingeri and Oryza rhizomatis, we examine the influence of transposable elements on genome structure and provide a detailed phylogeny and evolutionary history of the Oryza C genomes. The O. officinalis genome is 1.6 times larger than the A genome of cultivated Oryza sativa, mostly due to proliferation of Gypsy type long-terminal repeat transposable elements, but overall syntenic relationships are maintained with other Oryza genomes (A, B, and F). Draft genome assemblies of the two other C genome diploid species, Oryza eichingeri and Oryza rhizomatis, and short-read resequencing of a series of other C genome species and accessions reveal that after the divergence of the C genome progenitor, there was still a substantial degree of variation within the C genome species through proliferation and loss of both DNA and long-terminal repeat transposable elements. We provide a detailed phylogeny and evolutionary history of the Oryza C genomes and a genomic resource for the exploitation of the Oryza tertiary gene pool.
• Chen, E., Huang, X., Tian, Z., Wing, R. A., & Han, B. (2019). The Genomics of Species Provides Insights into Rice Domestication and Heterosis. Annual review of plant biology, 70, 639-665.
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  Here, we review recent progress in genetic and genomic studies of the diversity of species. In recent years, unlocking the genetic diversity of species has provided insights into the genomics of rice domestication, heterosis, and complex traits. Genome sequencing and analysis of numerous wild rice () and Asian cultivated rice () accessions have enabled the identification of genome-wide signatures of rice domestication and the unlocking of the origin of Asian cultivated rice. Moreover, similar studies on genome variations of African rice () cultivars and their closely related wild progenitor accessions have provided strong evidence to support a theory of independent domestication in African rice. Integrated genomic approaches have efficiently investigated many heterotic loci in hybrid rice underlying yield heterosis advantages and revealed the genomic architecture of rice heterosis. We conclude that in-depth unlocking of genetic variations among species will further enhance rice breeding.
• Fuentes, R. R., Chebotarov, D., Duitama, J., Smith, S., De la Hoz, J. F., Mohiyuddin, M., Wing, R. A., McNally, K. L., Tatarinova, T., Grigoriev, A., Mauleon, R., & Alexandrov, N. (2019). Structural variants in 3000 rice genomes. Genome research, 29(5), 870-880.
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  Investigation of large structural variants (SVs) is a challenging yet important task in understanding trait differences in highly repetitive genomes. Combining different bioinformatic approaches for SV detection, we analyzed whole-genome sequencing data from 3000 rice genomes and identified 63 million individual SV calls that grouped into 1.5 million allelic variants. We found enrichment of long SVs in promoters and an excess of shorter variants in 5' UTRs. Across the rice genomes, we identified regions of high SV frequency enriched in stress response genes. We demonstrated how SVs may help in finding causative variants in genome-wide association analysis. These new insights into rice genome biology are valuable for understanding the effects SVs have on gene function, with the prospect of identifying novel agronomically important alleles that can be utilized to improve cultivated rice.
• Hazzouri, K. M., Gros-Balthazard, M., Flowers, J. M., Copetti, D., Lemansour, A., Lebrun, M., Masmoudi, K., Ferrand, S., Dhar, M. I., Fresquez, Z. A., Rosas, U., Zhang, J., Talag, J., Lee, S., Kudrna, D., Powell, R. F., Leitch, I. J., Krueger, R. R., Wing, R. A., , Amiri, K. M., et al. (2019). Genome-wide association mapping of date palm fruit traits. Nature communications, 10(1), 4680.
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  Date palms (Phoenix dactylifera) are an important fruit crop of arid regions of the Middle East and North Africa. Despite its importance, few genomic resources exist for date palms, hampering evolutionary genomic studies of this perennial species. Here we report an improved long-read genome assembly for P. dactylifera that is 772.3 Mb in length, with contig N50 of 897.2 Kb, and use this to perform genome-wide association studies (GWAS) of the sex determining region and 21 fruit traits. We find a fruit color GWAS at the R2R3-MYB transcription factor VIRESCENS gene and identify functional alleles that include a retrotransposon insertion and start codon mutation. We also find a GWAS peak for sugar composition spanning deletion polymorphisms in multiple linked invertase genes. MYB transcription factors and invertase are implicated in fruit color and sugar composition in other crops, demonstrating the importance of parallel evolution in the evolutionary diversification of domesticated species.
• Jain, R., Jenkins, J., Shu, S., Chern, M., Martin, J. A., Copetti, D., Duong, P. Q., Pham, N. T., Kudrna, D. A., Talag, J., Schackwitz, W. S., Lipzen, A. M., Dilworth, D., Bauer, D., Grimwood, J., Nelson, C. R., Xing, F., Xie, W., Barry, K. W., , Wing, R. A., et al. (2019). Genome sequence of the model rice variety KitaakeX. BMC genomics, 20(1), 905.
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  The availability of thousands of complete rice genome sequences from diverse varieties and accessions has laid the foundation for in-depth exploration of the rice genome. One drawback to these collections is that most of these rice varieties have long life cycles, and/or low transformation efficiencies, which limits their usefulness as model organisms for functional genomics studies. In contrast, the rice variety Kitaake has a rapid life cycle (9 weeks seed to seed) and is easy to transform and propagate. For these reasons, Kitaake has emerged as a model for studies of diverse monocotyledonous species.
• Zhang, L., Ren, Y., Yang, T., Li, G., Chen, J., Gschwend, A. R., Yu, Y., Hou, G., Zi, J., Zhou, R., Wen, B., Zhang, J., Chougule, K., Wang, M., Copetti, D., Peng, Z., Zhang, C., Zhang, Y., Ouyang, Y., , Wing, R. A., et al. (2019). Rapid evolution of protein diversity by de novo origination in Oryza. Nature ecology & evolution, 3(4), 679-690.
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  New protein-coding genes that arise de novo from non-coding DNA sequences contribute to protein diversity. However, de novo gene origination is challenging to study as it requires high-quality reference genomes for closely related species, evidence for ancestral non-coding sequences, and transcription and translation of the new genes. High-quality genomes of 13 closely related Oryza species provide unprecedented opportunities to understand de novo origination events. Here, we identify a large number of young de novo genes with discernible recent ancestral non-coding sequences and evidence of translation. Using pipelines examining the synteny relationship between genomes and reciprocal-best whole-genome alignments, we detected at least 175 de novo open reading frames in the focal species O. sativa subspecies japonica, which were all detected in RNA sequencing-based transcriptomes. Mass spectrometry-based targeted proteomics and ribosomal profiling show translational evidence for 57% of the de novo genes. In recent divergence of Oryza, an average of 51.5 de novo genes per million years were generated and retained. We observed evolutionary patterns in which excess indels and early transcription were favoured in origination with a stepwise formation of gene structure. These data reveal that de novo genes contribute to the rapid evolution of protein diversity under positive selection.
• Kim, N. H., Jayakodi, M., Lee, S. C., Choi, B. S., Jang, W., Lee, J., Kim, H. H., Waminal, N. E., Lakshmanan, M., van Nguyen, B., Lee, Y. S., Park, H. S., Koo, H. J., Park, J. Y., Perumal, S., Joh, H. J., Lee, H., Kim, J., Kim, I. S., , Kim, K., et al. (2018). Genome and evolution of the shade-requiring medicinal herb Panax ginseng. Plant biotechnology journal.
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  Panax ginseng C. A. Meyer, reputed as the king of medicinal herbs, has slow growth, long generation time, low seed production and complicated genome structure that hamper its study. Here, we unveil the genomic architecture of tetraploid P. ginseng by de novo genome assembly, representing 2.98 Gbp with 59 352 annotated genes. Resequencing data indicated that diploid Panax species diverged in association with global warming in Southern Asia, and two North American species evolved via two intercontinental migrations. Two whole genome duplications (WGD) occurred in the family Araliaceae (including Panax) after divergence with the Apiaceae, the more recent one contributing to the ability of P. ginseng to overwinter, enabling it to spread broadly through the Northern Hemisphere. Functional and evolutionary analyses suggest that production of pharmacologically important dammarane-type ginsenosides originated in Panax and are produced largely in shoot tissues and transported to roots; that newly evolved P. ginseng fatty acid desaturases increase freezing tolerance; and that unprecedented retention of chlorophyll a/b binding protein genes enables efficient photosynthesis under low light. A genome-scale metabolic network provides a holistic view of Panax ginsenoside biosynthesis. This study provides valuable resources for improving medicinal values of ginseng either through genomics-assisted breeding or metabolic engineering.
• Lv, S., Wu, W., Wang, M., Meyer, R. S., Ndjiondjop, M. N., Tan, L., Zhou, H., Zhang, J., Fu, Y., Cai, H., Sun, C., Wing, R. A., & Zhu, Z. (2018). Genetic control of seed shattering during African rice domestication. Nature plants, 4(6), 331-337.
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  Domestication represents a unique opportunity to study the evolutionary process. The elimination of seed dispersal traits was a key step in the evolution of cereal crops under domestication. Here, we show that ObSH3, a YABBY transcription factor, is required for the development of the seed abscission layer. Moreover, selecting a genomic segment deletion containing SH3 resulted in the loss of seed dispersal in populations of African cultivated rice (Oryza glaberrima Steud.). Functional characterization of SH3 and SH4 (another gene controlling seed shattering on chromosome 4) revealed that multiple genes can lead to a spectrum of non-shattering phenotypes, affecting other traits such as ease of threshing that may be important to tune across different agroecologies and postharvest practices. The molecular evolution analyses of SH3 and SH4 in a panel of 93 landraces provided unprecedented geographical detail of the domestication history of African rice, tracing multiple dispersals from a core heartland and introgression from local wild rice. The cloning of ObSH3 not only provides new insights into a critical crop domestication process but also adds to the body of knowledge on the molecular mechanism of seed dispersal.
• Scheben, A., Chan, C. K., Mansueto, L., Mauleon, R., Larmande, P., Alexandrov, N., Wing, R. A., McNally, K. L., Quesneville, H., & Edwards, D. (2018). Progress in single-access information systems for wheat and rice crop improvement. Briefings in bioinformatics.
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  Improving productivity of the staple crops wheat and rice is essential to feed the growing global population, particularly in the context of a changing climate. However, current rates of yield gain are insufficient to support the predicted population growth. New approaches are required to accelerate the breeding process, and many of these are driven by the application of large-scale crop data. To leverage the substantial volumes and types of data that can be applied for precision breeding, the wheat and rice research communities are working towards the development of integrated systems to access and standardize the dispersed, heterogeneous available data. Here, we outline the initiatives of the International Wheat Information System (WheatIS) and the International Rice Informatics Consortium (IRIC) to establish Web-based single-access systems and data mining tools to make the available resources more accessible, drive discovery and accelerate the production of new crop varieties. We discuss the progress of WheatIS and IRIC towards unifying specialized wheat and rice databases and building custom software platforms to manage and interrogate these data. Single-access crop information systems will strengthen scientific collaboration, optimize the use of public research funds and help achieve the required yield gains in the two most important global food crops.
• Stein, J. C., Yu, Y., Copetti, D., Zwickl, D. J., Zhang, L., Zhang, C., Chougule, K., Gao, D., Iwata, A., Goicoechea, J. L., Wei, S., Wang, J., Liao, Y., Wang, M., Jacquemin, J., Becker, C., Kudrna, D., Zhang, J., Londono, C. E., , Song, X., et al. (2018). Genomes of 13 domesticated and wild rice relatives highlight genetic conservation, turnover and innovation across the genus Oryza. Nature genetics.
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  The genus Oryza is a model system for the study of molecular evolution over time scales ranging from a few thousand to 15 million years. Using 13 reference genomes spanning the Oryza species tree, we show that despite few large-scale chromosomal rearrangements rapid species diversification is mirrored by lineage-specific emergence and turnover of many novel elements, including transposons, and potential new coding and noncoding genes. Our study resolves controversial areas of the Oryza phylogeny, showing a complex history of introgression among different chromosomes in the young 'AA' subclade containing the two domesticated species. This study highlights the prevalence of functionally coupled disease resistance genes and identifies many new haplotypes of potential use for future crop protection. Finally, this study marks a milestone in modern rice research with the release of a complete long-read assembly of IR 8 'Miracle Rice', which relieved famine and drove the Green Revolution in Asia 50 years ago.
• Wang, W., Mauleon, R., Hu, Z., Chebotarov, D., Tai, S., Wu, Z., Li, M., Zheng, T., Fuentes, R. R., Zhang, F., Mansueto, L., Copetti, D., Sanciangco, M., Palis, K. C., Xu, J., Sun, C., Fu, B., Zhang, H., Gao, Y., , Zhao, X., et al. (2018). Genomic variation in 3,010 diverse accessions of Asian cultivated rice. Nature, 557(7703), 43-49.
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  Here we analyse genetic variation, population structure and diversity among 3,010 diverse Asian cultivated rice (Oryza sativa L.) genomes from the 3,000 Rice Genomes Project. Our results are consistent with the five major groups previously recognized, but also suggest several unreported subpopulations that correlate with geographic location. We identified 29 million single nucleotide polymorphisms, 2.4 million small indels and over 90,000 structural variations that contribute to within- and between-population variation. Using pan-genome analyses, we identified more than 10,000 novel full-length protein-coding genes and a high number of presence-absence variations. The complex patterns of introgression observed in domestication genes are consistent with multiple independent rice domestication events. The public availability of data from the 3,000 Rice Genomes Project provides a resource for rice genomics research and breeding.
• Wing, R. A., Purugganan, M. D., & Zhang, Q. (2018). The rice genome revolution: from an ancient grain to Green Super Rice. Nature reviews. Genetics.
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  Rice is a staple crop for half the world's population, which is expected to grow by 3 billion over the next 30 years. It is also a key model for studying the genomics of agroecosystems. This dual role places rice at the centre of an enormous challenge facing agriculture: how to leverage genomics to produce enough food to feed an expanding global population. Scientists worldwide are investigating the genetic variation among domesticated rice species and their wild relatives with the aim of identifying loci that can be exploited to breed a new generation of sustainable crops known as Green Super Rice.
• Choi, J. Y., Platts, A. E., Fuller, D. Q., Hsing, Y. I., Wing, R. A., & Purugganan, M. D. (2017). The rice paradox: Multiple origins but single domestication in Asian rice. Molecular biology and evolution.
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  The origin of domesticated Asian rice (Oryza sativa) has been a contentious topic, with conflicting evidence for either single or multiple domestication of this key crop species. We examined the evolutionary history of domesticated rice by analyzing de novo assembled genomes from domesticated rice and its wild progenitors. Our results indicate multiple origins, where each domesticated rice subpopulation (japonica, indica, and aus) arose separately from progenitor O. rufipogon and/or O. nivara Coalescence-based modeling of demographic parameters estimate that the first domesticated rice population to split off from O. rufipogon was O. sativa ssp. japonica, occurring at ∼13.1 - 24.1 kya, which is an order of magnitude older then the earliest archaeological date of domestication. This date is consistent, however, with the expansion of O. rufipogon populations after the Last Glacial Maximum ∼18 kya and archaeological evidence for early wild rice management in China. We also show that there is significant gene flow from japonica to both indica (∼17%) and aus (∼15%), which led to the transfer of domestication alleles from early-domesticated japonica to proto-indica and proto-aus populations. Our results provide support for a model in which different rice subspecies had separate origins, but that de novo domestication occurred only once, in O. sativa ssp. japonica, and introgressive hybridization from early japonica to proto-indica and proto-aus led to domesticated indica and aus rice.
• Copetti, D., Búrquez, A., Bustamante, E., Charboneau, J. L., Childs, K. L., Eguiarte, L. E., Lee, S., Liu, T. L., McMahon, M. M., Whiteman, N. K., Wing, R. A., Wojciechowski, M. F., & Sanderson, M. J. (2017). Extensive gene tree discordance and hemiplasy shaped the genomes of North American columnar cacti. Proceedings of the National Academy of Sciences of the United States of America.
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  Few clades of plants have proven as difficult to classify as cacti. One explanation may be an unusually high level of convergent and parallel evolution (homoplasy). To evaluate support for this phylogenetic hypothesis at the molecular level, we sequenced the genomes of four cacti in the especially problematic tribe Pachycereeae, which contains most of the large columnar cacti of Mexico and adjacent areas, including the iconic saguaro cactus (Carnegiea gigantea) of the Sonoran Desert. We assembled a high-coverage draft genome for saguaro and lower coverage genomes for three other genera of tribe Pachycereeae (Pachycereus, Lophocereus, and Stenocereus) and a more distant outgroup cactus, Pereskia We used these to construct 4,436 orthologous gene alignments. Species tree inference consistently returned the same phylogeny, but gene tree discordance was high: 37% of gene trees having at least 90% bootstrap support conflicted with the species tree. Evidently, discordance is a product of long generation times and moderately large effective population sizes, leading to extensive incomplete lineage sorting (ILS). In the best supported gene trees, 58% of apparent homoplasy at amino sites in the species tree is due to gene tree-species tree discordance rather than parallel substitutions in the gene trees themselves, a phenomenon termed "hemiplasy." The high rate of genomic hemiplasy may contribute to apparent parallelisms in phenotypic traits, which could confound understanding of species relationships and character evolution in cacti.
• Dos Santos, R. S., Farias, D. d., Pegoraro, C., Rombaldi, C. V., Fukao, T., Wing, R. A., & de Oliveira, A. C. (2017). Evolutionary analysis of the SUB1 locus across the Oryza genomes. Rice (New York, N.Y.), 10(1), 4.
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  Tolerance to complete submergence is recognized in a limited number of Asian rice (Oryza sativa L.) varieties, most of which contain submergence-inducible SUB1A on the polygenic SUBMERGENCE-1 (SUB1) locus. It has been shown that the SUB1 locus encodes two Ethylene-Responsive Factor (ERF) genes, SUB1B and SUB1C, in all O. sativa varieties. These genes were also found in O rufipogon and O nivara, wild relatives of O. sativa. However, detailed analysis of the polygenic locus in other Oryza species has not yet been made.
• Harkess, A., Zhou, J., Xu, C., Bowers, J. E., Van der Hulst, R., Ayyampalayam, S., Mercati, F., Riccardi, P., McKain, M. R., Kakrana, A., Tang, H., Ray, J., Groenendijk, J., Arikit, S., Mathioni, S. M., Nakano, M., Shan, H., Telgmann-Rauber, A., Kanno, A., , Yue, Z., et al. (2017). The asparagus genome sheds light on the origin and evolution of a young Y chromosome. Nature communications, 8(1), 1279.
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  Sex chromosomes evolved from autosomes many times across the eukaryote phylogeny. Several models have been proposed to explain this transition, some involving male and female sterility mutations linked in a region of suppressed recombination between X and Y (or Z/W, U/V) chromosomes. Comparative and experimental analysis of a reference genome assembly for a double haploid YY male garden asparagus (Asparagus officinalis L.) individual implicates separate but linked genes as responsible for sex determination. Dioecy has evolved recently within Asparagus and sex chromosomes are cytogenetically identical with the Y, harboring a megabase segment that is missing from the X. We show that deletion of this entire region results in a male-to-female conversion, whereas loss of a single suppressor of female development drives male-to-hermaphrodite conversion. A single copy anther-specific gene with a male sterile Arabidopsis knockout phenotype is also in the Y-specific region, supporting a two-gene model for sex chromosome evolution.
• Lee, C. R., Wang, B., Mojica, J. P., Mandáková, T., Prasad, K. V., Goicoechea, J. L., Perera, N., Hellsten, U., Hundley, H. N., Johnson, J., Grimwood, J., Barry, K., Fairclough, S., Jenkins, J. W., Yu, Y., Kudrna, D., Zhang, J., Talag, J., Golser, W., , Ghattas, K., et al. (2017). Young inversion with multiple linked QTLs under selection in a hybrid zone. Nature ecology & evolution, 1(5), 119.
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  Fixed chromosomal inversions can reduce gene flow and promote speciation in two ways: by suppressing recombination and by carrying locally favoured alleles at multiple loci. However, it is unknown whether favoured mutations slowly accumulate on older inversions or if young inversions spread because they capture pre-existing adaptive quantitative trait loci (QTLs). By genetic mapping, chromosome painting and genome sequencing, we have identified a major inversion controlling ecologically important traits in Boechera stricta. The inversion arose since the last glaciation and subsequently reached local high frequency in a hybrid speciation zone. Furthermore, the inversion shows signs of positive directional selection. To test whether the inversion could have captured existing, linked QTLs, we crossed standard, collinear haplotypes from the hybrid zone and found multiple linked phenology QTLs within the inversion region. These findings provide the first direct evidence that linked, locally adapted QTLs may be captured by young inversions during incipient speciation.
• Lee, C. R., Wang, B., Mojica, J. P., Mandáková, T., Prasad, K. V., Luis Goicoechea, J., Perera, N., Hellsten, U., Hundley, H. N., Johnson, J., Grimwood, J., Barry, K., Fairclough, S., Jenkins, J. W., Yu, Y., Kudrna, D., Zhang, J., Talag, J., Golser, W., , Ghattas, K., et al. (2017). Publisher correction: Young inversion with multiple linked QTLs under selection in a hybrid zone. Nature ecology & evolution, 1(10), 1585.
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  In Fig. 5 of the version of this Article originally published, the final number on the x axes of each panel was incorrectly written as 1.5; it should have read 7.5. This has now been corrected in all versions of the Article.
• Lee, C., Wang, B., Mojica, J., Mandakova, T., Prasad, K. V., Goicoechea, J. L., Perera, N., Hellsten, U., Hundley, H. N., Johnson, J., Grimwood, J., Barry, K., Fairclough, S., Jenkins, J. W., Yu, Y., Kudrna, D., Zhang, J., Talag, J., Golser, W., , Ghattas, K., et al. (2017). Selection in a hybrid zone: evidence for linked QTLs in a young inversion. Nature Evolution and Ecology. doi:doi:10.1038/s41559-017-0166
• Schmidt, M., Wing, R. A., Cotty, P., Zhang, J., & Thakare, D. (2016). Host induced gene silencing inhibits aflatoxin production in transgenic maize when challenged with Aspergillus. Science Advances.
• Thakare, D., Zhang, J., Wing, R. A., Cotty, P. J., & Schmidt, M. A. (2017). Aflatoxin-free transgenic maize using host-induced gene silencing. Science advances, 3(3), e1602382.
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  Aflatoxins, toxic secondary metabolites produced by some Aspergillus species, are a universal agricultural economic problem and a critical health issue. Despite decades of control efforts, aflatoxin contamination is responsible for a global loss of millions of tons of crops each year. We show that host-induced gene silencing is an effective method for eliminating this toxin in transgenic maize. We transformed maize plants with a kernel-specific RNA interference (RNAi) gene cassette targeting the aflC gene, which encodes an enzyme in the Aspergillus aflatoxin biosynthetic pathway. After pathogen infection, aflatoxin could not be detected in kernels from these RNAi transgenic maize plants, while toxin loads reached thousands of parts per billion in nontransgenic control kernels. A comparison of transcripts in developing aflatoxin-free transgenic kernels with those from nontransgenic kernels showed no significant differences between these two groups. These results demonstrate that small interfering RNA molecules can be used to silence aflatoxin biosynthesis in maize, providing an attractive and precise engineering strategy that could also be extended to other crops to improve food security.
• Wu, W., Liu, X., Wang, M., Meyer, R. S., Luo, X., Ndjiondjop, M. N., Tan, L., Zhang, J., Wu, J., Cai, H., Sun, C., Wang, X., Wing, R. A., & Zhu, Z. (2017). A single-nucleotide polymorphism causes smaller grain size and loss of seed shattering during African rice domestication. Nature plants, 3, 17064.
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  Grain size is one of the most important components of grain yield and selecting large seeds has been a main target during plant domestication. Surprisingly, the grain of African cultivated rice (Oryza glaberrima Steud.) typically is smaller than that of its progenitor, Oryza barthii. Here we report the cloning and characterization of a quantitative trait locus, GL4, controlling the grain length on chromosome 4 in African rice, which regulates longitudinal cell elongation of the outer and inner glumes. Interestingly, GL4 also controls the seed shattering phenotype like its orthologue SH4 gene in Asian rice. Our data show that a single-nucleotide polymorphism (SNP) mutation in the GL4 gene resulted in a premature stop codon and led to small seeds and loss of seed shattering during African rice domestication. These results provide new insights into diverse domestication practices in African rice, and also pave the way for enhancing crop yield to meeting the challenge of cereal demand in West Africa.
• Wu, W., Liu, X., Wang, M., Meyer, R. S., Luo, X., Ndjiondjop,, M., Tan, L., Zhang, J., Wu, J., Cai, H., Sun, C., Wang, X., Wing, R. A., & Zhu, Z. (2017). A single-nucleotide polymorphism causes smaller grain size and loss of seed shattering during African rice domestication. Nature Plants. doi:10.1038/nplants.2017.64
• dos Santos, R. S., Farias, D. R., Pegoraro, C., Rombaldi, C. V., Fukao, T., Wing, R. A., & Oliveira, A. C. (2017). Evolutionary analysis of the SUB1 locus across the Oryza genomes. Rice. doi:10.1186/s12284-016-0140-3
• Bai, Z., Chen, J., Liao, Y., Wang, M., Liu, R., Ge, S., Wing, R. A., & Chen, M. (2016). The impact and origin of copy number variations in the Oryza species. BMC genomics, 17, 261.
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  Copy number variation (CNV), a complex genomic rearrangement, has been extensively studied in humans and other organisms. In plants, CNVs of several genes were found to be responsible for various important traits; however, the cause and consequence of CNVs remains largely unknown. Recently released next-generation sequencing (NGS) data provide an opportunity for a genome-wide study of CNVs in rice.
• Brozynska, M., Copetti, D., Furtado, A., Wing, R. A., Crayn, D., Fox, G., Ishikawa, R., & Henry, R. J. (2016). Sequencing of Australian wild rice genomes reveals ancestral relationships with domesticated rice. Plant biotechnology journal.
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  The related A genome species of the Oryza genus are the effective gene pool for rice. Here we report draft genomes for two Australian wild A genome taxa: O. rufipogon-like population, referred to as Taxon A and O. meridionalis-like population, referred to as Taxon B. These two taxa were sequenced and assembled by integration of short and long read next generation sequencing (NGS) data to create a genomic platform for a wider rice gene pool. Here we report that, despite the distinct chloroplast genome, the nuclear genome of the Australian Taxon A, has a sequence that is much closer to that of domesticated rice (O. sativa) than to the other Australian wild populations. Analysis of 4,643 genes in the A genome clade showed that the Australian annual, O. meridionalis, and related perennial taxa have the most divergent (around 3 million years) genome sequences relative to domesticated rice. A test for admixture showed possible introgression into the Australian Taxa A (diverged around 1.6 million years ago) especially from the wild indica/O. nivara clade in Asia. These results demonstrate that northern Australia may be the centre of diversity of the A genome Oryza and suggest the possibility that this might also be the centre of origin of this group and represent an important resource for rice improvement. This article is protected by copyright. All rights reserved.
• Copetti, D., & Wing, R. A. (2016). The Dark Side of the Genome: Revealing the Native Transposable Element/Repeat Content of Eukaryotic Genomes. Molecular plant, 9(12), 1664-1666.
• Gan, X., Hay, A., Kwantes, M., Haberer, G., Hallab, A., Ioio, R. D., Hofhuis, H., Pieper, B., Cartolano, M., Neumann, U., Nikolov, L. A., Song, B., Hajheidari, M., Briskine, R., Kougioumoutzi, E., Vlad, D., Broholm, S., Hein, J., Meksem, K., , Lightfoot, D., et al. (2016). The Cardamine hirsuta genome offers insight into the evolution of morphological diversity. Nature plants, 2(11), 16167.
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  Finding causal relationships between genotypic and phenotypic variation is a key focus of evolutionary biology, human genetics and plant breeding. To identify genome-wide patterns underlying trait diversity, we assembled a high-quality reference genome of Cardamine hirsuta, a close relative of the model plant Arabidopsis thaliana. We combined comparative genome and transcriptome analyses with the experimental tools available in C. hirsuta to investigate gene function and phenotypic diversification. Our findings highlight the prevalent role of transcription factors and tandem gene duplications in morphological evolution. We identified a specific role for the transcriptional regulators PLETHORA5/7 in shaping leaf diversity and link tandem gene duplication with differential gene expression in the explosive seed pod of C. hirsuta. Our work highlights the value of comparative approaches in genetically tractable species to understand the genetic basis for evolutionary change.
• Mansueto, L., Fuentes, R. R., Borja, F. N., Detras, J., Abriol-Santos, J. M., Chebotarov, D., Sanciangco, M., Palis, K., Copetti, D., Poliakov, A., Dubchak, I., Solovyev, V., Wing, R. A., Hamilton, R. S., Mauleon, R., McNally, K. L., & Alexandrov, N. (2017). Rice SNP-seek database update: new SNPs, indels, and queries. Nucleic acids research.
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  We describe updates to the Rice SNP-Seek Database since its first release. We ran a new SNP-calling pipeline followed by filtering that resulted in complete, base, filtered and core SNP datasets. Besides the Nipponbare reference genome, the pipeline was run on genome assemblies of IR 64, 93-11, DJ 123 and Kasalath. New genotype query and display features are added for reference assemblies, SNP datasets and indels. JBrowse now displays BAM, VCF and other annotation tracks, the additional genome assemblies and an embedded VISTA genome comparison viewer. Middleware is redesigned for improved performance by using a hybrid of HDF5 and RDMS for genotype storage. Query modules for genotypes, varieties and genes are improved to handle various constraints. An integrated list manager allows the user to pass query parameters for further analysis. The SNP Annotator adds traits, ontology terms, effects and interactions to markers in a list. Web-service calls were implemented to access most data. These features enable seamless querying of SNP-Seek across various biological entities, a step toward semi-automated gene-trait association discovery. URL: http://snp-seek.irri.org.
• Ray, S., Bose, L. K., Ray, J., Ngangkham, U., Katara, J. L., Samantaray, S., Behera, L., Anumalla, M., Singh, O. N., Chen, M., Wing, R. A., & Mohapatra, T. (2016). Development and validation of cross-transferable and polymorphic DNA markers for detecting alien genome introgression in Oryza sativa from Oryza brachyantha. Molecular genetics and genomics : MGG, 291(4), 1783-94.
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  African wild rice Oryza brachyantha (FF), a distant relative of cultivated rice Oryza sativa (AA), carries genes for pests and disease resistance. Molecular marker assisted alien gene introgression from this wild species to its domesticated counterpart is largely impeded due to the scarce availability of cross-transferable and polymorphic molecular markers that can clearly distinguish these two species. Availability of the whole genome sequence (WGS) of both the species provides a unique opportunity to develop markers, which are cross-transferable. We observed poor cross-transferability (~0.75 %) of O. sativa specific sequence tagged microsatellite (STMS) markers to O. brachyantha. By utilizing the genome sequence information, we developed a set of 45 low cost PCR based co-dominant polymorphic markers (STS and CAPS). These markers were found cross-transferrable (84.78 %) between the two species and could distinguish them from each other and thus allowed tracing alien genome introgression. Finally, we validated a Monosomic Alien Addition Line (MAAL) carrying chromosome 1 of O. brachyantha in O. sativa background using these markers, as a proof of concept. Hence, in this study, we have identified a set molecular marker (comprising of STMS, STS and CAPS) that are capable of detecting alien genome introgression from O. brachyantha to O. sativa.
• Wang, J., Yu, Y., Tao, F., Zhang, J., Copetti, D., Kudrna, D., Talag, J., Lee, S., Wing, R. A., & Fan, C. (2016). DNA methylation changes facilitated evolution of genes derived from Mutator-like transposable elements. Genome biology, 17(1), 92.
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  Mutator-like transposable elements, a class of DNA transposons, exist pervasively in both prokaryotic and eukaryotic genomes, with more than 10,000 copies identified in the rice genome. These elements can capture ectopic genomic sequences that lead to the formation of new gene structures. Here, based on whole-genome comparative analyses, we comprehensively investigated processes and mechanisms of the evolution of putative genes derived from Mutator-like transposable elements in ten Oryza species and the outgroup Leersia perieri, bridging ~20 million years of evolutionary history.
• Wendel, J. F., Jackson, S. A., Meyers, B. C., & Wing, R. A. (2016). Evolution of plant genome architecture. Genome biology (an invited and peer-reviewed review article), 17, 37.
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  We have witnessed an explosion in our understanding of the evolution and structure of plant genomes in recent years. Here, we highlight three important emergent realizations: (1) that the evolutionary history of all plant genomes contains multiple, cyclical episodes of whole-genome doubling that were followed by myriad fractionation processes; (2) that the vast majority of the variation in genome size reflects the dynamics of proliferation and loss of lineage-specific transposable elements; and (3) that various classes of small RNAs help shape genomic architecture and function. We illustrate ways in which understanding these organism-level and molecular genetic processes can be used for crop plant improvement.
• Wicker, T., Yu, Y., Haberer, G., Mayer, K. F., Marri, P. R., Rounsley, S., Chen, M., Zuccolo, A., Panaud, O., Wing, R. A., & Roffler, S. (2016). DNA transposon activity is associated with increased mutation rates in genes of rice and other grasses. Nature communications, 7, 12790.
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  DNA (class 2) transposons are mobile genetic elements which move within their 'host' genome through excising and re-inserting elsewhere. Although the rice genome contains tens of thousands of such elements, their actual role in evolution is still unclear. Analysing over 650 transposon polymorphisms in the rice species Oryza sativa and Oryza glaberrima, we find that DNA repair following transposon excisions is associated with an increased number of mutations in the sequences neighbouring the transposon. Indeed, the 3,000 bp flanking the excised transposons can contain over 10 times more mutations than the genome-wide average. Since DNA transposons preferably insert near genes, this is correlated with increases in mutation rates in coding sequences and regulatory regions. Most importantly, we find this phenomenon also in maize, wheat and barley. Thus, these findings suggest that DNA transposon activity is a major evolutionary force in grasses which provide the basis of most food consumed by humankind.
• Zhang, J., Chen, L., Sun, S., Kudrna, D., Copetti, D., Li, W., Mu, T., Jiao, W., Xing, F., Lee, S., Talag, J., Song, J., Du, B., Xie, W., Luo, M., Maldonado, C. E., Goicoechea, J. L., Xiong, L., Wu, C., , Xing, Y., et al. (2016). Building two indica rice reference genomes with PacBio long-read and Illumina paired-end sequencing data. Scientific data, 3, 160076.
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  Over the past 30 years, we have performed many fundamental studies on two Oryza sativa subsp. indica varieties, Zhenshan 97 (ZS97) and Minghui 63 (MH63). To improve the resolution of many of these investigations, we generated two reference-quality reference genome assemblies using the most advanced sequencing technologies. Using PacBio SMRT technology, we produced over 108 (ZS97) and 174 (MH63) Gb of raw sequence data from 166 (ZS97) and 209 (MH63) pools of BAC clones, and generated ~97 (ZS97) and ~74 (MH63) Gb of paired-end whole-genome shotgun (WGS) sequence data with Illumina sequencing technology. With these data, we successfully assembled two platinum standard reference genomes that have been publicly released. Here we provide the full sets of raw data used to generate these two reference genome assemblies. These data sets can be used to test new programs for better genome assembly and annotation, aid in the discovery of new insights into genome structure, function, and evolution, and help to provide essential support to biological research in general.
• Zhang, J., Chen, L., Xing, F., Kudrna, D. A., Yao, W., Copetti, D., Mu, T., Li, W., Song, J., Xie, W., Lee, S., Talag, J., Shao, L., An, Y., Zhang, C., Ouyang, Y., Sun, S., Jiao, W., Lv, F., , Du, B., et al. (2016). Extensive sequence divergence between the reference genomes of two elite indica rice varieties Zhenshan 97 and Minghui 63. Proceedings of the National Academy of Sciences of the United States of America, 113(35), E5163-71.
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  Asian cultivated rice consists of two subspecies: Oryza sativa subsp. indica and O. sativa subsp. japonica Despite the fact that indica rice accounts for over 70% of total rice production worldwide and is genetically much more diverse, a high-quality reference genome for indica rice has yet to be published. We conducted map-based sequencing of two indica rice lines, Zhenshan 97 (ZS97) and Minghui 63 (MH63), which represent the two major varietal groups of the indica subspecies and are the parents of an elite Chinese hybrid. The genome sequences were assembled into 237 (ZS97) and 181 (MH63) contigs, with an accuracy >99.99%, and covered 90.6% and 93.2% of their estimated genome sizes. Comparative analyses of these two indica genomes uncovered surprising structural differences, especially with respect to inversions, translocations, presence/absence variations, and segmental duplications. Approximately 42% of nontransposable element related genes were identical between the two genomes. Transcriptome analysis of three tissues showed that 1,059-2,217 more genes were expressed in the hybrid than in the parents and that the expressed genes in the hybrid were much more diverse due to their divergence between the parental genomes. The public availability of two high-quality reference genomes for the indica subspecies of rice will have large-ranging implications for plant biology and crop genetic improvement.
• Zhang, J., Kudrna, D., Mu, T., Li, W., Copetti, D., Yu, Y., Goicoechea, J. L., Lei, Y., & Wing, R. A. (2016). Genome puzzle master (GPM): an integrated pipeline for building and editing pseudomolecules from fragmented sequences. Bioinformatics (Oxford, England), 32(20), 3058-3064.
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  Next generation sequencing technologies have revolutionized our ability to rapidly and affordably generate vast quantities of sequence data. Once generated, raw sequences are assembled into contigs or scaffolds. However, these assemblies are mostly fragmented and inaccurate at the whole genome scale, largely due to the inability to integrate additional informative datasets (e.g. physical, optical and genetic maps). To address this problem, we developed a semi-automated software tool-Genome Puzzle Master (GPM)-that enables the integration of additional genomic signposts to edit and build 'new-gen-assemblies' that result in high-quality 'annotation-ready' pseudomolecules.
• van Andel, T. R., Meyer, R. S., Aflitos, S. A., Carney, J. A., Veltman, M. A., Copetti, D., Flowers, J. M., Havinga, R. M., Maat, H., Purugganan, M. D., Wing, R. A., & Schranz, M. E. (2016). Tracing ancestor rice of Suriname Maroons back to its African origin. Nature plants, 2, 16149.
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  African rice (Oryza glaberrima) and African cultivation practices are said to have influenced emerging colonial plantation economies in the Americas(1,2). However, the level of impact of African rice practices is difficult to establish because of limited written or botanical records(2,3). Recent findings of O. glaberrima in rice fields of Suriname Maroons bear evidence of the high level of knowledge about rice among African slaves and their descendants, who consecrate it in ancestor rituals(4,5). Here we establish the strong similarity, and hence likely origin, of the first extant New World landrace of O. glaberrima to landraces from the Upper Guinean forests in West Africa. We collected African rice from a Maroon market in Paramaribo, Suriname, propagated it, sequenced its genome(6) and compared it with genomes of 109 accessions representing O. glaberrima diversity across West Africa. By analysing 1,649,769 single nucleotide polymorphisms (SNPs) in clustering analyses, the Suriname sample appears sister to an Ivory Coast landrace, and shows no evidence of introgression from Asian rice. Whereas the Dutch took most slaves from Ghana, Benin and Central Africa(7), the diaries of slave ship captains record the purchase of food for provisions when sailing along the West African Coast(8), offering one possible explanation for the patterns of genetic similarity. This study demonstrates the utility of genomics in understanding the largely unwritten histories of crop cultures of diaspora communities.
• Copetti, D., Zhang, J., El Baidouri, M., Gao, D., Wang, J., Barghini, E., Cossu, R. M., Angelova, A., Maldonado L, C. E., Roffler, S., Ohyanagi, H., Wicker, T., Fan, C., Zuccolo, A., Chen, M., Costa de Oliveira, A., Han, B., Henry, R., Hsing, Y., , Kurata, N., et al. (2015). RiTE database: a resource database for genus-wide rice genomics and evolutionary biology. BMC genomics, 16, 538.
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  Comparative evolutionary analysis of whole genomes requires not only accurate annotation of gene space, but also proper annotation of the repetitive fraction which is often the largest component of most if not all genomes larger than 50 kb in size.
• De Vega, J. J., Ayling, S., Hegarty, M., Kudrna, D., Goicoechea, J. L., Ergon, Å., Rognli, O. A., Jones, C., Swain, M., Geurts, R., Lang, C., Mayer, K. F., Rössner, S., Yates, S., Webb, K. J., Donnison, I. S., Oldroyd, G. E., Wing, R. A., Caccamo, M., , Powell, W., et al. (2015). Red clover (Trifolium pratense L.) draft genome provides a platform for trait improvement. Scientific reports, 5, 17394.
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  Red clover (Trifolium pratense L.) is a globally significant forage legume in pastoral livestock farming systems. It is an attractive component of grassland farming, because of its high yield and protein content, nutritional value and ability to fix atmospheric nitrogen. Enhancing its role further in sustainable agriculture requires genetic improvement of persistency, disease resistance, and tolerance to grazing. To help address these challenges, we have assembled a chromosome-scale reference genome for red clover. We observed large blocks of conserved synteny with Medicago truncatula and estimated that the two species diverged ~23 million years ago. Among the 40,868 annotated genes, we identified gene clusters involved in biochemical pathways of importance for forage quality and livestock nutrition. Genotyping by sequencing of a synthetic population of 86 genotypes show that the number of markers required for genomics-based breeding approaches is tractable, making red clover a suitable candidate for association studies and genomic selection.
• Gao, D., Jiang, N., Wing, R. A., Jiang, J., & Jackson, S. A. (2015). Transposons play an important role in the evolution and diversification of centromeres among closely related species. Frontiers in plant science, 6, 216.
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  Centromeres are important chromosomal regions necessary for eukaryotic cell segregation and replication. Due to high amounts of tandem repeats and transposons, centromeres have been difficult to sequence in most multicellular organisms, thus their sequence structure and evolution are poorly understood. In this study, we analyzed transposons in the centromere 8 (Cen8) from the African cultivated rice (O. glaberrima) and two subspecies of the Asian cultivated rice (O. sativa), indica and japonica. We detected much higher transposon contents (>69%) in centromere regions than in the whole genomes of O. sativa ssp. japonica and O. glaberrima (~35%). We compared the three Cen8s and identified numerous recent insertions of transposons that were frequently organized into multiple-layer nested blocks, similar to nested transposons in maize. Except for the Hopi retrotransposon, all LTR retrotransposons were shared but exhibit different abundances amongst the three Cen8s. Even though a majority of the transposons were located in intergenic regions, some gene-related transposons were found and may be involved in gene diversification. Chromatin immunoprecipitated (ChIP) data analysis revealed that 165 families from both Class I and Class II transposons were found in CENH3-associated chromatin sequences. These results indicate essential roles for transposons in centromeres and that the rapid divergence of the Cen8 sequences between the two cultivated rice species was primarily caused by recent transposon insertions.
• Kim, K., Lee, S., Lee, J., Yu, Y., Yang, K., Choi, B., Koh, H., Waminal, N. E., Choi, H., Kim, N., Jang, W., Park, H., Lee, J., Lee, H. O., Joh, H. J., Lee, H. J., Park, J. Y., Perumal, S., Jayakodi, M., , Lee, Y. S., et al. (2015). Complete chloroplast and ribosomal sequences for 30 accessions elucidate evolution of Oryza AA genome species. Scientific reports, 5, 15655.
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  Cytoplasmic chloroplast (cp) genomes and nuclear ribosomal DNA (nR) are the primary sequences used to understand plant diversity and evolution. We introduce a high-throughput method to simultaneously obtain complete cp and nR sequences using Illumina platform whole-genome sequence. We applied the method to 30 rice specimens belonging to nine Oryza species. Concurrent phylogenomic analysis using cp and nR of several of specimens of the same Oryza AA genome species provides insight into the evolution and domestication of cultivated rice, clarifying three ambiguous but important issues in the evolution of wild Oryza species. First, cp-based trees clearly classify each lineage but can be biased by inter-subspecies cross-hybridization events during speciation. Second, O. glumaepatula, a South American wild rice, includes two cytoplasm types, one of which is derived from a recent interspecies hybridization with O. longistminata. Third, the Australian O. rufipogan-type rice is a perennial form of O. meridionalis.
• Muñoz-Amatriaín, M., Lonardi, S., Luo, M., Madishetty, K., Svensson, J. T., Moscou, M. J., Wanamaker, S., Jiang, T., Kleinhofs, A., Muehlbauer, G. J., Wise, R. P., Stein, N., Ma, Y., Rodriguez, E., Kudrna, D., Bhat, P. R., Chao, S., Condamine, P., Heinen, S., , Resnik, J., et al. (2015). Sequencing of 15 622 gene-bearing BACs clarifies the gene-dense regions of the barley genome. The Plant journal : for cell and molecular biology, 84(1), 216-27.
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  Barley (Hordeum vulgare L.) possesses a large and highly repetitive genome of 5.1 Gb that has hindered the development of a complete sequence. In 2012, the International Barley Sequencing Consortium released a resource integrating whole-genome shotgun sequences with a physical and genetic framework. However, because only 6278 bacterial artificial chromosome (BACs) in the physical map were sequenced, fine structure was limited. To gain access to the gene-containing portion of the barley genome at high resolution, we identified and sequenced 15 622 BACs representing the minimal tiling path of 72 052 physical-mapped gene-bearing BACs. This generated ~1.7 Gb of genomic sequence containing an estimated 2/3 of all Morex barley genes. Exploration of these sequenced BACs revealed that although distal ends of chromosomes contain most of the gene-enriched BACs and are characterized by high recombination rates, there are also gene-dense regions with suppressed recombination. We made use of published map-anchored sequence data from Aegilops tauschii to develop a synteny viewer between barley and the ancestor of the wheat D-genome. Except for some notable inversions, there is a high level of collinearity between the two species. The software HarvEST:Barley provides facile access to BAC sequences and their annotations, along with the barley-Ae. tauschii synteny viewer. These BAC sequences constitute a resource to improve the efficiency of marker development, map-based cloning, and comparative genomics in barley and related crops. Additional knowledge about regions of the barley genome that are gene-dense but low recombination is particularly relevant.
• Sanderson, M. J., Copetti, D., Búrquez, A., Bustamante, E., Charboneau, J. L., Eguiarte, L. E., Kumar, S., Lee, H. O., Lee, J., McMahon, M., Steele, K., Wing, R., Yang, T., Zwickl, D., & Wojciechowski, M. F. (2015). Exceptional reduction of the plastid genome of saguaro cactus (Carnegiea gigantea): Loss of the ndh gene suite and inverted repeat. American journal of botany, 102(7), 1115-27.
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• Wicker, T., Wing, R. A., & Schubert, I. (2015). Recurrent sequence exchange between homeologous grass chromosomes. The Plant journal : for cell and molecular biology, 84(4), 747-59.
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  All grass species evolved from an ancestor that underwent a whole-genome duplication (WGD) approximately 70 million years ago. Interestingly, the short arms of rice chromosomes 11 and 12 (and independently their homologs in sorghum) were found to be much more similar to each other than other homeologous regions within the duplicated genome. Based on detailed analysis of rice chromosomes 11 and 12 and their homologs in seven grass species, we propose a mechanism that explains the apparently 'younger' age of the duplication in this region of the genome, assuming a small number of reciprocal translocations at the chromosome termini. In each case the translocations were followed by unbalanced transmission and subsequent lineage sorting of the involved chromosomes to offspring. Molecular dating of these translocation events also allowed us to date major chromosome 'fusions' in the evolutionary lineages that led to Brachypodium and Triticeae. Furthermore, we provide evidence that rice is exceptional regarding the evolution of chromosomes 11 and 12, inasmuch as in other species the process of sequence exchange between homeologous chromosomes ceased much earlier than in rice. We presume that random events rather than selective forces are responsible for the observed high similarity between the short arm ends of rice chromosomes 11 and 12.
• Wing, R. A. (2015). Harvesting rice's dispensable genome. Genome biology, 16, 217.
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  A rapid and cost-effective approach has been developed to harvest and map the dispensable genome, that is, population-level natural sequence variation within a species that is not present in static genome assemblies.
• Zhang, Y., Zhang, S., Liu, H., Fu, B., Li, L., Xie, M., Song, Y., Li, X., Cai, J., Wan, W., Kui, L., Huang, H., Lyu, J., Dong, Y., Wang, W., Huang, L., Zhang, J., Yang, Q., Shan, Q., , Li, Q., et al. (2015). Genome and Comparative Transcriptomics of African Wild Rice Oryza longistaminata Provide Insights into Molecular Mechanism of Rhizomatousness and Self-Incompatibility. Molecular plant.
• Geering, A. D., Maumus, F., Copetti, D., Choisne, N., Zwickl, D. J., Zytnicki, M., McTaggart, A. R., Scalabrin, S., Vezzulli, S., Wing, R. A., Quesneville, H., & Teycheney, P. (2014). Endogenous florendoviruses are major components of plant genomes and hallmarks of virus evolution. Nature communications, 5, 5269.
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  The extent and importance of endogenous viral elements have been extensively described in animals but are much less well understood in plants. Here we describe a new genus of Caulimoviridae called 'Florendovirus', members of which have colonized the genomes of a large diversity of flowering plants, sometimes at very high copy numbers (>0.5% total genome content). The genome invasion of Oryza is dated to over 1.8 million years ago (MYA) but phylogeographic evidence points to an even older age of 20-34 MYA for this virus group. Some appear to have had a bipartite genome organization, a unique characteristic among viral retroelements. In Vitis vinifera, 9% of the endogenous florendovirus loci are located within introns and therefore may influence host gene expression. The frequent colocation of endogenous florendovirus loci with TA simple sequence repeats, which are associated with chromosome fragility, suggests sequence capture during repair of double-stranded DNA breaks.
• Jacquemin, J., Ammiraju, J. S., Haberer, G., Billheimer, D. D., Yu, Y., Liu, L. C., Rivera, L. F., Mayer, K., Chen, M., & Wing, R. A. (2014). Fifteen million years of evolution in the Oryza genus shows extensive gene family expansion. Molecular plant, 7(4), 642-56.
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  In analyzing gene families in the whole-genome sequences available for O. sativa (AA), O. glaberrima (AA), and O. brachyantha (FF), we observed large size expansions in the AA genomes compared to FF genomes for the super-families F-box and NB-ARC, and five additional families: the Aspartic proteases, BTB/POZ proteins (BTB), Glutaredoxins, Trypsin α-amylase inhibitor proteins, and Zf-Dof proteins. Their evolutionary dynamic was investigated to understand how and why such important size variations are observed between these closely related species. We show that expansions resulted from both amplification, largely by tandem duplications, and contraction by gene losses. For the F-box and NB-ARC gene families, the genes conserved in all species were under strong purifying selection while expanded orthologous genes were under more relaxed purifying selection. In F-box, NB-ARC, and BTB, the expanded groups were enriched in genes with little evidence of expression, in comparison with conserved groups. We also detected 87 loci under positive selection in the expanded groups. These results show that most of the duplicated copies in the expanded groups evolve neutrally after duplication because of functional redundancy but a fraction of these genes were preserved following neofunctionalization. Hence, the lineage-specific expansions observed between Oryza species were partly driven by directional selection.
• Ortiz, M., Legatzki, A., Neilson, J., Fryslie, B., Nelson, W., Wing, R., Soderlund, C., Pryor, B., & Maier, R. (2014). Making a living while starving in the dark: metagenomic insights into the energy dynamics of a carbonate cave. ISME Journal, 8, 478-491.
• Pan, Y., Deng, Y., Lin, H., Kudrna, D., Wing, R., Li, L., Zhang, Q., & Luo, M. (2014). Comparative BAC-based physical mapping of Oryza sativa ssp. indica var. 93-11 and evaluation of the two rice reference sequence assemblies. Plant Journal, 77, 795-805.
• Schmutz, J., McClean, P. E., Mamidi, S., Wu, G. A., Cannon, S. B., Grimwood, J., Jenkins, J., Shu, S., Song, Q., Chavarro, C., Torres-Torres, M., Geffroy, V., Moghaddam, S. M., Gao, D., Abernathy, B., Barry, K., Blair, M., Brick, M. A., Chovatia, M., , Gepts, P., et al. (2014). A reference genome for common bean and genome-wide analysis of dual domestications. NATURE GENETICS, 46(7), 707-713.
• Schmutz, J., McClean, P. E., Mamidi, S., Wu, G. A., Cannon, S. B., Grimwood, J., Jenkins, J., Shu, S., Song, Q., Chavarro, C., Torres-Torres, M., Geffroy, V., Moghaddam, S. M., Gao, D., Abernathy, B., Barry, K., Blair, M., Brick, M. A., Chovatia, M., , Gepts, P., et al. (2014). A reference genome for common bean and genome-wide analysis of dual domestications. Nature genetics, 46(7), 707-13.
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  Common bean (Phaseolus vulgaris L.) is the most important grain legume for human consumption and has a role in sustainable agriculture owing to its ability to fix atmospheric nitrogen. We assembled 473 Mb of the 587-Mb genome and genetically anchored 98% of this sequence in 11 chromosome-scale pseudomolecules. We compared the genome for the common bean against the soybean genome to find changes in soybean resulting from polyploidy. Using resequencing of 60 wild individuals and 100 landraces from the genetically differentiated Mesoamerican and Andean gene pools, we confirmed 2 independent domestications from genetic pools that diverged before human colonization. Less than 10% of the 74 Mb of sequence putatively involved in domestication was shared by the two domestication events. We identified a set of genes linked with increased leaf and seed size and combined these results with quantitative trait locus data from Mesoamerican cultivars. Genes affected by domestication may be useful for genomics-enabled crop improvement.
• Wang, M., Yu, Y., Haberer, G., Marri, P. R., Fan, C., Goicoechea, J. L., Zuccolo, A., Song, X., Kudrna, D., Ammiraju, J. S., Cossu, R. M., Maldonado, C., Chen, J., Lee, S., Sisneros, N., de Baynast, K., Golser, W., Wissotski, M., Kim, W., , Sanchez, P., et al. (2014). The genome sequence of African rice (Oryza glaberrima) and evidence for independent domestication. Nature genetics, 46(9), 982-8.
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  The cultivation of rice in Africa dates back more than 3,000 years. Interestingly, African rice is not of the same origin as Asian rice (Oryza sativa L.) but rather is an entirely different species (i.e., Oryza glaberrima Steud.). Here we present a high-quality assembly and annotation of the O. glaberrima genome and detailed analyses of its evolutionary history of domestication and selection. Population genomics analyses of 20 O. glaberrima and 94 Oryza barthii accessions support the hypothesis that O. glaberrima was domesticated in a single region along the Niger river as opposed to noncentric domestication events across Africa. We detected evidence for artificial selection at a genome-wide scale, as well as with a set of O. glaberrima genes orthologous to O. sativa genes that are known to be associated with domestication, thus indicating convergent yet independent selection of a common set of genes during two geographically and culturally distinct domestication processes.
• Wang, M., Yu, Y., Haberer, G., Marri, P. R., Fan, C., Goicoechea, J. L., Zuccolo, A., Song, X., Kudrna, D., Ammiraju, J., Cossu, R. M., Maldonado, C., Chen, J., Lee, S., Sisneros, N., de, B. K., Golser, W., Wissotski, M., Kim, W., , Sanchez, P., et al. (2014). The genome sequence of African rice (Oryza glaberrima) and evidence for independent domestication. NATURE GENETICS, 46(9), 982-+.
• Wang, X., Kudrna, D., Pan, Y., Wang, H., Liu, L., Lin, H., Zhang, J., Song, X., Goicoechea, J., Wing, R., Zhang, Q., & Luo, M. (2014). Global Genomic Diversity of Oryza sativa Varieties Revealed by Comparative Physical Mapping. Genetics, 196, 937-+.
• Zwickl, D. J., Stein, J. C., Wing, R. A., Ware, D., & Sanderson, M. J. (2014). Disentangling methodological and biological sources of gene tree discordance on Oryza (Poaceae) chromosome 3. Systematic biology, 63(5), 645-59.
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  We describe new methods for characterizing gene tree discordance in phylogenomic data sets, which screen for deviations from neutral expectations, summarize variation in statistical support among gene trees, and allow comparison of the patterns of discordance induced by various analysis choices. Using an exceptionally complete set of genome sequences for the short arm of chromosome 3 in Oryza (rice) species, we applied these methods to identify the causes and consequences of differing patterns of discordance in the sets of gene trees inferred using a panel of 20 distinct analysis pipelines. We found that discordance patterns were strongly affected by aspects of data selection, alignment, and alignment masking. Unusual patterns of discordance evident when using certain pipelines were reduced or eliminated by using alternative pipelines, suggesting that they were the product of methodological biases rather than evolutionary processes. In some cases, once such biases were eliminated, evolutionary processes such as introgression could be implicated. Additionally, patterns of gene tree discordance had significant downstream impacts on species tree inference. For example, inference from supermatrices was positively misleading when pipelines that led to biased gene trees were used. Several results may generalize to other data sets: we found that gene tree and species tree inference gave more reasonable results when intron sequence was included during sequence alignment and tree inference, the alignment software PRANK was used, and detectable "block-shift" alignment artifacts were removed. We discuss our findings in the context of well-established relationships in Oryza and continuing controversies regarding the domestication history of O. sativa.
• Albert, V. A., Barbazuk, W. B., dePamphilis, C. W., Der, J. P., Leebens-Mack, J., Ma, H., Palmer, J. D., Rounsley, S., Sankoff, D., Schuster, S. C., Soltis, D. E., Soltis, P. S., Wessler, S. R., Wing, R. A., Albert, V. A., Ammiraju, J., Barbazuk, W. B., Chamala, S., Chanderbali, A. S., , dePamphilis, C. W., et al. (2013). The Amborella Genome and the Evolution of Flowering Plants. SCIENCE, 342(6165), 1467-+.
• Chamala, S., Chanderbali, A. S., Der, J. P., Lan, T., Walts, B., Albert, V. A., DePamphilis, C. W., Leebens-Mack, J., Rounsley, S., Schuster, S. C., Wing, R. A., Xiao, N., Moore, R., Soltis, P. S., Soltis, D. E., & Barbazuk, W. B. (2013). Assembly and validation of the genome of the nonmodel basal angiosperm Amborella. Science, 342(6165), 1516-1517.
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  PMID: 24357320;Abstract: Genome sequencing with next-generation sequence (NGS) technologies can now be applied to organisms pivotal to addressing fundamental biological questions, but with genomes previously considered intractable or too expensive to undertake. However, for species with large and complex genomes, extensive genetic and physical map resources have, until now, been required to direct the sequencing effort and sequence assembly. As these resources are unavailable for most species, assembling high-quality genome sequences from NGS data remains challenging. We describe a strategy that uses NGS, fluorescence in situ hybridization, and whole-genome mapping to assemble a high-quality genome sequence for Amborella trichopoda, a nonmodel species crucial to understanding flowering plant evolution. These methods are applicable to many other organisms with limited genomic resources.
• Chen, J., Huang, Q., Gao, D., Wang, J., Lang, Y., Liu, T., Bo, L. i., Bai, Z., Goicoechea, J. L., Liang, C., Chen, C., Zhang, W., Sun, S., Liao, Y., Zhang, X., Yang, L., Song, C., Wang, M., Shi, J., , Liu, G., et al. (2013). Whole-genome sequencing of Oryza brachyantha reveals mechanisms underlying Oryza genome evolution. Nature Communications, 4.
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  PMID: 23481403;PMCID: PMC3615480;Abstract: The wild species of the genus Oryza contain a largely untapped reservoir of agronomically important genes for rice improvement. Here we report the 261-Mb de novo assembled genome sequence of Oryza brachyantha. Low activity of long-terminal repeat retrotransposons and massive internal deletions of ancient long-terminal repeat elements lead to the compact genome of Oryza brachyantha. We model 32,038 protein-coding genes in the Oryza brachyantha genome, of which only 70% are located in collinear positions in comparison with the rice genome. Analysing breakpoints of non-collinear genes suggests that double-strand break repair through non-homologous end joining has an important role in gene movement and erosion of collinearity in the Oryza genomes. Transition of euchromatin to heterochromatin in the rice genome is accompanied by segmental and tandem duplications, further expanded by transposable element insertions. The high-quality reference genome sequence of Oryza brachyantha provides an important resource for functional and evolutionary studies in the genus Oryza. © 2013 Macmillan Publishers Limited. All rights reserved.
• DePamphilis, C. W., Palmer, J. D., Rounsley, S., Sankoff, D., Schuster, S. C., S., J., Barbazuk, W. B., Chamala, S., Chanderbali, A. S., Determann, R., Hong, M., Ralph, P., Talag, J., Tomsho, L., Walts, B., Wanke, S., Wing, R. A., Chang, T., Lan, T., , Soltis, D. E., et al. (2013). The Amborella genome and the evolution of flowering plants. Science, 342(6165).
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  Abstract: Amborella trichopoda is strongly supported as the single living species of the sister lineage to all other extant flowering plants, providing a unique reference for inferring the genome content and structure of the most recent common ancestor (MRCA) of living angiosperms. Sequencing the Amborella genome, we identified an ancient genome duplication predating angiosperm diversification, without evidence of subsequent, lineage-specific genome duplications. Comparisons between Amborella and other angiosperms facilitated reconstruction of the ancestral angiosperm gene content and gene order in the MRCA of core eudicots. We identify new gene families, gene duplications, and floral protein-protein interactions that first appeared in the ancestral angiosperm. Transposable elements in Amborella are ancient and highly divergent, with no recent transposon radiations. Population genomic analysis across Amborella's native range in New Caledonia reveals a recent genetic bottleneck and geographic structure with conservation implications.
• Dereeper, A., Guyot, R., Tranchant-Dubreuil, C., Anthony, F., Argout, X., Bellis, F. d., Combes, M., Gavory, F., Kochko, A. d., Kudrna, D., Leroy, T., Poulain, J., Rondeau, M., Song, X., Wing, R., & Lashermes, P. (2013). BAC-end sequences analysis provides first insights into coffee (Coffea canephora P.) genome composition and evolution. Plant Molecular Biology, 83(3), 177-189.
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  PMID: 23708951;Abstract: Coffee is one of the world's most important agricultural commodities. Coffee belongs to the Rubiaceae family in the euasterid I clade of dicotyledonous plants, to which the Solanaceae family also belongs. Two bacterial artificial chromosome (BAC) libraries of a homozygous doubled haploid plant of Coffea canephora were constructed using two enzymes, HindIII and BstYI. A total of 134,827 high quality BAC-end sequences (BESs) were generated from the 73,728 clones of the two libraries, and 131,412 BESs were conserved for further analysis after elimination of chloroplast and mitochondrial sequences. This corresponded to almost 13 % of the estimated size of the C. canephora genome. 6.7 % of BESs contained simple sequence repeats, the most abundant (47.8 %) being mononucleotide motifs. These sequences allow the development of numerous useful marker sites. Potential transposable elements (TEs) represented 11.9 % of the full length BESs. A difference was observed between the BstYI and HindIII libraries (14.9 vs. 8.8 %). Analysis of BESs against known coding sequences of TEs indicated that 11.9 % of the genome corresponded to known repeat sequences, like for other flowering plants. The number of genes in the coffee genome was estimated at 41,973 which is probably overestimated. Comparative genome mapping revealed that microsynteny was higher between coffee and grapevine than between coffee and tomato or Arabidopsis. BESs constitute valuable resources for the first genome wide survey of coffee and provide new insights into the composition and evolution of the coffee genome. © 2013 Springer Science+Business Media Dordrecht.
• Horvath, D., Kudrna, D., Talag, J., Anderson, J., Chao, W., Wing, R., Foley, M., & Dogramaci, M. (2013). BAC library development and clone characterization for dormancy-responsive DREB4A, DAM, and FT from leafy spurge (Euphorbia esula) identifies differential splicing and conserved promoter motifs. Weed Science, 61.
• Koo, H. J., McDowell, E. T., Ma, X., Greer, K. A., Kapteyn, J., Xie, Z., Descour, A., Kim, H., Yu, Y., Kudrna, D., Wing, R. A., Soderlund, C. A., & Gang, D. R. (2013). Ginger and turmeric expressed sequence tags identify signature genes for rhizome identity and development and the biosynthesis of curcuminoids, gingerols and terpenoids. BMC plant biology, 13, 27.
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  Ginger (Zingiber officinale) and turmeric (Curcuma longa) accumulate important pharmacologically active metabolites at high levels in their rhizomes. Despite their importance, relatively little is known regarding gene expression in the rhizomes of ginger and turmeric.
• Maron, L. G., Guimaraes, C. T., Kirst, M., Albert, P. S., Birchler, J. A., Bradbury, P. J., Buckler, E. S., Coluccio, A. E., Danilova, T. V., Kudrna, D., Magalhaes, J. V., Pineros, M. A., Schatz, M. C., Wing, R. A., & Kochian, L. V. (2013). Aluminum tolerance in maize is associated with higher MATE1 gene copy number. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 110(13), 5241-5246.
• Matsuba, Y., Nguyen, T. T., Wiegert, K., Falara, V., Gonzales-Vigil, E., Leong, B., Schäfer, P., Kudrna, D., Wing, R. A., Bolger, A. M., Usadel, B., Tissier, A., Fernie, A. R., Barry, C. S., & Pichersky, E. (2013). Evolution of a complex locus for terpene biosynthesis in Solanum. Plant Cell, 25(6), 2022-2036.
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  PMID: 23757397;PMCID: PMC3723610;Abstract: Functional gene clusters, containing two or more genes encoding different enzymes for the same pathway, are sometimes observed in plant genomes, most often when the genes specify the synthesis of specialized defensive metabolites. Here, we show that a cluster of genes in tomato (Solanum lycopersicum; Solanaceae) contains genes for terpene synthases (TPSs) that specify the synthesis of monoterpenes and diterpenes from cis-prenyl diphosphates, substrates that are synthesized by enzymes encoded by cis-prenyl transferase (CPT) genes also located within the same cluster. The monoterpene synthase genes in the cluster likely evolved from a diterpene synthase gene in the cluster by duplication and divergence. In the orthologous cluster in Solanum habrochaites, a new sesquiterpene synthase gene was created by a duplication event of a monoterpene synthase followed by a localized gene conversion event directed by a diterpene synthase gene. The TPS genes in the Solanum cluster encoding cis-prenyl diphosphate-utilizing enzymes are closely related to a tobacco (Nicotiana tabacum; Solanaceae) diterpene synthase encoding Z-abienol synthase (Nt-ABS). Nt-ABS uses the substrate copal-8-ol diphosphate, which is made from the all-trans geranylgeranyl diphosphate by copal-8-ol diphosphate synthase (Nt-CPS2). The Solanum gene cluster also contains an ortholog of Nt-CPS2, but it appears to encode a nonfunctional protein. Thus, the Solanum functional gene cluster evolved by duplication and divergence of TPS genes, together with alterations in substrate specificity to utilize cis-prenyl diphosphates and through the acquisition of CPT genes. © 2013 American Society of Plant Biologists. All rights reserved.
• Ortiz, M., Neilson, J. W., Nelson, W. M., Legatzki, A., Byrne, A., Yu, Y., Wing, R. A., Soderlund, C. A., Pryor, B. M., Pierson, L. S., & Maier, R. M. (2013). Profiling bacterial diversity and taxonomic composition on speleothem surfaces in Kartchner Caverns, AZ. Microbial ecology, 65(2), 371-83.
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  Caves are relatively accessible subterranean habitats ideal for the study of subsurface microbial dynamics and metabolisms under oligotrophic, non-photosynthetic conditions. A 454-pyrotag analysis of the V6 region of the 16S rRNA gene was used to systematically evaluate the bacterial diversity of ten cave surfaces within Kartchner Caverns, a limestone cave. Results showed an average of 1,994 operational taxonomic units (97 % cutoff) per speleothem and a broad taxonomic diversity that included 21 phyla and 12 candidate phyla. Comparative analysis of speleothems within a single room of the cave revealed three distinct bacterial taxonomic profiles dominated by either Actinobacteria, Proteobacteria, or Acidobacteria. A gradient in observed species richness along the sampling transect revealed that the communities with lower diversity corresponded to those dominated by Actinobacteria while the more diverse communities were those dominated by Proteobacteria. A 16S rRNA gene clone library from one of the Actinobacteria-dominated speleothems identified clones with 99 % identity to chemoautotrophs and previously characterized oligotrophs, providing insights into potential energy dynamics supporting these communities. The robust analysis conducted for this study demonstrated a rich bacterial diversity on speleothem surfaces. Further, it was shown that seemingly comparable speleothems supported divergent phylogenetic profiles suggesting that these communities are very sensitive to subtle variations in nutritional inputs and environmental factors typifying speleothem surfaces in Kartchner Caverns.
• Pan, Y., Deng, Y., Lin, H., Kudrna, D. A., Wing, R. A., Li, L., Zhang, Q., & Luo, M. (2013). Detection and correction of assembly errors of rice Nipponbare reference sequence. Plant Biology.
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  Abstract: A complete and high-quality genome reference sequence of an organism provides a solid foundation for a wide research community and determines the outcomes of relevant genomic, genetic, molecular and evolutionary research. Rice is an important food crop and a model plant for grasses, and therefore was the first chosen crop plant for whole genome sequencing. The genome of the japonica representative rice variety, Nipponbare, was sequenced using a gold standard, map-based clone-by-clone strategy. However, although the Nipponbare reference sequence (RefSeq) has the best quality for existing crop genome sequences, it still contains many assembly errors and gaps. To improve the Nipponbare RefSeq, first a robust method is required to detect the hidden assembly errors. Through alignments between BAC-end sequences (BESs) embedded in the Nipponbare bacterial artificial chromosome (BAC) physical map and the Nipponbare RefSeq, we detected locations on the Nipponbare RefSeq that were inversely matched with BESs and could therefore be candidates for spurious inversions of assembly. We performed further analysis of five potential locations and confirmed assembly errors at those locations; four of them, two on chr4 and two on chr11 of the Nipponbare RefSeq (IRGSP build 5), were found to be caused by reverse repetitive sequences flanking the locations. Our approach is effective in detecting spurious inversions in the Nipponbare RefSeq and can be applied for improving the sequence qualities of other genomes as well. © 2013 German Botanical Society and The Royal Botanical Society of the Netherlands.
• Singh, R., Ong-Abdullah, M., Low, E. L., Manaf, M., Rosli, R., Nookiah, R., Ooi, L. C., Ooi, S., Chan, K., Halim, M. A., Azizi, N., Nagappan, J., Bacher, B., Lakey, N., Smith, S. W., He, D., Hogan, M., Budiman, M. A., Lee, E. K., , DeSalle, R., et al. (2013). Oil palm genome sequence reveals divergence of interfertile species in Old and New Worlds. NATURE, 500(7462), 335-+.
• Wang, C., Shi, X., Liu, L., Haiyan, L. i., Ammiraju, J. S., Kudrna, D. A., Xiong, W., Wang, H., Dai, Z., Zheng, Y., Lai, J., Jin, W., Messing, J., Bennetzen, J. L., Wing, R. A., & Luo, M. (2013). Genomic resources for gene discovery, functional genome annotation, and evolutionary studies of maize and its close relatives. Genetics, 195(3), 723-737.
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  PMID: 24037269;PMCID: PMC3813860;Abstract: Maize is one of the most important food crops and a key model for genetics and developmental biology. A genetically anchored and high-quality draft genome sequence of maize inbred B73 has been obtained to serve as a reference sequence. To facilitate evolutionary studies in maize and its close relatives, much like the Oryza Map Alignment Project (OMAP) (www.OMAP.org) bacterial artificial chromosome (BAC) resource did for the rice community, we constructed BAC libraries for maize inbred lines Zheng58, Chang7-2, and Mo17 and maize wild relatives Zea mays ssp. parviglumis and Tripsacum dactyloides. Furthermore, to extend functional genomic studies to maize and sorghum, we also constructed binary BAC (BIBAC) libraries for the maize inbred B73 and the sorghum landrace Nengsi-1. The BAC/BIBAC vectors facilitate transfer of large intact DNA inserts from BAC clones to the BIBAC vector and functional complementation of large DNA fragments. These seven Zea Map Alignment Project (ZMAP) BAC/BIBAC libraries have average insert sizes ranging from 92 to 148 kb, organellar DNA from 0.17 to 2.3%, empty vector rates between 0.35 and 5.56%, and genome equivalents of 4.7-to 8.4-fold. The usefulness of the Parviglumis and Tripsacum BAC libraries was demonstrated by mapping clones to the reference genome. Novel genes and alleles present in these ZMAP libraries can now be used for functional complementation studies and positional or homology-based cloning of genes for translational genomics. © 2013 by the Genetics Society of America.
• Wing, R., Jacquemin, J., Bhatia, D., Singh, K., & Wing, R. A. (2013). The International Oryza Map Alignment Project: development of a genus-wide comparative genomics platform to help solve the 9 billion-people question. Current opinion in plant biology, 16(2).
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  The wild relatives of rice contain a virtually untapped reservoir of traits that can be used help drive the 21st century green revolution aimed at solving world food security issues by 2050. To better understand and exploit the 23 species of the Oryza genus the rice research community is developing foundational resources composed of: 1) reference genomes and transcriptomes for all 23 species; 2) advanced mapping populations for functional and breeding studies; and 3) in situ conservation sites for ecological, evolutionary and population genomics. To this end, 16 genome sequencing projects are currently underway, and all completed assemblies have been annotated; and several advanced mapping populations have been developed, and more will be generated, mapped, and phenotyped, to uncover useful alleles. As wild Oryza populations are threatened by human activity and climate change, we also discuss the urgent need for sustainable in situ conservation of the genus.
• Yang, R., Jarvis, D. E., Chen, H., Beilstein, M. A., Grimwood, J., Jenkins, J., Shu, S. Q., Prochnik, S., Xin, M., Ma, C., Schmutz, J., Wing, R. A., Mitchell-Olds, T., Schumaker, K. S., & Wang, X. (2013). The reference genome of the halophytic plant Eutrema salsugineum. FRONTIERS IN PLANT SCIENCE, 4.
• Yang, R., Jarvis, D. E., Chen, H., Beilstein, M. A., Grimwood, J., Jenkins, J., Shu, S., Prochnik, S., Xin, M., Ma, C., Schmutz, J., Wing, R. A., Mitchell-Olds, T., Schumaker, K. S., & Wang, X. -. (2013). The reference genome of the halophytic plant Eutrema salsugineum. Frontiers in Plant Genetics and Genomics, doi: 10.3389/fpls.2013.00046.
• Zhang, C., Wang, J., Marowsky, N. C., Long, M., Wing, R. A., & Fan, C. (2013). High occurrence of functional new chimeric genes in survey of rice chromosome 3 short arm genome sequences. Genome Biology and Evolution, 5(5), 1038-1048.
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  PMID: 23651622;Abstract: In an effort to identify newly evolved genes in rice, we searched the genomes of Asian-cultivated rice Oryza sativa ssp. japonica and its wild progenitors, looking for lineage-specific genes. Using genome pairwise comparison of approximately 20-Mb DNA sequences from the chromosome 3 short arm (Chr3s) in six rice species, O. sativa, O. nivara, O. rufipogon, O. glaberrima, O. barthii, and O. punctata, combined with synonymous substitution rate tests and other evidence, we were able to identify potential recently duplicated genes, which evolved within the last 1 Myr. We identified 28 functional O. sativa genes, which likely originated after O. sativa diverged from O. glaberrima. These genes account for around 1% (28/3,176) of all annotated genes on O. sativa's Chr3s. Among the28 newgenes, two recentlyduplicatedsegments contained eightgenes. Fourteen ofthe28 newgenes consist of chimeric gene structure derived from one or multiple parental genes and flanking targeting sequences. Although the majority of these 28 new genes were formed by single or segmental DNA-based gene duplication and recombination, we found two genes that were likely originated partially through exon shuffling. Sequence divergence tests between new genes and their putative progenitors indicated that new genes were most likely evolving under natural selection. We showed all 28 new genes appeared to be functional, as suggested by Ka/Ksanalysis and the presence of RNA-seq, cDNA, expressed sequence tag, massively parallel signature sequencing, and/or small RNA data. The high rate of new gene origination and of chimeric gene formation in rice may demonstrate rice's broad diversification, domestication, its environmental adaptation, and the role of new genes in rice speciation © The Author(s) 2013. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.
• Zhang, Q., & Wing, R. (2013). Genome studies and molecular genetics: Understanding the functional genome based on the rice model. Current Opinion in Plant Biology, 16(2), 129-132.
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  PMID: 23706861;
• F., K., Waugh, R., Langridge, P., Close, T. J., Wise, R. P., Graner, A., Matsumoto, T., Sato, K., Schulman, A., Ariyadasa, R., Schulte, D., Poursarebani, N., Zhou, R., Steuernagel, B., Mascher, M., Scholz, U., Shi, B., Madishetty, K., Svensson, J. T., , Bhat, P., et al. (2012). A physical, genetic and functional sequence assembly of the barley genome. Nature, 491(7426), 711-716.
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  PMID: 23075845;Abstract: Barley (Hordeum vulgare L.) is among the world's earliest domesticated and most important crop plants. It is diploid with a large haploid genome of 5.1 gigabases (Gb). Here we present an integrated and ordered physical, genetic and functional sequence resource that describes the barley gene-space in a structured whole-genome context. We developed a physical map of 4.98 Gb, with more than 3.90 Gb anchored to a high-resolution genetic map. Projecting a deep whole-genome shotgun assembly, complementary DNA and deep RNA sequence data onto this framework supports 79,379 transcript clusters, including 26,159 'high-confidence' genes with homology support from other plant genomes. Abundant alternative splicing, premature termination codons and novel transcriptionally active regions suggest that post-transcriptional processing forms an important regulatory layer. Survey sequences from diverse accessions reveal a landscape of extensive single-nucleotide variation. Our data provide a platform for both genome-assisted research and enabling contemporary crop improvement. © 2012 Macmillan Publishers Limited. All rights reserved.
• Figueira, T., Okura, V., Silva, F., Silva, M., Kudrna, D., Ammiraju, J., Talag, J., Wing, R., & Arruda, P. (2012). A BAC library of the SP80-3280 sugarcane variety (Saccharum sp.) and its inferred microsynteny with the sorghum genome. BMC Genomics.
• Jungmin, H. a., Abernathy, B., Nelson, W., Grant, D., Xiaolei, W. u., Nguyen, H. T., Stacey, G., Yeisoo, Y. u., Wing, R. A., Shoemaker, R. C., & Jackson, S. A. (2012). Integration of the draft sequence and physical map as a framework for genomic research in soybean (glycine max (l.) merr.) and wild soybean (glycine soja sieb. and zucc.). G3: Genes, Genomes, Genetics, 2(3), 321-329.
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  PMID: 22413085;PMCID: PMC3291501;Abstract: Soybean is a model for the legume research community because of its importance as a crop, densely populated genetic maps, and the availability of a genome sequence. Even though a whole-genome shotgun sequence and bacterial artificial chromosome (BAC) libraries are available, a high-resolution, chromosome-based physical map linked to the sequence assemblies is still needed for whole-genome alignments and to facilitate map-based gene cloning. Three independent G. max BAC libraries combined with genetic and gene-based markers were used to construct a minimum tiling path (MTP) of BAC clones. A total of 107,214 clones were assembled into 1355 FPC (FingerPrinted Contigs) contigs, incorporating 4628 markers and aligned to the G. max reference genome sequence using BAC end-sequence information. Four different MTPs were made for G. max that covered from 92.6% to 95.0% of the soybean draft genome sequence (gmax1.01). Because our purpose was to pick the most reliable and complete MTP, and not the MTP with the minimal number of clones, the FPC map and draft sequence were integrated and clones with unpaired BES were added to build a high-quality physical map with the fewest gaps possible (http://soybase.org). A physical map was also constructed for the undomesticated ancestor (G. soja) of soybean to explore genome variation between G. max and G. soja. 66,028 G. soja clones were assembled into 1053 FPC contigs covering approximately 547Mbp of the G. max genome sequence. These physical maps for G. max and its undomesticated ancestor, G. soja, will serve as a framework for ordering sequence fragments, comparative genomics, cloning genes, and evolutionary analyses of legume genomes. © 2012 Ha et al.
• Li, H., Li, X., Xiao, J., Wing, R. A., & Wang, S. (2012). Ortholog alleles at Xa3/Xa26 locus confer conserved race-specific resistance against xanthomonas oryzae in rice. Molecular Plant, 5(1), 281-290.
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  PMID: 21930802;PMCID: PMC3261417;Abstract: The rice disease resistance (R) gene Xa3/Xa26 (having also been named Xa3 and Xa26) against Xanthomonas oryzae pv. oryzae (Xoo), which causes bacterial blight disease, belongs to a multiple gene family clustered in chromosome 11 and is from an AA genome rice cultivar (Oryza sativa L.). This family encodes leucine-rich repeat (LRR) receptor kinase-type proteins. Here, we show that the orthologs (alleles) of Xa3/Xa26, Xa3/Xa26-2, and Xa3/Xa26-3, from wild Oryza species O. officinalis (CC genome) and O. minuta (BBCC genome), respectively, were also R genes against Xoo. Xa3/Xa26-2 and Xa3/Xa26-3 conferred resistance to 16 of the 18 Xoo strains examined. Comparative sequence analysis of the Xa3/Xa26 families in the two wild Oryza species showed that Xa3/Xa26-3 appeared to have originated from the CC genome of O. minuta. The predicted proteins encoded by Xa3/Xa26, Xa3/Xa26-2, and Xa3/Xa26-3 share 91-99% sequence identity and 94-99% sequence similarity. Transgenic plants carrying a single copy of Xa3/Xa26, Xa3/Xa26-2, or Xa3/Xa26-3, in the same genetic background, showed a similar resistance spectrum to a set of Xoo strains, although plants carrying Xa3/Xa26-2 or Xa3/Xa26-3 showed lower resistance levels than the plants carrying Xa3/Xa26. These results suggest that the Xa3/Xa26 locus predates the speciation of A and C genome, which is approximately 7.5million years ago. Thus, the resistance specificity of this locus has been conserved for a long time. © 2011 The Author.
• Lin, H., Xia, P., Wing, R., Zhang, Q., & Luo, M. (2012). Dynamic intra-japonica subspecies variation and resource application. Molecular Plant, 5, 218-230.
• Neilson, J. W., Quade, J., Ortiz, M., Nelson, W. M., Legatzki, A., Tian, F., LaComb, M., Betancourt, J. L., Wing, R. A., Soderlund, C. A., & Maier, R. M. (2012). Life at the hyperarid margin: novel bacterial diversity in arid soils of the Atacama Desert, Chile. Extremophiles : life under extreme conditions, 16(3), 553-66.
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  Nearly half the earth's surface is occupied by dryland ecosystems, regions susceptible to reduced states of biological productivity caused by climate fluctuations. Of these regions, arid zones located at the interface between vegetated semiarid regions and biologically unproductive hyperarid zones are considered most vulnerable. The objective of this study was to conduct a deep diversity analysis of bacterial communities in unvegetated arid soils of the Atacama Desert, to characterize community structure and infer the functional potential of these communities based on observed phylogenetic associations. A 454-pyrotag analysis was conducted of three unvegetated arid sites located at the hyperarid-arid margin. The analysis revealed communities with unique bacterial diversity marked by high abundances of novel Actinobacteria and Chloroflexi and low levels of Acidobacteria and Proteobacteria, phyla that are dominant in many biomes. A 16S rRNA gene library of one site revealed the presence of clones with phylogenetic associations to chemoautotrophic taxa able to obtain energy through oxidation of nitrite, carbon monoxide, iron, or sulfur. Thus, soils at the hyperarid margin were found to harbor a wealth of novel bacteria and to support potentially viable communities with phylogenetic associations to non-phototrophic primary producers and bacteria capable of biogeochemical cycling.
• Sato, S., Tabata, S., Hirakawa, H., Asamizu, E., Shirasawa, K., Isobe, S., Kaneko, T., Nakamura, Y., Shibata, D., Aoki, K., Egholm, M., Knight, J., Bogden, R., Li, C., Shuang, Y., Xu, X., Pan, S., Cheng, S., Liu, X., , Ren, Y., et al. (2012). The tomato genome sequence provides insights into fleshy fruit evolution. NATURE, 485(7400), 635-641.
• Wing, R. A. (2012). The genomes that make tomatoes. Nature, 485, 635-641.
• Yang, L., Liu, T., Bo, L. i., Sui, Y., Chen, J., Shi, J., Wing, R. A., & Chen, M. (2012). Comparative Sequence Analysis of the Ghd7 Orthologous Regions Revealed Movement of Ghd7 in the Grass Genomes. PLoS ONE, 7(11).
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  PMID: 23185584;PMCID: PMC3503983;Abstract: Ghd7 is an important rice gene that has a major effect on several agronomic traits, including yield. To reveal the origin of Ghd7 and sequence evolution of this locus, we performed a comparative sequence analysis of the Ghd7 orthologous regions from ten diploid Oryza species, Brachypodium distachyon, sorghum and maize. Sequence analysis demonstrated high gene collinearity across the genus Oryza and a disruption of collinearity among non-Oryza species. In particular, Ghd7 was not present in orthologous positions except in Oryza species. The Ghd7 regions were found to have low gene densities and high contents of repetitive elements, and that the sizes of orthologous regions varied tremendously. The large transposable element contents resulted in a high frequency of pseudogenization and gene movement events surrounding the Ghd7 loci. Annotation information and cytological experiments have indicated that Ghd7 is a heterochromatic gene. Ghd7 orthologs were identified in B. distachyon, sorghum and maize by phylogenetic analysis; however, the positions of orthologous genes differed dramatically as a consequence of gene movements in grasses. Rather, we identified sequence remnants of gene movement of Ghd7 mediated by illegitimate recombination in the B. distachyon genome. © 2012 Yang et al.
• Argout, X., Salse, J., Aury, J., Guiltinan, M. J., Droc, G., Gouzy, J., Allegre, M., Chaparro, C., Legavre, T., Maximova, S. N., Abrouk, M., Murat, F., Fouet, O., Poulain, J., Ruiz, M., Roguet, Y., Rodier-Goud, M., Barbosa-Neto, J. F., Sabot, F., , Kudrna, D., et al. (2011). The genome of Theobroma cacao. NATURE GENETICS, 43(2), 101-108.
• Argout, X., Salse, J., Aury, J., Guiltinan, M. J., Droc, G., Gouzy, J., Allegre, M., Chaparro, C., Legavre, T., Maximova, S. N., Abrouk, M., Murat, F., Fouet, O., Poulain, J., Ruiz, M., Roguet, Y., Rodier-Goud, M., Barbosa-Neto, J. F., Sabot, F., , Kudrna, D., et al. (2011). The genome of Theobroma cacao. Nature Genetics, 43(2), 101-108.
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  PMID: 21186351;Abstract: We sequenced and assembled the draft genome of Theobroma cacao, an economically important tropical-fruit tree crop that is the source of chocolate. This assembly corresponds to 76% of the estimated genome size and contains almost all previously described genes, with 82% of these genes anchored on the 10 T. cacao chromosomes. Analysis of this sequence information highlighted specific expansion of some gene families during evolution, for example, flavonoid-related genes. It also provides a major source of candidate genes for T. cacao improvement. Based on the inferred paleohistory of the T. cacao genome, we propose an evolutionary scenario whereby the ten T. cacao chromosomes were shaped from an ancestor through eleven chromosome fusions. © 2011 Nature America, Inc. All rights reserved.
• Blair, M., Hurtado, N., Chavarro, C., , M., Giraldo, M., Pedraza, F., Tomkins, J., & Wing, R. (2011). Gene-based SSR markers for common bean (Phaseolus vulgaris L.) derived from root and leaf tissue ESTs: an integration of the BMc series. BMC Plant Biol, 11, 50.
• Fan, C., Walling, J. G., Zhang, J., Hirsch, C. D., Jiang, J., & Wing, R. A. (2011). Conservation and purifying selection of transcribed genes located in a rice centromere. Plant Cell, 23(8), 2821-2830.
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  PMID: 21856794;PMCID: PMC3180794;Abstract: Recombination is strongly suppressed in centromeric regions. In chromosomal regions with suppressed recombination, deleterious mutations can easily accumulate and cause degeneration of genes and genomes. Surprisingly, the centromere of chromosome8 (Cen8) of rice (Oryza sativa) contains several transcribed genes. However, it remains unclear as to what selective forces drive the evolution and existence of transcribed genes in Cen8. Sequencing of orthologous Cen8 regions from two additional Oryza species, Oryza glaberrima and Oryza brachyantha, which diverged from O. sativa 1 and 10 million years ago, respectively, revealed a set of seven transcribed Cen8 genes conserved across all three species. Chromatin immunoprecipitation analysis with the centromere-specific histone CENH3 confirmed that the sequenced orthologous regions are part of the functional centromere. All seven Cen8genes have undergone purifying selection, representing a striking phenomenon of active gene survival within a recombination-free zone over a long evolutionary time. The coding sequences of the Cen8 genes showed sequence divergence and mutation rates that were significantly reduced from those of genes located on the chromosome arms. This suggests that Oryza has a mechanism to maintain the fidelity and functionality of Cen8 genes, even when embedded in a sea of repetitive sequences and transposable elements. © 2011 American Society of Plant Biologists. All rights reserved.
• Gao, D., Gong, Z., Wing, R. A., Jiang, J., & Jackson, S. A. (2011). Molecular and Cytological Characterization of Centromeric Retrotransposons in a Wild Relative of Rice, Oryza granulata. Tropical Plant Biology, 4(3-4), 217-227.
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  Abstract: Centromeric retrotransposons (CRs) are important component of the functional centromeres of rice chromosomes. To track the evolution of the CR elements in genus Oryza, we sequenced the orthologous region of the rice centromere 8 (Cen8) in O. granulata and analyzed transposons in this region. A total of 12 bacterial artificial chromosomes (BACs) that span the centromeric region in O. granulata were sequenced. The O. granulate centromeric sequences are composed of as much as 85% of transposons, higher than any other reported eukaryotic centromeres. Ten novel LTR retrotransposon families were identified but a single retrotransposon, Gran3, constitutes nearly 43% of the centromeric sequences. Integration times of complete LTR retrotransposons indicate that the centromeric region had a massive insertion of LTR retrotransposons within 4.5 million year (Myr), which indicates a recent expansion of the centromere in O. granulata after the radiation of the Oryza genus. Two retrotransposon families, OGRetro7 and OGRetro9, show sequence similarity with the canonical CRs from rice and maize. Both OGRetro7 and OGRetro9 are highly concentrated in the centromeres of O. granulata chromosomes. Furthermore, strong hybridization signals were detected in all Oryza species but in O. brachyantha with the OGRetro7 and OGRetro9 probes. Characterization of the centromeric retrotransposons in O. granulata confirms the conservation of the CRs in the Oryza genus and provides a resource for comparative analysis of centromeres and centromere evolution among the Oryza genus and other genomes. © 2011 Springer Science+Business Media, LLC.
• Jacquemin, J., Chaparro, C., Laudié, M., Berger, A., Gavory, F., Goicoechea, J. L., Wing, R. A., & Cooke, R. (2011). Long-range and targeted ectopic recombination between the two homeologous chromosomes 11 and 12 in Oryza species. Molecular Biology and Evolution, 28(11), 3139-3150.
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  PMID: 21616911;Abstract: Whole genome duplication (WGD) and subsequent evolution of gene pairs have been shown to have shaped the present day genomes of most, if not all, plants and to have played an essential role in the evolution of many eukaryotic genomes. Analysis of the rice (Oryza sativa ssp. japonica) genome sequence suggested an ancestral WGD ∼50-70 Ma common to all cereals and a segmental duplication between chromosomes 11 and 12 as recently as 5 Ma. More recent studies based on coding sequences have demonstrated that gene conversion is responsible for the high sequence conservation which suggested such a recent duplication. We previously showed that gene conversion has been a recurrent process throughout the Oryza genus and in closely related species and that orthologous duplicated regions are also highly conserved in other cereal genomes. We have extended these studies to compare megabase regions of genomic (coding and noncoding) sequences between two cultivated (O. sativa, Oryza glaberrima) and one wild (Oryza brachyantha) rice species using a novel approach of topological incongruency. The high levels of intraspecies conservation of both gene and nongene sequences, particularly in O. brachyantha, indicate long-range conversion events less than 4 Ma in all three species. These observations demonstrate megabase-scale conversion initiated within a highly rearranged region located at ∼2.1 Mb from the chromosome termini and emphasize the importance of gene conversion in cereal genome evolution. The Author 2011. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved.
• Paiva, J. A., Prat, E., Vautrin, S., Santos, M. D., San-Clemente, H., Brommonschenkel, S., Fonseca, P. G., Grattapaglia, D., Song, X., Ammiraju, J. S., Kudrna, D., Wing, R. A., Freitas, A. T., Bergès, H., & Grima-Pettenati, J. (2011). Advancing Eucalyptus genomics: Identification and sequencing of lignin biosynthesis genes from deep-coverage BAC libraries. BMC Genomics, 12.
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  PMID: 21375742;PMCID: PMC3060884;Abstract: Background: Eucalyptus species are among the most planted hardwoods in the world because of their rapid growth, adaptability and valuable wood properties. The development and integration of genomic resources into breeding practice will be increasingly important in the decades to come. Bacterial artificial chromosome (BAC) libraries are key genomic tools that enable positional cloning of important traits, synteny evaluation, and the development of genome framework physical maps for genetic linkage and genome sequencing.Results: We describe the construction and characterization of two deep-coverage BAC libraries EG_Ba and EG_Bb obtained from nuclear DNA fragments of E. grandis (clone BRASUZ1) digested with HindIII and BstYI, respectively. Genome coverages of 17 and 15 haploid genome equivalents were estimated for EG_Ba and EG_Bb, respectively. Both libraries contained large inserts, with average sizes ranging from 135 Kb (Eg_Bb) to 157 Kb (Eg_Ba), very low extra-nuclear genome contamination providing a probability of finding a single copy gene ≥ 99.99%. Libraries were screened for the presence of several genes of interest via hybridizations to high-density BAC filters followed by PCR validation. Five selected BAC clones were sequenced and assembled using the Roche GS FLX technology providing the whole sequence of the E. grandis chloroplast genome, and complete genomic sequences of important lignin biosynthesis genes.Conclusions: The two E. grandis BAC libraries described in this study represent an important milestone for the advancement of Eucalyptus genomics and forest tree research. These BAC resources have a highly redundant genome coverage (> 15×), contain large average inserts and have a very low percentage of clones with organellar DNA or empty vectors. These publicly available BAC libraries are thus suitable for a broad range of applications in genetic and genomic research in Eucalyptus and possibly in related species of Myrtaceae, including genome sequencing, gene isolation, functional and comparative genomics. Because they have been constructed using the same tree (E. grandis BRASUZ1) whose full genome is being sequenced, they should prove instrumental for assembly and gap filling of the upcoming Eucalyptus reference genome sequence. © 2011 Paiva et al; licensee BioMed Central Ltd.
• Piegu, B., Guyot, R., Picault, N., Roulin, A., Sanyal, A., Kim, H., Collura, K., Brar, D. S., Jackson, S., Wing, R. A., & Panaud, O. (2011). Doubling genome size without polyploidization: Dynamics of retrotransposition-driven genomic expansions in Oryza australiensis, a wild relative of rice (Genome Research (2006) 16, (1262-1269)). Genome Research, 21(7), 1201-.
• Tian, Z., Yu, Y., Feng, L., Yu, Y., SanMiguel, P., Wing, R., McCouch, S., Ma, J., & Jackson, S. (2011). Exceptional lability of a genomic complex in rice and its close relatives revealed by interspecific and intraspecific comparison and population analysis. BMC Genomics, 12, 142.
• Wing, R., Fan, C., Walling, J. G., Zhang, J., Hirsch, C. D., Jiang, J., & Wing, R. A. (2011). Conservation and purifying selection of transcribed genes located in a rice centromere. The Plant cell, 23(8).
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  Recombination is strongly suppressed in centromeric regions. In chromosomal regions with suppressed recombination, deleterious mutations can easily accumulate and cause degeneration of genes and genomes. Surprisingly, the centromere of chromosome8 (Cen8) of rice (Oryza sativa) contains several transcribed genes. However, it remains unclear as to what selective forces drive the evolution and existence of transcribed genes in Cen8. Sequencing of orthologous Cen8 regions from two additional Oryza species, Oryza glaberrima and Oryza brachyantha, which diverged from O. sativa 1 and 10 million years ago, respectively, revealed a set of seven transcribed Cen8 genes conserved across all three species. Chromatin immunoprecipitation analysis with the centromere-specific histone CENH3 confirmed that the sequenced orthologous regions are part of the functional centromere. All seven Cen8 genes have undergone purifying selection, representing a striking phenomenon of active gene survival within a recombination-free zone over a long evolutionary time. The coding sequences of the Cen8 genes showed sequence divergence and mutation rates that were significantly reduced from those of genes located on the chromosome arms. This suggests that Oryza has a mechanism to maintain the fidelity and functionality of Cen8 genes, even when embedded in a sea of repetitive sequences and transposable elements.
• Wing, R., Lin, J., Kudrna, D., & Wing, R. A. (2011). Construction, characterization, and preliminary BAC-end sequence analysis of a bacterial artificial chromosome library of the tea plant (Camellia sinensis). Journal of biomedicine & biotechnology, 2011.
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  We describe the construction and characterization of a publicly available BAC library for the tea plant, Camellia sinensis. Using modified methods, the library was constructed with the aim of developing public molecular resources to advance tea plant genomics research. The library consists of a total of 401,280 clones with an average insert size of 135 kb, providing an approximate coverage of 13.5 haploid genome equivalents. No empty vector clones were observed in a random sampling of 576 BAC clones. Further analysis of 182 BAC-end sequences from randomly selected clones revealed a GC content of 40.35% and low chloroplast and mitochondrial contamination. Repetitive sequence analyses indicated that LTR retrotransposons were the most predominant sequence class (86.93%-87.24%), followed by DNA retrotransposons (11.16%-11.69%). Additionally, we found 25 simple sequence repeats (SSRs) that could potentially be used as genetic markers.
• Wing, R., Song, X., Goicoechea, J. L., Ammiraju, J. S., Luo, M., He, R., Lin, J., Lee, S., Sisneros, N., Watts, T., Kudrna, D. A., Golser, W., Ashley, E., Collura, K., Braidotti, M., Yu, Y., Matzkin, L. M., McAllister, B. F., Markow, T. A., & Wing, R. A. (2011). The 19 genomes of Drosophila: a BAC library resource for genus-wide and genome-scale comparative evolutionary research. Genetics, 187(4).
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  The genus Drosophila has been the subject of intense comparative phylogenomics characterization to provide insights into genome evolution under diverse biological and ecological contexts and to functionally annotate the Drosophila melanogaster genome, a model system for animal and insect genetics. Recent sequencing of 11 additional Drosophila species from various divergence points of the genus is a first step in this direction. However, to fully reap the benefits of this resource, the Drosophila community is faced with two critical needs: i.e., the expansion of genomic resources from a much broader range of phylogenetic diversity and the development of additional resources to aid in finishing the existing draft genomes. To address these needs, we report the first synthesis of a comprehensive set of bacterial artificial chromosome (BAC) resources for 19 Drosophila species from all three subgenera. Ten libraries were derived from the exact source used to generate 10 of the 12 draft genomes, while the rest were generated from a strategically selected set of species on the basis of salient ecological and life history features and their phylogenetic positions. The majority of the new species have at least one sequenced reference genome for immediate comparative benefit. This 19-BAC library set was rigorously characterized and shown to have large insert sizes (125-168 kb), low nonrecombinant clone content (0.3-5.3%), and deep coverage (9.1-42.9×). Further, we demonstrated the utility of this BAC resource for generating physical maps of targeted loci, refining draft sequence assemblies and identifying potential genomic rearrangements across the phylogeny.
• Zuccolo, A., Bowers, J., Estill, J., Xiong, Z., Luo, M., Sebastian, A., Goicoechea, J., Collura, K., Yu, Y., Jiao, Y., Duarte, J., Tang, H., Ayyampalayam, S., Rounsley, S., Kudrna, D., Paterson, A., Pires, J., Chanderbali, A., Soltis, D., , Chamala, S., et al. (2011). A physical map for the Amborella trichopoda genome sheds light on the evolution of angiosperm genome structure. Genome Biology, 12, R48.
• den, C. R., Streng, A., Mita, S., Cao, Q., Polone, E., Liu, W., Ammiraju, J., Kudrna, D., Wing, R., Untergasser, A., Bisseling, T., & Geurts, R. (2011). LysM-Type Mycorrhizal Receptor Recruited for Rhizobium Symbiosis in Nonlegume Parasponia. Science, 331, 909-912.
• Ammiraju, J. S., Song, X., Luo, M., Sisneros, N., Angelova, A., Kudrna, D., Kim, H., Yeisoo, Y. u., Goicoechea, J. L., Lorieux, M., Kurata, N., Brar, D., Ware, D., Jackson, S., & Wing, R. A. (2010). The Oryza BAC resource: A genus-wide and genome scale tool for exploring rice genome evolution and leveraging useful genetic diversity from wild relatives. Breeding Science, 60(5), 536-543.
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  Abstract: Rice was the first crop to have a high-quality reference genome sequence and is now at the forefront of intense functional and evolutionary research for two reasons-its central role in world food security, and its status as a model system for grasses. A thorough characterization of the rice genome cannot be accomplished without a deep understanding of its evolutionary history. The genus Oryza contains two cultivated and 22 wild rice species that represent 10 distinct genome types embedded within a robust phylogeny spanning a ~15 million year time span. The genus contains an untapped reservoir of agriculturally important traits and a historical record of genomic changes (especially those related to domestication, polyploidy, speciation and adaption).The two main objectives of the 'Oryza Map Alignment Project' (OMAP) were to functionally characterize the rice genome from a comparative standpoint and to provide essential tools to leverage the novel genetic diversity from wild relatives for rice improvement. The objective of this review is to summarize our efforts towards developing the most comprehensive genus-wide set of publicly available BAC resources for the genus Oryza, the first of its kind among plants (and perhaps higher eukaryotes), and their applications.
• Dai, L., Jun, W. u., Xunbo, L. i., Wang, X., Liu, X., Jantasuriyarat, C., Kudrna, D., Yeisoo, Y. u., Wing, R. A., Han, B., Zhou, B., & Wang, G. (2010). Genomic structure and evolution of the Pi2/9 locus in wild rice species. Theoretical and Applied Genetics, 121(2), 295-309.
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  PMID: 20229250;Abstract: Rice blast, caused by the fungal pathogen Magnaporthe oryzae, is a devastating disease of rice worldwide. Among the 85 mapped resistance (R) genes against blast, 13 have been cloned and characterized. However, how these genes originated and how they evolved in the Oryza genus remains unclear. We previously cloned the rice blast R-genes Pi2, Pi9, and Piz-t, and analyzed their genomic structure and evolution in cultivated rice. In this study, we determined the genomic sequences of the Pi2/9 locus in four wild Oryza species representing three genomes (AA, BB and CC). The number of Pi2/9 family members in the four wild species ranges from two copies to 12 copies. Although these genes are conserved in structure and categorized into the same subfamily, sequence duplications and subsequent inversions or uneven crossing overs were observed, suggesting that the locus in different wild species has undergone dynamic changes. Positive selection was found in the leucine-rich repeat region of most members, especially in the largest clade where Pi9 is included. We also provide evidence that the Pi9 gene is more related to its homologues in the recurrent line and other rice cultivars than to those in its alleged donor species O. minuta, indicating a possible origin of the Pi9 gene from O. sativa. Comparative sequence analysis between the four wild Oryza species and the previously established reference sequences in cultivated rice species at the Pi2/9 locus has provided extensive and unique information on the genomic structure and evolution of a complex R-gene cluster in the Oryza genus. © 2010 Springer-Verlag.
• Febrer, M., Goicoechea, J. L., Wright, J., McKenzie, N., Song, X., Lin, J., Collura, K., Wissotski, M., Yeisoo, Y. u., S., J., Wolny, E., Idziak, D., Betekhtin, A., Kudrna, D., Hasterok, R., Wing, R. A., & Bevan, M. W. (2010). An integrated physical, genetic and cytogenetic map of Brachypodium distachyon, a model system for grass research. PLoS ONE, 5(10).
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  PMID: 20976139;PMCID: PMC2956642;Abstract: The pooid subfamily of grasses includes some of the most important crop, forage and turf species, such as wheat, barley and Lolium. Developing genomic resources, such as whole-genome physical maps, for analysing the large and complex genomes of these crops and for facilitating biological research in grasses is an important goal in plant biology. We describe a bacterial artificial chromosome (BAC)-based physical map of the wild pooid grass Brachypodium distachyon and integrate this with whole genome shotgun sequence (WGS) assemblies using BAC end sequences (BES). The resulting physical map contains 26 contigs spanning the 272 Mb genome. BES from the physical map were also used to integrate a genetic map. This provides an independent vaildation and confirmation of the published WGS assembly. Mapped BACs were used in Fluorescence In Situ Hybridisation (FISH) experiments to align the integrated physical map and sequence assemblies to chromosomes with high resolution. The physical, genetic and cytogenetic maps, integrated with whole genome shotgun sequence assemblies, enhance the accuracy and durability of this important genome sequence and will directly facilitate gene isolation. © 2010 Febrer et al.
• Geraldes, A., Rambo, T., Wing, R. A., Ferrand, N., & Nachman, M. W. (2010). Extensive gene conversion drives the concerted evolution of paralogous copies of the SRY gene in European rabbits. Molecular Biology and Evolution, 27(11), 2437-2440.
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  PMID: 20525900;PMCID: PMC2955732;Abstract: The human Y chromosome consists of ampliconic genes, which are located in palindromes and undergo frequent gene conversion, and single-copy genes including the primary sex-determining locus, SRY. Here, we demonstrate that SRY is duplicated in a large palindrome in the European rabbit (Oryctolagus cuniculus). Furthermore, we show through comparative sequencing that orthologous palindrome arms have diverged 0.40% between rabbit subspecies over at least 2 My, but paralogous palindrome arms have remained nearly identical. This provides clear evidence of gene conversion on the rabbit Y chromosome. Together with previous observations in humans, these results suggest that gene conversion is a general feature of the evolution of the mammalian Y chromosome. © 2010 The Author.
• Gill, N., SanMiguel, P., Deep, B., Abernathy, B., Kim, H., Stein, L., Ware, D., Wing, R., & Jackson, S. A. (2010). Dynamic Oryza genomes: Repetitive DNA sequences as genome modeling agents. Rice, 3(4), 251-269.
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  Abstract: Repetitive sequences, primarily transposable elements form an indispensable part of eukaryotic genomes. However, little is known about how these sequences originate, evolve and function in context of a genome. In an attempt to address this question, we performed a comparative analysis of repetitive DNA sequences in the genus Oryza, representing ~15 million years of evolution. Both Class I and Class II transposable elements, through their expansion, loss and movement in the genome, were found to influence genome size variation in this genus. We identified 38 LTRretrotransposon families that are present in 1,500 or more copies throughout Oryza, and many are preferentially amplified in specific lineages. The data presented here, besides furthering our understanding of genome organization in the genus Oryza, will aid in the assembly, annotation and analysis of genomic data, as part of the future genome sequencing projects of O. sativa wild relatives. © Springer Science+Business Media, LLC 2010.
• Goicoechea, J. L., Siva, J., Marri, P. R., Chen, M., Jackson, S., Yeisoo, Y. u., Rounsley, S., & Wing, R. A. (2010). The future of rice genomics: Sequencing the collective Oryza genome. Rice, 3(2-3), 89-97.
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  Abstract: The main objectives of the "Oryza Map Alignment Project" (OMAP) are to characterize the rice genome from a comparative standpoint by establishing a genuswide and genome-scale comparative framework from representative species. Here, we report our progress in the analyses of these datasets and emerging "comparative phylogenomics" insights into Oryza evolution at two different resolutions-chromosomal and sequence levels. We demonstrate the abundance and impact of structural variations (SV) on genome diversity using African Oryza as a model. The molecular basis of SV was inferred using three genus-wide vertical sequence datasets. Combined, these data demonstrate that a single reference genome sequence for the genus Oryza is insufficient to comprehensively capture the genomic and allelic diversity present within the genus. Towards this end, we present a strategy to generate high-quality and cost-effective de novo reference sequences of collective Oryza. The application and broader scientific impact of the OMAP resources under an international cooperative effort (I-OMAP) are discussed. © Springer Science + Business Media, LLC 2010.
• Henry, R. J., Rice, N., L., D., Kasem, S., Ishikawa, R., Hao, Y., Dillon, S., Crayn, D., Wing, R., & Vaughan, D. (2010). Australian Oryza: Utility and conservation. Rice, 3(4), 235-241.
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  Abstract: Australian Oryza are an understudied and underexploited genetic resource for rice improvement. Four species are indigenous: Oryza rufipogon, Oryza meridionalis, Oryza australiensis are widespread across northern Australia, whereas Oryza officinalis is known from two localities only. Molecular analysis of these wild populations is required to better define the distinctness of the taxa and the extent of any gene flow between them and rice. Limited collections of these wild populations are held in seed and DNA banks. These species have potential for domestication in some cases but also have many traits of potential value in the improvement of domesticated rice. Stress tolerance (biotic and abiotic) and grain quality characteristics in these populations may be useful. © Springer Science+Business Media, LLC 2010.
• Hurwitz, B. L., Kudrna, D., Yeisoo, Y. u., Sebastian, A., Zuccolo, A., Jackson, S. A., Ware, D., Wing, R. A., & Stein, L. (2010). Rice structural variation: A comparative analysis of structural variation between rice and three of its closest relatives in the genus Oryza. Plant Journal, 63(6), 990-1003.
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  PMID: 20626650;Abstract: Rapid progress in comparative genomics among the grasses has revealed similar gene content and order despite exceptional differences in chromosome size and number. Large- and small-scale genomic variations are of particular interest, especially among cultivated and wild species, as they encode rapidly evolving features that may be important in adaptation to particular environments. We present a genome-wide study of intermediate-sized structural variation (SV) among rice (Oryza sativa) and three of its closest relatives in the genus Oryza (Oryza nivara, Oryza rufipogon and Oryza glaberrima). We computationally identified regional expansions, contractions and inversions in the Oryza species genomes relative to O. sativa by combining data from paired-end clone alignments to the O. sativa reference genome and physical maps. A subset of the computational predictions was validated using a new approach for BAC size determination. The result was a confirmed catalog of 674 expansions (25-38 Mb) and 611 (4-19 Mb) contractions, and 140 putative inversions (14-19 Mb) between the three Oryza species and O. sativa. In the expanded regions unique to O. sativa we found enrichment in transposable elements (TEs): long terminal repeats (LTRs) were randomly located across the chromosomes, and their insertion times corresponded to the date of the A genome radiation. Also, rice-expanded regions contained an over-representation of single-copy genes related to defense factors in the environment. This catalog of confirmed SV in reference to O. sativa provides an entry point for future research in genome evolution, speciation, domestication and novel gene discovery. © 2010 Blackwell Publishing Ltd.
• Lin, L., Pierce, G. J., Bowers, J. E., Estill, J. C., Compton, R. O., Rainville, L. K., Kim, C., Lemke, C., Rong, J., Tang, H., Wang, X., Braidotti, M., Chen, A. H., Chicola, K., Collura, K., Epps, E., Golser, W., Grover, C., Ingles, J., , Karunakaran, S., et al. (2010). A draft physical map of a D-genome cotton species (Gossypium raimondii). BMC Genomics, 11(1).
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  PMID: 20569427;PMCID: PMC2996926;Abstract: Background: Genetically anchored physical maps of large eukaryotic genomes have proven useful both for their intrinsic merit and as an adjunct to genome sequencing. Cultivated tetraploid cottons, Gossypium hirsutum and G. barbadense, share a common ancestor formed by a merger of the A and D genomes about 1-2 million years ago. Toward the long-term goal of characterizing the spectrum of diversity among cotton genomes, the worldwide cotton community has prioritized the D genome progenitor Gossypium raimondii for complete sequencing.Results: A whole genome physical map of G. raimondii, the putative D genome ancestral species of tetraploid cottons was assembled, integrating genetically-anchored overgo hybridization probes, agarose based fingerprints and 'high information content fingerprinting' (HICF). A total of 13,662 BAC-end sequences and 2,828 DNA probes were used in genetically anchoring 1585 contigs to a cotton consensus genetic map, and 370 and 438 contigs, respectively to Arabidopsis thaliana (AT) and Vitis vinifera (VV) whole genome sequences.Conclusion: Several lines of evidence suggest that the G. raimondii genome is comprised of two qualitatively different components. Much of the gene rich component is aligned to the Arabidopsis and Vitis vinifera genomes and shows promise for utilizing translational genomic approaches in understanding this important genome and its resident genes. The integrated genetic-physical map is of value both in assembling and validating a planned reference sequence. © 2010 Lin et al; licensee BioMed Central Ltd.
• S., J., Fan, C., Yeisoo, Y. u., Song, X., Cranston, K. A., Pontaroli, A. C., Fei, L. u., Sanyal, A., Jiang, N., Rambo, T., Currie, J., Collura, K., Talag, J., Bennetzen, J. L., Chen, M., Jackson, S., & Wing, R. A. (2010). Spatio-temporal patterns of genome evolution in allotetraploid species of the genus Oryza. Plant Journal, 63(3), 430-442.
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  Abstract: Despite knowledge that polyploidy is widespread and a major evolutionary force in flowering plant diversification, detailed comparative molecular studies on polyploidy have been confined to only a few species and families. The genus Oryza is composed of 23 species that are classified into ten distinct 'genome types' (six diploid and four polyploid), and is emerging as a powerful new model system to study polyploidy. Here we report the identification, sequence and comprehensive comparative annotation of eight homoeologous genomes from a single orthologous region (Adh1-Adh2) from four allopolyploid species representing each of the known Oryza genome types (BC, CD, HJ and KL). Detailed comparative phylogenomic analyses of these regions within and across species and ploidy levels provided several insights into the spatio-temporal dynamics of genome organization and evolution of this region in 'natural' polyploids of Oryza. The major findings of this study are that: (i) homoeologous genomic regions within the same nucleus experience both independent and parallel evolution, (ii) differential lineage-specific selection pressures do not occur between polyploids and their diploid progenitors, (iii) there have been no dramatic structural changes relative to the diploid ancestors, (iv) a variation in the molecular evolutionary rate exists between the two genomes in the BC complex species even though the BC and CD polyploid species appear to have arisen
• Sanyal, A., Ammiraju, J. S., Fei, L. u., Yeisoo, Y. u., Rambo, T., Currie, J., Kollura, K., Kim, H., Chen, J., Jianxin, M. a., Miguel, P. S., Mingsheng, C., Wing, R. A., & Jackson, S. A. (2010). Orthologous comparisons of the Hd1 region across genera reveal hd1 gene lability within diploid oryza species and disruptions to microsynteny in sorghum. Molecular Biology and Evolution, 27(11), 2487-2506.
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  PMID: 20522726;Abstract: Heading date is one of the most important quantitative traits responsible for the domestication of rice. We compared a 155-kb reference segment of the Oryza sativa ssp. japonica cv. Nipponbare genome surrounding Hd1, a major heading date gene in rice, with orthologous regions from nine diploid Oryza species that diverged over a relatively short time frame (∼16 My) to study sequence evolution around a domestication locus. The orthologous Hd1 region from Sorghum bicolor was included to compare and contrast the evolution in a more distant relative of rice. Consistent with other observations at the adh1/adh2, monoculm1, and sh2/a1 loci in grass species, we found high gene colinearity in the Hd1 region amidst size differences that were lineage specific and long terminal repeat retrotransposon driven. Unexpectedly, the Hd1 gene was deleted in O. glaberrima, whereas the O. rufipogon and O. punctata copies had degenerative mutations, suggesting that other heading date loci might compensate for the loss or nonfunctionality of Hd1 in these species. Compared with the japonica Hd1 region, the orthologous region in sorghum exhibited micro-rearrangements including gene translocations, seven additional genes, and a gene triplication and truncation event predating the divergence from Oryza. © 2010 The Author.
• Schmutz, J., Cannon, S. B., Schlueter, J., Jianxin, M. a., Mitros, T., Nelson, W., Hyten, D. L., Song, Q., Thelen, J. J., Cheng, J., Dong, X. u., Hellsten, U., May, G. D., Yeisoo, Y. u., Sakurai, T., Umezawa, T., Bhattacharyya, M. K., Sandhu, D., Valliyodan, B., , Lindquist, E., et al. (2010). Erratum: Genome sequence of the palaeopolyploid soybean (Nature (2010) 463 (178-183)). Nature, 465(7294), 120-.
• Schmutz, J., Cannon, S. B., Schlueter, J., Ma, J., Mitros, T., Nelson, W., Hyten, D. L., Song, Q., Thelen, J. J., Cheng, J., Xu, D., Hellsten, U., May, G. D., Yu, Y., Sakurai, T., Umezawa, T., Bhattacharyya, M. K., Sandhu, D., Valliyodan, B., , Lindquist, E., et al. (2010). Genome sequence of the palaeopolyploid soybean. NATURE, 463(7278), 178-183.
• Zuccolo, A., Sebastian, A., Yeisoo, Y. u., Jackson, S., Rounsley, S., Billheimer, D., & Wing, R. A. (2010). Assessing the extent of substitution rate variation of retrotransposon Long Terminal Repeat sequences in Oryza sativa and Oryza glaberrima. Rice, 3(4), 242-250.
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  Abstract: Long Terminal Repeat retrotransposons (LTRRTs) are a major component of several plant genomes. Important insights into the evolutionary dynamics of these elements in a genome are provided by the comparative study of their insertion times. These can be inferred by the comparison of pairs of LTRs flanking intact LTR-RTs in combination with an estimated substitution rate. Over the past several years, different substitution rates have been proposed for LTRs in crop plants. However, very little is known about the extent of substitution rate variation and the factors contributing to this variation, so the rates currently used are generally considered rough estimators of actual rates. To evaluate the extent of substitution rate variation in LTRs, we identified 70 orthologous LTRs on the short arms of chromosome 3 of both Oryza sativa and Oryza glaberrima, species that diverged ~0.64 Ma. Since these orthologous sequences were present in a common ancestor prior to species divergence, nucleotide differences identified in comparing these regions must correspond to mutations accumulated post-speciation, thereby giving us the opportunity to study LTR substitution rate variation in different elements across these short arms. As a control, we analyzed a similar amount of non-repeat-related sequences collected near the orthologous LTRs. Our analysis showed that substitution rate variation in LTRs is greater than 5- fold, is positively correlated with G+C content, and tends to increase near centromeric regions. We confirmed that in the vast majority of cases, LTRs mutate faster than their corresponding non-repeat-related neighboring sequences. © Springer Science+Business Media, LLC 2010.
• Degnan, P. H., Yeisoo, Y. u., Sisneros, N., Wing, R. A., & Moran, N. A. (2009). Hamiltonella defensa, genome evolution of protective bacterial endosymbiont from pathogenic ancestors. Proceedings of the National Academy of Sciences of the United States of America, 106(22), 9063-9068.
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  PMID: 19451630;PMCID: PMC2690004;Abstract: Eukaryotes engage in a multitude of beneficial and deleterious interactions with bacteria. Hamiltonella defensa, an endosymbiont of aphids and other sap-feeding insects, protects its aphid host from attack by parasitoid wasps. Thus H. defensa is only conditionally beneficial to hosts, unlike ancient nutritional symbionts, such as Buchnera, that are obligate. Similar to pathogenic bacteria, H. defensa is able to invade naive hosts and circumvent host immune responses. We have sequenced the genome of H. defensa to identify possible mechanisms that underlie its persistence in healthy aphids and protection from parasitoids. The 2.1-Mb genome has undergone significant reduction in size relative to its closest free-living relatives, which include Yersinia and Serratia species (4.6-5.4 Mb). Auxotrophic for 8 of the 10 essential amino acids, H. defensa is reliant upon the essential amino acids produced by Buchnera. Despite these losses, the H. defensa genome retains more genes and pathways for a variety of cell structures and processes than do obligate symbionts, such as Buchnera. Furthermore, putative pathogenicity loci, encoding type-3 secretion systems, and toxin homologs, which are absent in obligate symbionts, are abundant in the H. defensa genome, as are regulatory genes that likely control the timing of their expression. The genome is also littered with mobile DNA, including phage-derived genes, plasmids, and insertion-sequence elements, highlighting its dynamic nature and the continued role horizontal gene transfer plays in shaping it.
• Degnan, P. H., Yu, Y., Sisneros, N., Wing, R. A., & Moran, N. A. (2009). Hamiltonella defensa, genome evolution of protective bacterial endosymbiont from pathogenic ancestors. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 106(22), 9063-9068.
• Fei, L. u., S., J., Sanyal, A., Zhang, S., Son, R., Chen, J., Guisheng, L. i., Sui, Y., Song, X., Cheng, Z., Costa, A., Bennetzen, J. L., Jackson, S. A., Wing, R. A., & Chena, M. (2009). Comparative sequence analysis of MONOCULM1-orthologous regions in 14 Oryza genomes. Proceedings of the National Academy of Sciences of the United States of America, 106(6), 2071-2076.
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  PMID: 19164767;PMCID: PMC2629783;Abstract: Comparative genomics is a powerful tool to decipher gene and genome evolution. Placing multiple genome comparisons in a phylogenetic context improves the sensitivity of evolutionary inferences. In the genus Oryza, this comparative approach can be used to investigate gene function, genome evolution, domestication, polyploidy, and ecological adaptation. A large genomic region surrounding the MONOCULM1 (MOC1) locus was chosen for study in 14 Oryza species, including 10 diploids and 4 allotetraploids. Sequencing and annotation of 18 bacterial artificial chromosome clones for these species revealed highly conserved gene colinearity and structure in the MOC1 region. Since the Oryza radiation about 14 Mya, differences in transposon amplification appear to be responsible for the different current sizes of the Oryza genomes. In the MOC1 region, transposons were only conserved between genomes of the same type (e.g., AA or BB). In addition to the conserved gene content, several apparent genes have been generated de novo or uniquely retained in the AA lineage. Two different 3-gene segments have been inserted into the MOC1 region of O. coarctata (KK) or O. sativa by unknown mechanism(s). Large and apparently noncoding sequences flanking the MOC1 gene were observed to be under strong purifying selection. The allotetraploids Oryza alta and Oryza minuta were found to be products of recent polyploidization, less than 1.6 and 0.4 Mya, respectively. In allotetraploids, pseudogenization of duplicated genes was common, caused by large deletions, small frame-shifting insertions/deletions, or nonsense mutations. © 2009 by The National Academy of Sciences of the USA.
• Gao, D., Gill, N., Kim, H., Walling, J. G., Zhang, W., Fan, C., Yeisoo, Y. u., Jianxin, M. a., Sanmiguel, P., Jiang, N., Cheng, Z., Wing, R. A., Jiang, J., & Jackson, S. A. (2009). A lineage-specific centromere retrotransposon in Oryza brachyantha. Plant Journal, 60(5), 820-831.
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  PMID: 19702667;Abstract: Most eukaryotic centromeres contain large quantities of repetitive DNA, such as satellite repeats and retrotransposons. Unlike most transposons in plant genomes, the centromeric retrotransposon (CR) family is conserved over long evolutionary periods among a majority of the grass species. CR elements are highly concentrated in centromeres, and are likely to play a role in centromere function. In order to study centromere evolution in the Oryza (rice) genus, we sequenced the orthologous region to centromere 8 of Oryza sativa from a related species, Oryza brachyantha. We found that O. brachyantha does not have the canonical CRR (CR of rice) found in the centromeres of all other Oryza species. Instead, a new Ty3-gypsy (Metaviridae) retroelement (FRetro3) was found to colonize the centromeres of this species. This retroelement is found in high copy numbers in the O. brachyantha genome, but not in other Oryza genomes, and based on the dating of long terminal repeats (LTRs) of FRetro3 it was amplified in the genome in the last few million years. Interestingly, there is a high level of removal of FRetro3 based on solo-LTRs to full-length elements, and this rapid turnover may have played a role in the replacement of the canonical CRR with the new element by active deletion. Comparison with previously described ChIP cloning data revealed that FRetro3 is found in CENH3-associated chromatin sequences. Thus, within a single lineage of the Oryza genus, the canonical component of grass centromeres has been replaced with a new retrotransposon that has all the hallmarks of a centromeric retroelement. © 2009 Blackwell Publishing Ltd.
• Lu, F., Ammiraju, J., Sanyal, A., Zhang, S., Song, R., Chen, J., Li, G., Sui, Y. i., Song, X., Cheng, Z., de, O., Bennetzen, J. L., Jackson, S. A., Wing, R. A., & Chen, M. (2009). Comparative sequence analysis of MONOCULM1-orthologous regions in 14 Oryza genomes. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 106(6), 2071-2076.
• Rounsley, S., Marri, P. R., Yeisoo, Y. u., Ruifeng, H. e., Sisneros, N., Goicoechea, J. L., Lee, S. J., Angelova, A., Kudrna, D., Luo, M., Affourtit, J., Desany, B., Knight, J., Niazi, F., Egholm, M., & Wing, R. A. (2009). De novo next generation sequencing of plant genomes. Rice, 2(1), 35-43.
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  Abstract: The genome sequencing of all major food and bioenergy crops is of critical importance in the race to improve crop production to meet the future food and energy security needs of the world. Next generation sequencing technologies have brought about great improvements in sequencing throughput and cost, but do not yet allow for de novo sequencing of large repetitive genomes as found in most crop plants.We present a strategy that combines cutting edge next generation sequencing with "old school" genomics resources and allows rapid cost-effective sequencing of plant genomes. © Springer Science + Business Media, LLC 2009.
• Schnable, P. S., Ware, D., Fulton, R. S., Stein, J. C., Wei, F., Pasternak, S., Liang, C., Zhang, J., Fulton, L., Graves, T. A., Minx, P., Reily, A. D., Courtney, L., Kruchowski, S. S., Tomlinson, C., Strong, C., Delehaunty, K., Fronick, C., Courtney, B., , Rock, S. M., et al. (2009). The B73 Maize Genome: Complexity, Diversity, and Dynamics. SCIENCE, 326(5956), 1112-1115.
• Schnable, P. S., Ware, D., Fulton, R. S., Stein, J. C., Wei, F., Pasternak, S., Liang, C., Zhang, J., Fulton, L., Graves, T. A., Minx, P., Reily, A. D., Courtney, L., Kruchowski, S. S., Tomlinson, C., Strong, C., Delehaunty, K., Fronick, C., Courtney, B., , Rock, S. M., et al. (2009). The B73 maize genome: Complexity, diversity, and dynamics. Science, 326(5956), 1112-1115.
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  PMID: 19965430;Abstract: We report an improved draft nucleotide sequence of the 2.3-gigabase genome of maize, an important crop plant and model for biological research. Over 32,000 genes were predicted, of which 99.8% were placed on reference chromosomes. Nearly 85% of the genome is composed of hundreds of families of transposable elements, dispersed nonuniformly across the genome. These were responsible for the capture and amplification of numerous gene fragments and affect the composition, sizes, and positions of centromeres. We also report on the correlation of methylationpoor regions with Mu transposon insertions and recombination, and copy number variants with insertions and/or deletions, as well as how uneven gene losses between duplicated regions were involved in returning an ancient allotetraploid to a genetically diploid state. These analyses inform and set the stage for further investigations to improve our understanding of the domestication and agricultural improvements of maize.
• Wing, R., Cranston, K. A., Hurwitz, B., Ware, D., Stein, L., & Wing, R. A. (2009). Species trees from highly incongruent gene trees in rice. Systematic biology, 58(5).
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  Several methods have recently been developed to infer multilocus phylogenies by incorporating information from topological incongruence of the individual genes. In this study, we investigate 2 such methods, Bayesian concordance analysis and Bayesian estimation of species trees. Our test data are a collection of genes from cultivated rice (genus Oryza) and the most closely related wild species, generated using a high-throughput sequencing protocol and bioinformatics pipeline. Trees inferred from independent genes display levels of topological incongruence that far exceed that seen in previous data sets analyzed with these species tree methods. We identify differences in phylogenetic results between inference methods that incorporate gene tree incongruence. Finally, we discuss the challenges of scaling these analyses for data sets with thousands of gene trees and extensive levels of missing data.
• Wing, R., Wei, F., Stein, J. C., Liang, C., Zhang, J., Fulton, R. S., Baucom, R. S., De Paoli, E., Zhou, S., Yang, L., Han, Y., Pasternak, S., Narechania, A., Zhang, L., Yeh, C., Ying, K., Nagel, D. H., Collura, K., Kudrna, D., , Currie, J., et al. (2009). Detailed analysis of a contiguous 22-Mb region of the maize genome. PLoS genetics, 5(11).
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  Most of our understanding of plant genome structure and evolution has come from the careful annotation of small (e.g., 100 kb) sequenced genomic regions or from automated annotation of complete genome sequences. Here, we sequenced and carefully annotated a contiguous 22 Mb region of maize chromosome 4 using an improved pseudomolecule for annotation. The sequence segment was comprehensively ordered, oriented, and confirmed using the maize optical map. Nearly 84% of the sequence is composed of transposable elements (TEs) that are mostly nested within each other, of which most families are low-copy. We identified 544 gene models using multiple levels of evidence, as well as five miRNA genes. Gene fragments, many captured by TEs, are prevalent within this region. Elimination of gene redundancy from a tetraploid maize ancestor that originated a few million years ago is responsible in this region for most disruptions of synteny with sorghum and rice. Consistent with other sub-genomic analyses in maize, small RNA mapping showed that many small RNAs match TEs and that most TEs match small RNAs. These results, performed on approximately 1% of the maize genome, demonstrate the feasibility of refining the B73 RefGen_v1 genome assembly by incorporating optical map, high-resolution genetic map, and comparative genomic data sets. Such improvements, along with those of gene and repeat annotation, will serve to promote future functional genomic and phylogenomic research in maize and other grasses.
• Wing, R., Wei, F., Zhang, J., Zhou, S., He, R., Schaeffer, M., Collura, K., Kudrna, D., Faga, B. P., Wissotski, M., Golser, W., Rock, S. M., Graves, T. A., Fulton, R. S., Coe, E., Schnable, P. S., Schwartz, D. C., Ware, D., Clifton, S. W., , Wilson, R. K., et al. (2009). The physical and genetic framework of the maize B73 genome. PLoS genetics, 5(11).
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  Maize is a major cereal crop and an important model system for basic biological research. Knowledge gained from maize research can also be used to genetically improve its grass relatives such as sorghum, wheat, and rice. The primary objective of the Maize Genome Sequencing Consortium (MGSC) was to generate a reference genome sequence that was integrated with both the physical and genetic maps. Using a previously published integrated genetic and physical map, combined with in-coming maize genomic sequence, new sequence-based genetic markers, and an optical map, we dynamically picked a minimum tiling path (MTP) of 16,910 bacterial artificial chromosome (BAC) and fosmid clones that were used by the MGSC to sequence the maize genome. The final MTP resulted in a significantly improved physical map that reduced the number of contigs from 721 to 435, incorporated a total of 8,315 mapped markers, and ordered and oriented the majority of FPC contigs. The new integrated physical and genetic map covered 2,120 Mb (93%) of the 2,300-Mb genome, of which 405 contigs were anchored to the genetic map, totaling 2,103.4 Mb (99.2% of the 2,120 Mb physical map). More importantly, 336 contigs, comprising 94.0% of the physical map ( approximately 1,993 Mb), were ordered and oriented. Finally we used all available physical, sequence, genetic, and optical data to generate a golden path (AGP) of chromosome-based pseudomolecules, herein referred to as the B73 Reference Genome Sequence version 1 (B73 RefGen_v1).
• Zhang, H., DiBaise, J. K., Zuccolo, A., Kudrna, D., Braidotti, M., Yeisoo, Y. u., Parameswaran, P., Crowell, M. D., Wing, R., Rittmann, B. E., & Krajmalnik-Brown, R. (2009). Human gut microbiota in obesity and after gastric bypass. Proceedings of the National Academy of Sciences of the United States of America, 106(7), 2365-2370.
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  PMID: 19164560;PMCID: PMC2629490;Abstract: Recent evidence suggests that the microbial community in the human intestine may play an important role in the pathogenesis of obesity. We examined 184,094 sequences of microbial 16S rRNA genes from PCR amplicons by using the 454 pyrosequencing technology to compare the microbial community structures of 9 individuals, 3 in each of the categories of normal weight, morbidly obese, and post-gastric-bypass surgery. Phylogenetic analysis demonstrated that although the Bacteria in the human intestinal community were highly diverse, they fell mainly into 6 bacterial divisions that had distinct differences in the 3 study groups. Specifically, Firmicutes were dominant in normal-weight and obese individuals but significantly decreased in post-gastric-bypass individuals, who had a proportional increase of Gammaproteobacteria. Numbers of the H 2-producing Prevotellaceae were highly enriched in the obese individuals. Unlike the highly diverse Bacteria, the Archaea comprised mainly members of the order Methanobacteriales, which are H2-oxidizing methanogens. Using real-time PCR, we detected significantly higher numbers of H2-utilizing methanogenic Archaea in obese individuals than in normal-weight or post-gastric-bypass individuals. The coexistence of H 2-producing bacteria with relatively high numbers of H 2-utilizing methanogenic Archaea in the gastrointestinal tract of obese individuals leads to the hypothesis that interspecies H2 transfer between bacterial and archaeal species is an important mechanism for increasing energy uptake by the human large intestine in obese persons. The large bacterial population shift seen in the post-gastric-bypass individuals may reflect the double impact of the gut alteration caused by the surgical procedure and the consequent changes in food ingestion and digestion. © 2009 by The National Academy of Sciences of the USA.
• Zhang, H., DiBaise, J. K., Zuccolo, A., Kudrna, D., Braidotti, M., Yu, Y., Parameswaran, P., Crowell, M. D., Wing, R., Rittmann, B. E., & Krajmalnik-Brown, R. (2009). Human gut microbiota in obesity and after gastric bypass. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 106(7), 2365-2370.
• Zhou, S., Wei, F., Nguyen, J., Bechner, M., Potamousis, K., Goldstein, S., Pape, L., Mehan, M. R., Churas, C., Pasternak, S., Forrest, D. K., Wise, R., Ware, D., Wing, R. A., Waterman, M. S., Livny, M., & Schwartz, D. C. (2009). A Single Molecule Scaffold for the Maize Genome. PLOS GENETICS, 5(11).
• Zhou, S., Wei, F., Nguyen, J., Bechner, M., Potamousis, K., Goldstein, S., Pape, L., Mehan, M. R., Churas, C., Pasternak, S., Forrest, D. K., Wise, R., Ware, D., Wing, R. A., Waterman, M. S., Livny, M., & Schwartz, D. C. (2009). A single molecule scaffold for the maize genome. PLoS Genetics, 5(11).
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  PMID: 19936062;PMCID: PMC2774507;Abstract: About 85% of the maize genome consists of highly repetitive sequences that are interspersed by low-copy, gene-coding sequences. The maize community has dealt with this genomic complexity by the construction of an integrated genetic and physical map (iMap), but this resource alone was not sufficient for ensuring the quality of the current sequence build. For this purpose, we constructed a genome-wide, high-resolution optical map of the maize inbred line B73 genome containing >91,000 restriction sites (averaging 1 site/∼23 kb) accrued from mapping genomic DNA molecules. Our optical map comprises 66 contigs, averaging 31.88 Mb in size and spanning 91.5% (2,103.93 Mb/∼2,300 Mb) of the maize genome. A new algorithm was created that considered both optical map and unfinished BAC sequence data for placing 60/66 (2,032.42 Mb) optical map contigs onto the maize iMap. The alignment of optical maps against numerous data sources yielded comprehensive results that proved revealing and productive. For example, gaps were uncovered and characterized within the iMap, the FPC (fingerprinted contigs) map, and the chromosome-wide pseudomolecules. Such alignments also suggested amended placements of FPC contigs on the maize genetic map and proactively guided the assembly of chromosome-wide pseudomolecules, especially within complex genomic regions. Lastly, we think that the full integration of B73 optical maps with the maize iMap would greatly facilitate maize sequence finishing efforts that would make it a valuable reference for comparative studies among cereals, or other maize inbred lines and cultivars. © 2009 Zhou et al.
• Ammiraju, J., Lu, F., Sanyal, A., Yu, Y., Song, X., Jiang, N., Pontaroli, A. C., Rambo, T., Currie, J., Collura, K., Talag, J., Fan, C., Goicoechea, J. L., Zuccolo, A., Chen, J., Bennetzen, J. L., Chen, M., Jackson, S., & Wing, R. A. (2008). Dynamic Evolution of Oryza Genomes Is Revealed by Comparative Genomic Analysis of a Genus-Wide Vertical Data Set. PLANT CELL, 20(12), 3191-3209.
• Cunff, L. L., Garsmeur, O., Raboin, L. M., Pauquet, J., Telismart, H., Selvi, A., Grivet, L., Philippe, R., Begum, D., Deu, M., Costet, L., Wing, R., Glaszmann, J. C., & D'Hont, A. (2008). Diploid/polyploid syntenic shuttle mapping and haplotype-specific chromosome walking toward a rust resistance gene (Bru1) in highly polyploid sugarcane (2n ∼ 12x ∼ 115). Genetics, 180(1), 649-660.
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  PMID: 18757946;PMCID: PMC2535714;Abstract: The genome of modern sugarcane cultivars is highly polyploid (∼12x), aneuploid, of interspecific origin, and contains 10 Gb of DNA. Its size and complexity represent a major challenge for the isolation of agronomically important genes. Here we report on the first attempt to isolate a gene from sugarcane by map-based cloning, targeting a durable major rust resistance gene (Bru1). We describe the genomic strategies that we have developed to overcome constraints associated with high polyploidy in the successive steps of map-based cloning approaches, including diploid/polyploid syntenic shuttle mapping with two model diploid species (sorghum and rice) and haplotype-specific chromosome walking. Their applications allowed us (i) to develop a high-resolution map including markers at 0.28 and 0.14 cM on both sides and 13 markers cosegregating with Bru1 and (ii) to develop a physical map of the target haplotype that still includes two gaps at this stage due to the discovery of an insertion specific to this haplotype. These approaches will pave the way for the development of future map-based cloning approaches for sugarcane and other complex polyploid species. Copyright © 2008 by the Genetics Society of America.
• Fan, C., Zhang, Y., Yeisoo, Y. u., Rounsley, S., Long, M., & Wing, R. A. (2008). The subtelomere of Oryza sativa chromosome 3 short arm as a hot bed of new gene origination in rice. Molecular Plant, 1(5), 839-850.
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  PMID: 19825586;PMCID: PMC2902912;Abstract: Despite general observations of non-random genomic distribution of new genes, it is unclear whether or not new genes preferentially occur in certain genomic regions driven by related molecular mechanisms. Using 1.5 Mb of genomic sequences from short arms of chromosome 3 of Oryza glaberrima and O. punctata, we conducted a comparative genomic analysis with the reference O. sativa ssp. japonica genome. We identified a 60-kb segment located in the middle of the subtelomeric region of chromosome 3, which is unique to the species O. sativa. The region contained gene duplicates that occurred in Asian cultivated rice species that diverged from the ancestor of Asian and African cultivated rice one million years ago (MYA). For the 12 genes and one complete retrotransposon identified in this segment in O. sativa ssp. japonica, we searched for their parental genes. The high similarity between duplicated paralogs further supports the recent origination of these genes. We found that this segment was recently generated through multiple independent gene recombination and transposon insertion events. Among the 12 genes, we found that five had chimeric gene structures derived from multiple parental genes. Nine out of the 12 new genes seem to be functional, as suggested by Ka/Ks analysis and the presence of cDNA and/or MPSS data. Furthermore, for the eight transcribed genes, at least two genes could be classified as defense or stress response-related genes. Given these findings, and the fact that subtelomeres are associated with high rates of recombination and transcription, it is likely that subtelomeres may facilitate gene recombination and transposon insertions and serve as hot spots for new gene origination in rice genomes. © The Author 2008.
• Grover, C. E., Yeisoo, Y. u., Wing, R. A., Paterson, A. H., & Wendel, J. F. (2008). A phylogenetic analysis of indel dynamics in the cotton genus. Molecular Biology and Evolution, 25(7), 1415-1428.
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  PMID: 18400789;Abstract: Genome size evolution is a dynamic process involving counterbalancing mechanisms whose actions vary across lineages and over time. Whereas the primary mechanism of expansion, transposable element (TE) amplification, has been widely documented, the evolutionary dynamics of genome contraction have been less thoroughly explored. To evaluate the relative impact and evolutionary stability of the mechanisms that affect genome size, we conducted a phylogenetic analysis of indel rates for 2 genomic regions in 4 Gossypium genomes: the 2 coresident genomes (AT and DT) of tetraploid cotton and its model diploid progenitors, Gossypium arboreum (A) and Gossypium raimondii (D). We determined the rates of sequence gain or loss along each branch, partitioned by mechanism, and how these changed during species divergence. In general, there has been a propensity toward growth of the diploid genomes and contraction in the polyploid. Most of the size difference between the diploid species occurred prior to polyploid divergence and was largely attributable to TE amplification in the A/AT genome. After separating from the true parents of the polyploid genomes, both diploid genomes experienced slower sequence gain than in the ancestor, due to fewer TE insertions in the A genome and a combination of increased deletions and decreased TE insertions in the D genome. Both genomes of the polyploid displayed increased rates of deletion and decreased rates of insertion, leading to a rate of near stasis in DT and overall contraction in AT resulting in polyploid genome contraction. As expected, TE insertions contributed significantly to the genome size differences; however, intrastrand homologous recombination, although rare, had the most significant impact on the rate of deletion. Small indel data for the diploids suggest the possibility of a bias as the smaller genomes add less or delete more sequence through small indels than do the larger genomes, whereas data for the polyploid suggest increased sequence turnover in general (both as small deletions and small insertions). Illegitimate recombination, although not demonstrated to be a dominant mechanism of genome size change, was biased in the polyploid toward deletions, which may provide a partial explanation of polyploid genomic downsizing. © The Author 2008. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved.
• Maughan, P. J., Sisneros, N., Luo, M., Kudrna, D., S., J., & Wing, R. A. (2008). Construction of an Amaranthus hypochondriacus bacterial artificial chromosome library and genomic sequencing of herbicide target genes. Crop Science, 48(SUPPL. 1), S85-S94.
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  Abstract: Before the Spanish conquest of the ancient Americas, the grain amaranths (Amaranthus caudatus L., A. cruentus L., A. hypochondriacus L.) were a staple food of the New World. Recently, the grain amaranths have regained international attention for their nuttitional quality and importance as a symbol of indigenous cultures. Here we report the development of a bacterial artificial chromosome (BAC) library constructed from the cultivar 'Plainsman' (A. hypochondriacus; 2n = 2x = 32). The library consists of a total of 36,864 clones with an average insert size of 147 kb with less than 1.8% of the clones containing empty vectors. The frequency of BAC clones carrying inserts derived from chloroplast and mitochondrial DNA was estimated to be 6.9%. Thus, based on a haploid genome size of 466 Mb per haploid nucleus, the BAC library coverage is approximately 10.6 times the haploid genome content. The genome coverage estimate was empirically confirmed by screening the library with seven low copy amaranth probe sequences. The utility of the amaranth BAC library was demonstrated by identification and full-length genomic sequencing of the acetolactate synthase and protoporphyrinogen oxidase genes - both major targets for several classes of important herbicides. The quality of the BAC library for BAC end sequencing projects was evaluated by bidirectional end sequencing of 384 random clones. End sequences were annotated using BLAST searches and queries to plant transposable element databases. © Crop Science Society of America.
• Meier, S., Gehring, C., MacPherson, C. R., Kaur, M., Maqungo, M., Reuben, S., Muyanga, S., Shih, M., Wei, F., Wanchana, S., Mauleon, R., Radovanovic, A., Bruskiewich, R., Tanaka, T., Mohanty, B., Itoh, T., Wing, R., Gojobori, T., Sasaki, T., , Swarup, S., et al. (2008). The promoter signatures in rice LEA genes can be used to build a co-expressing LEA gene network. Rice, 1(2), 177-187.
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  Abstract: Coordinated transcriptional modulation of large gene sets depends on the combinatorial use of cis-regulatory motifs in promoters. We postulate that promoter content similarities are diagnostic for co-expressing genes that function coherently during specific cellular responses. To find the coexpressing genes we propose an ab initio method that identifies motif families in promoters of target gene groups, map these families to the promoters of all genes in the genome, and determine the best matches of each of the target group gene promoters with all other promoters. When the method was tested in rice starting from a group of co-expressing Late Embryogenesis Abundant (LEA) genes, we obtained a promoter similarity-based network that contained candidate genes that could plausibly complement the function of LEA genes. Importantly, 73.36% of 244 genes predicted by our method were experimentally confirmed to co-express with the LEA genes inmaturing rice embryos,making thismethodology a promising tool for biological systems analyses. © The Author(s) 2008.
• Nelson, W., Luo, M., Jianxin, M. a., Estep, M., Estill, J., Ruifeng, H. e., Talag, J., Sisneros, N., Kudrna, D., Kim, H., Ammiraju, J. S., Collura, K., Bharti, A. K., Messing, J., Wing, R. A., SanMiguel, P., Bennetzen, J. L., & Soderlund, C. (2008). Methylation-sensitive linking libraries enhance gene-enriched sequencing of complex genomes and map DNA methylation domains. BMC Genomics, 9.
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  PMID: 19099592;PMCID: PMC2628917;Abstract: Background: Many plant genomes are resistant to whole-genome assembly due to an abundance of repetitive sequence, leading to the development of gene-rich sequencing techniques. Two such techniques are hypomethylated partial restriction (HMPR) and methylation spanning linker libraries (MSLL). These libraries differ from other gene-rich datasets in having larger insert sizes, and the MSLL clones are designed to provide reads localized to "epigenetic boundaries" where methylation begins or ends. Results: A large-scale study in maize generated 40,299 HMPR sequences and 80,723 MSLL sequences, including MSLL clones exceeding 100 kb. The paired end reads of MSLL and HMPR clones were shown to be effective in linking existing gene-rich sequences into scaffolds. In addition, it was shown that the MSLL clones can be used for anchoring these scaffolds to a BAC-based physical map. The MSLL end reads effectively identified epigenetic boundaries, as indicated by their preferential alignment to regions upstream and downstream from annotated genes. The ability to precisely map long stretches of fully methylated DNA sequence is a unique outcome of MSLL analysis, and was also shown to provide evidence for errors in gene identification. MSLL clones were observed to be significantly more repeat-rich in their interiors than in their end reads, confirming the correlation between methylation and retroelement content. Both MSLL and HMPR reads were found to be substantially gene-enriched, with the SalI MSLL libraries being the most highly enriched (31% align to an EST contig), while the HMPR clones exhibited exceptional depletion of repetitive DNA (to ∼11%). These two techniques were compared with other gene-enrichment methods, and shown to be complementary. Conclusion: MSLL technology provides an unparalleled approach for mapping the epigenetic status of repetitive blocks and for identifying sequences mis-identified as genes. Although the types and natures of epigenetic boundaries are barely understood at this time, MSLL technology flags both approximate boundaries and methylated genes that deserve additional investigation. MSLL and HMPR sequences provide a valuable resource for maize genome annotation, and are a uniquely valuable complement to any plant genome sequencing project. In order to make these results fully accessible to the community, a web display was developed that shows the alignment of MSLL, HMPR, and other gene-rich sequences to the BACs; this display is continually updated with the latest ESTs and BAC sequences. © 2008 Nelson et al; licensee BioMed Central Ltd.
• Roulin, A., Piegu, B., Wing, R. A., & Panaud, O. (2008). Evidence of multiple horizontal transfers of the long terminal repeat retrotransposon RIRE1 within the genus Oryza. Plant Journal, 53(6), 950-959.
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  PMID: 18088314;Abstract: Horizontal gene transfer, defined as the transmission of genetic material between reproductively isolated species, has been considered for a long time to be a rare phenomenon. Most well-documented cases of horizontal gene transfer have been described in prokaryotes or in animals and they often involve transposable elements. The most abundant class of transposable elements in plant genomes are the long terminal repeat (LTR) retrotransposons. Because of their propensity to increase their copy number while active, LTR retrotransposons can have a significant impact on genomics changes during evolution. In a previous study, we showed that in the wild rice species Oryza australiensis, 60% of the genome is composed of only three families of LTR retrotransposons named RIRE1, Wallabi and Kangourou. In the present study, using both in silico and experimental approaches, we show that one of these three families, RIRE1, has been transferred horizontally between O. australiensis and seven other reproductively isolated Oryza species. This constitutes a new case of horizontal transfer in plants. © 2008 The Authors.
• Shaohong, Q. u., Desai, A., Wing, R., & Sundaresan, V. (2008). A versatile transposon-based activation tag vector system for functional genomics in cereals and other monocot plants. Plant Physiology, 146(1), 189-199.
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  PMID: 17993541;PMCID: PMC2230568;Abstract: Transposon insertional mutagenesis is an effective alternative to T-DNA mutagenesis when transformation through tissue culture is inefficient as is the case for many crop species. When used as activation tags, transposons can be exploited to generate novel gain-of-function phenotypes without transformation and are of particular value in the study of polyploid plants where gene knockouts will not have phenotypes. We have developed an in cis-activation- tagging Ac-Ds transposon system in which a T-DNA vector carries a Dissociation (Ds) element containing 4X cauliflower mosaic virus enhancers along with the Activator (Ac) transposase gene. Stable Ds insertions were selected using green fluorescent protein and red fluorescent protein genes driven by promoters that are functional in maize (Zea mays) and rice (Oryza sativa). The system has been tested in rice, where 638 stable Ds insertions were selected from an initial set of 26 primary transformants. By analysis of 311 flanking sequences mapped to the rice genome, we could demonstrate the wide distribution of the elements over the rice chromosomes. Enhanced expression of rice genes adjacent to Ds insertions was detected in the insertion lines using semiquantitative reverse transcription-PCR method. The in cis-two-element vector system requires minimal number of primary transformants and eliminates the need for crossing, while the use of fluorescent markers instead of antibiotic or herbicide resistance increases the applicability to other plants and eliminates problems with escapes. Because Ac-Ds has been shown to transpose widely in the plant kingdom, the activation vector system developed in this study should be of utility more generally to other monocots. © 2007 American Society of Plant Biologists.
• Shoemaker, R. C., Grant, D., Olson, T., Warren, W. C., Wing, R., Yeisoo, Y. u., Kim, H., Cregan, P., Joseph, B., Futrell-Griggs, M., Nelson, W., Davito, J., Walker, J., Wallis, J., Kremitski, C., Scheer, D., Clifton, S. W., Graves, T., Nguyen, H., , Xiaolei, W. u., et al. (2008). Microsatellite discovery from BAC end sequences and genetic mapping to anchor the soybean physical and genetic maps. Genome, 51(4), 294-302.
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  PMID: 18356965;Abstract: Whole-genome sequencing of the soybean (Glycine max (L.) Merr. 'Williams 82') has made it important to integrate its physical and genetic maps. To facilitate this integration of maps, we screened 3290 microsatellites (SSRs) identified from BAC end sequences of clones comprising the 'Williams 82' physical map. SSRs were screened against 3 mapping populations. We found the AAT and ACT motifs produced the greatest frequency of length polymorphisms, ranging from 17.2% to 32.3% and from 11.8% to 33.3%, respectively. Other useful motifs include the dinucleotide repeats AG, AT, and AG, with frequency of length polymorphisms ranging from 11.2% to 18.4% (AT), 12.4% to 20.6% (AG), and 11.3% to 16.4% (GT). Repeat lengths less than 16 bp were generally less useful than repeat lengths of 40-60 bp. Two hundred and sixty-five SSRs were genetically mapped in at least one population. Of the 265 mapped SSRs, 60 came from BAC singletons not yet placed into contigs of the physical map. One hundred and ten originated in BACs located in contigs for which no genetic map location was previously known. Ninety-five SSRs came from BACs within contigs for which one or more other BACs had already been mapped. For these fingerprinted contigs (FPC) a high percentage of the mapped markers showed inconsistent map locations. A strategy is introduced by which physical and genetic map inconsistencies can be resolved using the preliminary 4x assembly of the whole genome sequence of soybean. © 2008 NRC.
• Soltis, D. E., Albert, V. A., Leebens-Mack, J., Palmer, J. D., Wing, R. A., dePamphilis, C. W., Hong, M. a., Carlson, J. E., Altman, N., Kim, S., Kerr, P. K., Zuccolo, A., & Soltis, P. S. (2008). The Amborella genome: An evolutionary reference for plant biology. Genome Biology, 9(3).
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  PMID: 18341710;PMCID: PMC2397498;Abstract: The nuclear genome sequence of Amborella trichopoda, the sister species to all other extant angiosperms, will be an exceptional resource for plant genomics. © 2008 BioMed Central Ltd.
• Tanaka, T., Antonio, B. A., Kikuchi, S., Matsumoto, T., Nagamura, Y., Numa, H., Sakai, H., Wu, J., Itoh, T., Sasaki, T., Aono, R., Fujii, Y., Habara, T., Harada, E., Kanno, M., Kawahara, Y., Kawashima, H., Kubooka, H., Matsuya, A., , Nakaoka, H., et al. (2008). The rice annotation project database (RAP-DB): 2008 update. NUCLEIC ACIDS RESEARCH, 36, D1028-D1033.
• Wei, F., & Wing, R. A. (2008). A fruitful outcome to the papaya genome project. Genome Biology, 9(6).
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  PMID: 18557995;PMCID: PMC2481413;Abstract: The draft genome sequence of a transgenic virus-resistant papaya marks the first genome sequence of a commercially important transgenic crop plant. © 2008 BioMed Central Ltd.
• Wing, R., Ammiraju, J. S., Lu, F., Sanyal, A., Yu, Y., Song, X., Jiang, N., Pontaroli, A. C., Rambo, T., Currie, J., Collura, K., Talag, J., Fan, C., Goicoechea, J. L., Zuccolo, A., Chen, J., Bennetzen, J. L., Chen, M., Jackson, S., & Wing, R. A. (2008). Dynamic evolution of oryza genomes is revealed by comparative genomic analysis of a genus-wide vertical data set. The Plant cell, 20(12).
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  Oryza (23 species; 10 genome types) contains the world's most important food crop - rice. Although the rice genome serves as an essential tool for biological research, little is known about the evolution of the other Oryza genome types. They contain a historical record of genomic changes that led to diversification of this genus around the world as well as an untapped reservoir of agriculturally important traits. To investigate the evolution of the collective Oryza genome, we sequenced and compared nine orthologous genomic regions encompassing the Adh1-Adh2 genes (from six diploid genome types) with the rice reference sequence. Our analysis revealed the architectural complexities and dynamic evolution of this region that have occurred over the past approximately 15 million years. Of the 46 intact genes and four pseudogenes in the japonica genome, 38 (76%) fell into eight multigene families. Analysis of the evolutionary history of each family revealed independent and lineage-specific gain and loss of gene family members as frequent causes of synteny disruption. Transposable elements were shown to mediate massive replacement of intergenic space (>95%), gene disruption, and gene/gene fragment movement. Three cases of long-range structural variation (inversions/deletions) spanning several hundred kilobases were identified that contributed significantly to genome diversification.
• Wing, R., Kim, H., Hurwitz, B., Yu, Y., Collura, K., Gill, N., SanMiguel, P., Mullikin, J. C., Maher, C., Nelson, W., Wissotski, M., Braidotti, M., Kudrna, D., Goicoechea, J. L., Stein, L., Ware, D., Jackson, S. A., Soderlund, C., & Wing, R. A. (2008). Construction, alignment and analysis of twelve framework physical maps that represent the ten genome types of the genus Oryza. Genome biology, 9(2).
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  We describe the establishment and analysis of a genus-wide comparative framework composed of 12 bacterial artificial chromosome fingerprint and end-sequenced physical maps representing the 10 genome types of Oryza aligned to the O. sativa ssp. japonica reference genome sequence. Over 932 Mb of end sequence was analyzed for repeats, simple sequence repeats, miRNA and single nucleotide variations, providing the most extensive analysis of Oryza sequence to date.
• Zuccolo, A., S., J., Kim, H., Sanyal, A., Jackson, S., & Wing, R. A. (2008). Rapid and differential proliferation of the Ty3-Gypsy LTR retrotransposon Atlantys in the genus Oryza. Rice, 1(1), 85-99.
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  Abstract: Here, we present the results of a comprehensive study of the distribution, evolution, heterogeneity, and phylogenetic relationships of the Ty3-Gypsy Atlantys long terminal repeat retrotransposable element family in Oryza. Atlantys element-related sequences make up a significant fraction of the genomes of species from the Officinalis complex as well as the Oryza ridleyi and O. granulata genomes. The proliferation of Atlantys elements, in many cases, took place after respective speciation events occurred. Most of the retrotranspositional events occurred within the last three million years. Atlantys is an ancient and ubiquitous component of the genus Oryza and has made significant contributions to genome size variation across the genus. Its structure is unusual when compared to other Ty3-Gypsy elements and its proliferation in the different Oryza species has been rapid and differential. © Springer Science + Business Media, LLC 2008.
• Chen, K., Cong, B., Wing, R., Vrebalov, J., & Tanksley, S. D. (2007). Changes in regulation of a transcription factor lead to autogamy in cultivated tomatoes. SCIENCE, 318(5850), 643-645.
• Chen, K., Cong, B., Wing, R., Vrebalov, J., & Tanksley, S. D. (2007). Changes in regulation of a transcription factor lead to autogamy in cultivated tomatoes. Science, 318(5850), 643-645.
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  PMID: 17962563;Abstract: We report the cloning of Style2.1, the major quantitative trait locus responsible for a key floral attribute (style length) associated with the evolution of self-pollination in cultivated tomatoes. The gene encodes a putative transcription factor that regulates cell elongation in developing styles. The transition from cross-pollination to self-pollination was accompanied, not by a change in the STYLE2.1 protein, but rather by a mutation in the Style2.1 promoter that results in a down-regulation of Style2.1 expression during flower development.
• Clark, A. G., Eisen, M. B., Smith, D. R., Bergman, C. M., Oliver, B., Markow, T. A., Kaufman, T. C., Kellis, M., Gelbart, W., Iyer, V. N., Pollard, D. A., Sackton, T. B., Larracuente, A. M., Singh, N. D., Abad, J. P., Abt, D. N., Adryan, B., Aguade, M., Akashi, H., , Anderson, W. W., et al. (2007). Evolution of genes and genomes on the Drosophila phylogeny. NATURE, 450(7167), 203-218.
• Gowda, M., Venu, R. -., Huameng, L. i., Jantasuriyarat, C., Chen, S., Bellizzi, M., Pampanwar, V., Kim, H., Dean, R. A., Stahlberg, E., Wing, R., Soderlund, C., & Wang, G. (2007). Magnaporthe grisea infection triggers RNA variation and antisense transcript expression in rice. Plant Physiology, 144(1), 524-533.
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  PMID: 17351054;PMCID: PMC1913787;Abstract: Rice blast disease, caused by the fungal pathogen Magnaporthe grisea, is an excellent model system to study plant-fungal interactions and host defense responses. In this study, comprehensive analysis of the rice (Oryza sativa) transcriptome after M. grisea infection was conducted using robust-long serial analysis of gene expression. A total of 83,382 distinct 21-bp robust-long serial analysis of gene expression tags were identified from 627,262 individual tags isolated from the resistant (R), susceptible (S), and control (C) libraries. Sequence analysis revealed that the tags in the R and S libraries had a significant reduced matching rate to the rice genomic and expressed sequences in comparison to the C library. The high level of one-nucleotide mismatches of the R and S library tags was due to nucleotide conversions. The A-to-G and U-to-C nucleotide conversions were the most predominant types, which were induced in the M. grisea-infected plants. Reverse transcription-polymerase chain reaction analysis showed that expression of the adenine deaminase and cytidine deaminase genes was highly induced after inoculation. In addition, many antisense transcripts were induced in infected plants and expression of four antisense transcripts was confirmed by strand-specific reverse transcription-polymerase chain reaction. These results demonstrate that there is a series of dynamic and complex transcript modifications and changes in the rice transcriptome at the M. grisea early infection stages. © 2007 American Society of Plant Biologists.
• Grover, C. E., Kim, H., Wing, R. A., Paterson, A. H., & Wendel, J. F. (2007). Microcolinearity and genome evolution in the AdhA region of diploid and polyploid cotton (Gossypium). Plant Journal, 50(6), 995-1006.
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  PMID: 17461788;Abstract: Genome sizes vary by several orders of magnitude, driven by mechanisms such as illegitimate recombination and transposable element proliferation. Prior analysis of the CesA region in two cotton genomes that diverged 5-10 million years ago (Ma), and acquired a twofold difference in genome size, revealed extensive local conservation of genic and intergenic regions, with no evidence of the global genome size difference. The present study extends the comparison to include BAC sequences surrounding the gene encoding alcohol dehydrogenase A (AdhA) from four cotton genomes: the two co-resident genomes (AT and DT) of the allotetraploid, Gossypium hirsutum, as well as the model diploid progenitors, Gossypium arboreum (A) and Gossypium raimondii (D). In contrast to earlier work, evolution in the AdhA region reflects, in a microcosm, the overall difference in genome size, with a nearly twofold difference in aligned sequence length. Most size differences may be attributed to differential accumulation of retroelements during divergence of the genome diploids from their common ancestor, but in addition there has been a biased accumulation of small deletions, such that those in the smaller D genome are on average twice as large as those in the larger A genome. The data also provide evidence for the global phenomenon of 'genomic downsizing' in polyploids shortly after formation. This in part reflects a higher frequency of small deletions post-polyploidization, and increased illegitimate recombination. In conjunction with previous work, the data here confirm the conclusion that genome size evolution reflects many forces that collectively operate heterogeneously among genomic regions. © 2007 The Authors.
• Itoh, T., Tanaka, T., Barrero, R. A., Yamasaki, C., Fujii, Y., Hilton, P. B., Antonio, B. A., Aono, H., Apweiler, R., Bruskiewich, R., Bureau, T., Burr, F., Costa, A., Fuks, G., Habara, T., Haberer, G., Han, B., Harada, E., Hiraki, A. T., , Hirochika, H., et al. (2007). Curated genome annotation of Oryza sativa ssp. japonica and comparative genome analysis with Arabidopsis thaliana: The Rice Annotation Project. Genome Research, 17(2), 175-183.
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  PMID: 17210932;PMCID: PMC1781349;Abstract: We present here the annotation of the complete genome of rice Oryza sativa L. ssp. japonica cultivar Nipponbare. All functional annotations for proteins and non-protein-coding RNA (npRNA) candidates were manually curated. Functions were identified or inferred in 19,969 (70%) of the proteins, and 131 possible npRNAs (including 58 antisense transcripts) were found. Almost 5000 annotated protein-coding genes were found to be disrupted in insertional mutant lines, which will accelerate future experimental validation of the annotations. The rice loci were determined by using cDNA sequences obtained from rice and other representative cereals. Our conservative estimate based on these loci and an extrapolation suggested that the gene number of rice is ∼32,000, which is smaller than previous estimates. We conducted comparative analyses between rice and Arabidopsis thaliana and found that both genomes possessed several lineage-specific genes, which might account for the observed differences between these species, while they had similar sets of predicted functional domains among the protein sequences. A system to control translational efficiency seems to be conserved across large evolutionary distances. Moreover, the evolutionary process of protein-coding genes was examined. Our results suggest that natural selection may have played a role for duplicated genes in both species, so that duplication was suppressed or favored in a manner that depended on the function of a gene. ©2007 by Cold Spring Harbor Laboratory Press.
• Jianxin, M. a., Wing, R. A., Bennetzen, J. L., & Jackson, S. A. (2007). Evolutionary history and positional shift of a rice centromere. Genetics, 177(2), 1217-1220.
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  PMID: 17660534;PMCID: PMC2034625;Abstract: Rice centromere 8 was previously proposed to be an "immature" centromere that recently arose from a genic region. Our comparative genomics analysis indicates that Cen8 was formed at its current location at least 7-9 million years ago and was physically shifted by a more recent inversion of a segment spanning centromeric and pericentromeric regions. Copyright © 2007 by the Genetics Society of America.
• Jianxin, M. a., Wing, R. A., Bennetzen, J. L., & Jackson, S. A. (2007). Plant centromere organization: a dynamic structure with conserved functions. Trends in Genetics, 23(3), 134-139.
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  PMID: 17275131;Abstract: Although the structural features of centromeres from most multicellular eukaryotes remain to be characterized, recent analyses of the complete sequences of two centromeric regions of rice, together with data from Arabidopsis thaliana and maize, have illuminated the considerable size variation and sequence divergence of plant centromeres. Despite the severe suppression of meiotic chromosomal exchange in centromeric and pericentromeric regions of rice, the centromere core shows high rates of unequal homologous recombination in the absence of chromosomal exchange, resulting in frequent and extensive DNA rearrangement. Not only is the sequence of centromeric tandem and non-tandem repeats highly variable but also the copy number, spacing, order and orientation, providing ample natural variation as the basis for selection of superior centromere performance. This review article focuses on the structural and evolutionary dynamics of plant centromere organization and the potential molecular mechanisms responsible for the rapid changes of centromeric components. © 2007 Elsevier Ltd. All rights reserved.
• Jung, J., Won, S. Y., Suh, S. C., Kim, H., Wing, R., Jeong, Y., Hwang, I., & Kim, M. (2007). The barley ERF-type transcription factor HvRAF confers enhanced pathogen resistance and salt tolerance in Arabidopsis. PLANTA, 225(3), 575-588.
• Liang, H., Fang, E. G., Tomkins, J. P., Luo, M., Kudrna, D., Kim, H. R., Arumuganathan, K., Zhao, S., Leebens-Mack, J., Schlarbaum, S. E., Banks, J. A., Depamphilis, C. W., Mandoli, D. F., Wing, R. A., & Carlson, J. E. (2007). Development of a BAC library for yellow-poplar (Liriodendron tulipifera) and the identification of genes associated with flower development and lignin biosynthesis. Tree Genetics and Genomes, 3(3), 215-225.
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  Abstract: Liriodendron tulipifera L., a member of the Magnoliaceae, occupies an important phylogenetic position as a basal angiosperm that has retained numerous putatively ancestral morphological characters, and thus has often been used in studies of the evolution of flowering plants and of specific gene families. However, genomic resources for these early branching angiosperm lineages are very limited. In this study, we describe the construction of a large-insert bacterial artificial chromosome (BAC) library from L. tulipifera. Flow cytometry estimates that this nuclear genome is approximately 1,802 Mbp per haploid genome (±16 SD). The BAC library contains 73,728 clones, a 4.8-fold genome coverage, with an average insert size of 117 kb, a chloroplast DNA content of 0.2%, and little to no bacterial sequences nor empty vector content clones. As a test of the utility of this BAC library, we screened the library with six single/low-copy genic probes. We obtained at least two positive clones for each gene and confirmed the clones by DNA sequencing. A total of 182 paired end sequences were obtained from 96 of the BAC clones. Using BLAST searches, we found that 25% of the BAC end sequences were similar to DNA sequences in GenBank. Of these, 68% shared sequence with transposable elements and 25% with genes from other taxa. This result closely reflected the content of random sequences obtained from a small insert genomic library for L. tulipifera, indicating that the BAC library construction process was not biased. The first genomic DNA sequences for Liriodendron genes are also reported. All the Liriodendron genomic sequences described in this paper have been deposited in the GenBank data library. The end sequences from shotgun genomic clones and BAC clones are under accession DU169330-DU169684. Partial sequences of Gigantea, Frigida, LEAFY, cinnamyl alcohol dehydrogenase, 4-coumarate:CoA ligase, and phenylalanine ammonia-lyase genes are under accession DQ223429-DQ223434. © Springer-Verlag 2007.
• Ma, J., Wing, R. A., Bennetzen, J. L., & Jackson, S. A. (2007). Plant centromere organization: a dynamic structure with conserved functions. TRENDS IN GENETICS, 23(3), 134-139.
• Nelson, W. M., Dvorak, J., Luo, M., Messing, J., Wing, R. A., & Soderlund, C. (2007). Efficacy of clone fingerprinting methodologies. Genomics, 89(1), 160-165.
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  PMID: 17011744;Abstract: With the development of new high-information content fingerprinting techniques for constructing BAC-based physical maps, physical map construction is accelerating and it is important to determine which methodologies work best. In a recent publication (Z. Xu et al., 2004, Genomics 84:941-951), Xu et al. evaluated five different techniques (one agarose-based and four using multiple enzymes) and concluded that a two-enzyme technique was superior. In addition, they found that no benefit was gained from fingerprinting more than 10× coverage. In this paper we report our own extensive simulation results, which lead to contrasting conclusions. Our data indicate that the five-enzyme method known as SNaPshot is the most effective and that the assembly can in fact be significantly improved with greater than 10× coverage. © 2006 Elsevier Inc. All rights reserved.
• S., J., Zuccolo, A., Yeisoo, Y. u., Song, X., Piegu, B., Chevalier, F., Walling, J. G., Jianxin, M. a., Talag, J., Brar, D. S., SanMiguel, P. J., Jiang, N., Jackson, S. A., Panaud, O., & Wing, R. A. (2007). Evolutionary dynamics of an ancient retrotransposon family provides insights into evolution of genome size in the genus Oryza. Plant Journal, 52(2), 342-351.
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  PMID: 17764506;Abstract: Long terminal repeat (LTR) retrotransposons constitute a significant portion of most eukaryote genomes and can dramatically change genome size and organization. Although LTR retrotransposon content variation is well documented, the dynamics of genomic flux caused by their activity are poorly understood on an evolutionary time scale. This is primarily because of the lack of an experimental system composed of closely related species whose divergence times are within the limits of the ability to detect ancestrally related retrotransposons. The genus Oryza, with 24 species, ten genome types, different ploidy levels and over threefold genome size variation, constitutes an ideal experimental system to explore genus-level transposon dynamics. Here we present data on the discovery and characterization of an LTR retrotransposon family named RWG in the genus Oryza. Comparative analysis of transposon content (approximately 20 to 27 000 copies) and transpositional history of this family across the genus revealed a broad spectrum of independent and lineage-specific changes that have implications for the evolution of genome size and organization. In particular, we provide evidence that the basal GG genome of Oryza (O. granulata) has expanded by nearly 25% by a burst of the RWG lineage Gran3 subsequent to speciation. Finally we describe the recent evolutionary origin of Dasheng, a large retrotransposon derivative of the RWG family, specifically found in the A, B and C genome lineages of Oryza. © 2007 The Authors.
• Venu, R. C., Jia, Y., Gowda, M., Jia, M. H., Jantasuriyarat, C., Stahlberg, E., Huameng, L. i., Rhineheart, A., Boddhireddy, P., Singh, P., Rutger, N., Kudrna, D., Wing, R., Nelson, J. C., & Wang, G. (2007). RL-SAGE and microarray analysis of the rice transcriptome after Rhizoctonia solani infection. Molecular Genetics and Genomics, 278(4), 421-431.
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  PMID: 17579886;Abstract: Sheath blight caused by the fungal pathogen Rhizoctonia solani is an emerging problem in rice production worldwide. To elucidate the molecular basis of rice defense to the pathogen, RNA isolated from R. solani-infected leaves of Jasmine 85 was used for both RL-SAGE library construction and microarray hybridization. RL-SAGE sequence analysis identified 20,233 and 24,049 distinct tags from the control and inoculated libraries, respectively. Nearly half of the significant tags (≥2 copies) from both libraries matched TIGR annotated genes and KOME full-length cDNAs. Among them, 42% represented sense and 7% antisense transcripts, respectively. Interestingly, 60% of the library-specific (≥10 copies) and differentially expressed (>4.0-fold change) tags were novel transcripts matching genomic sequence but not annotated genes. About 70% of the genes identified in the SAGE libraries showed similar expression patterns (up or down-regulated) in the microarray data obtained from three biological replications. Some candidate RL-SAGE tags and microarray genes were located in known sheath blight QTL regions. The expression of ten differentially expressed RL-SAGE tags was confirmed with RT-PCR. The defense genes associated with resistance to R. solani identified in this study are useful genomic materials for further elucidation of the molecular basis of the defense response to R. solani and fine mapping of target sheath blight QTLs. © 2007 Springer-Verlag.
• Wei, F., Coe, E. d., Nelson, W., Bharti, A. K., Engler, F., Butler, E. d., Kim, H., Goicoechea, J. L., Chen, M., Lee, S., Fuks, G., Sanchez-Villeda, H., Schroeder, S., Fang, Z., McMullen, M., Davis, G., Bowers, J. E., Paterson, A. H., Schaeffer, M., , Gardiner, J., et al. (2007). Physical and genetic structure of the maize genome reflects its complex evolutionary history. PLOS GENETICS, 3(7), 1254-1263.
• Wei, F., Coe, E., Nelson, W., Bharti, A. K., Engler, F., Butler, E., Kim, H., Goicoechea, J. L., Chen, M., Lee, S., Fuks, G., Sanchez-Villeda, H., Schroeder, S., Fang, Z., McMullen, M., Davis, G., Bowers, J. E., Paterson, A. H., Schaeffer, M., , Gardiner, J., et al. (2007). Physical and genetic structure of the maize genome reflects its complex evolutionary history. PLoS Genetics, 3(7), 1254-1263.
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  Abstract: Maize (Zea mays L.) is one of the most important cereal crops and a model for the study of genetics, evolution, and domestication. To better understand maize genome organization and to build a framework for genome sequencing, we constructed a sequence-ready fingerprinted contig-based physical map that covers 93.5% of the genome, of which 86.1% is aligned to the genetic map. The fingerprinted contig map contains 25,908 genic markers that enabled us to align nearly 73% of the anchored maize genome to the rice genome. The distribution pattern of expressed sequence tags correlates to that of recombination. In collinear regions, 1 kb in rice corresponds to an average of 3.2 kb in maize, yet maize has a 6-fold genome size expansion. This can be explained by the fact that most rice regions correspond to two regions in maize as a result of its recent polyploid origin. Inversions account for the majority of chromosome structural variations during subsequent maize diploidization. We also find clear evidence of ancient genome duplication predating the divergence of the progenitors of maize and rice. Reconstructing the paleoethnobotany of the maize genome indicates that the progenitors of modern maize contained ten chromosomes.
• Wing, R., Kim, H., San Miguel, P., Nelson, W., Collura, K., Wissotski, M., Walling, J. G., Kim, J. P., Jackson, S. A., Soderlund, C., & Wing, R. A. (2007). Comparative physical mapping between Oryza sativa (AA genome type) and O. punctata (BB genome type). Genetics, 176(1).
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  A comparative physical map of the AA genome (Oryza sativa) and the BB genome (O. punctata) was constructed by aligning a physical map of O. punctata, deduced from 63,942 BAC end sequences (BESs) and 34,224 fingerprints, onto the O. sativa genome sequence. The level of conservation of each chromosome between the two species was determined by calculating a ratio of BES alignments. The alignment result suggests more divergence of intergenic and repeat regions in comparison to gene-rich regions. Further, this characteristic enabled localization of heterochromatic and euchromatic regions for each chromosome of both species. The alignment identified 16 locations containing expansions, contractions, inversions, and transpositions. By aligning 40% of the punctata BES on the map, 87% of the punctata FPC map covered 98% of the O. sativa genome sequence. The genome size of O. punctata was estimated to be 8% larger than that of O. sativa with individual chromosome differences of 1.5-16.5%. The sum of expansions and contractions observed in regions >500 kb were similar, suggesting that most of the contractions/expansions contributing to the genome size difference between the two species are small, thus preserving the macro-collinearity between these species, which diverged approximately 2 million years ago.
• Wing, R., Zuccolo, A., Sebastian, A., Talag, J., Yu, Y., Kim, H., Collura, K., Kudrna, D., & Wing, R. A. (2007). Transposable element distribution, abundance and role in genome size variation in the genus Oryza. BMC evolutionary biology, 7.
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  The genus Oryza is composed of 10 distinct genome types, 6 diploid and 4 polyploid, and includes the world's most important food crop - rice (Oryza sativa [AA]). Genome size variation in the Oryza is more than 3-fold and ranges from 357 Mbp in Oryza glaberrima [AA] to 1283 Mbp in the polyploid Oryza ridleyi [HHJJ]. Because repetitive elements are known to play a significant role in genome size variation, we constructed random sheared small insert genomic libraries from 12 representative Oryza species and conducted a comprehensive study of the repetitive element composition, distribution and phylogeny in this genus. Particular attention was paid to the role played by the most important classes of transposable elements (Long Terminal Repeats Retrotransposons, Long interspersed Nuclear Elements, helitrons, DNA transposable elements) in shaping these genomes and in their contributing to genome size variation.
• Adema, C. M., Luo, M., Hanelt, B., Hertel, L. A., Marshall, J. J., Zhang, S., DeJong, R. J., Kim, H., Kudrna, D., Wing, R. A., Soderlund, C., Knight, M., Lewis, F. A., Caldeira, R. L., Jannotti-Passos, L. K., Dos, O., & Loker, E. S. (2006). A bacterial artificial chromosome library for Biomphalaria glabrata, intermediate snail host of Schistosoma mansoni. Memorias do Instituto Oswaldo Cruz, 101(SUPPL. 1), 167-177.
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  PMID: 17308766;Abstract: To provide a novel resource for analysis of the genome of Biomphalaria glabrata, members of the international Biomphalaria glabrata Genome Initiative (biology.unm.edu/biomphalaria-genome.html), working with the Arizona Genomics Institute (AGI) and supported by the National Human Genome Research Institute (NHGRI), produced a high quality bacterial artificial chromosome (BAC) library. The BB02 strain B. glabrata, a field isolate (Belo Horizonte, Minas Gerais, Brasil) that is susceptible to several strains of Schistosoma mansoni, was selfed for two generations to reduce haplotype diversity in the offspring. High molecular weight DNA was isolated from ovotestes of 40 snails, partially digested with HindIII, and ligated into pAGIBAC1 vector. The resulting B. glabrata BAC library (BG_BBa) consists of 61824 clones (136.3 kb average insert size) and provides 9.05 x coverage of the 931 Mb genome. Probing with single/low copy number genes from B. glabrata and fingerprinting of selected BAC clones indicated that the BAC library sufficiently represents the gene complement. BAC end sequence data (514 reads, 299860 nt) indicated that the genome of B. glabrata contains ∼ 63% AT, and disclosed several novel genes, transposable elements, and groups of high frequency sequence elements. This BG_BBa BAC library, available from AGI at cost to the research community, gains in relevance because BB02 strain B. glabrata is targeted whole genome sequencing by NHGRI.
• Bi, I. V., McMullen, M. D., Sanchez-Villeda, H., Schroeder, S., Gardiner, J., Polacco, M., Soderlund, C., Wing, R., Fang, Z., & Coe Jr., E. H. (2006). Single nucleotide polymorphisms and insertion-deletions for genetic markers and anchoring the maize fingerprint contig physical map. Crop Science, 46(1), 12-21.
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  Abstract: Single nucleotide polymorphisms (SNPs) and insertion-deletions (InDels) are becoming important genetic markers for major crop species. In this study we demonstrate their utility for locating fingerprint contigs (FPCs) to the genetic map. To derive SNP and InDel markers, we amplified genomic regions corresponding to 3000 unigenes across 12 maize (Zea mays L.) lines, of which 194 unigenes (6.4%) showed size polymorphism InDels between B73 and Mo17 on agarose gels. The analysis of these InDels in 83 diverse inbred lines showed that InDels are often multiallelic markers in maize. Single nucleotide polymorphism discovery conducted on 592 unigenes revealed that 44% of the unigenes contained B73/Mo17 SNPs, while 8% showed no sequence variation among the 12 inbred lines. On average, SNPs and InDels occurred every 73 and 309 bp, respectively. Multiple SNPs within unigenes led to a SNP haplotype genetic diversity of 0.61 among inbreds. The unigenes were previously assigned to maize FPCs by overgo hybridization. From this set of unigenes, 311 (133 SNP and 178 InDel) loci were mapped on the intermated B73 X Mo17 (IBM) high-resolution mapping population. These markers provided unambiguous anchoring of 129 FPCs and orientation for 30 contigs. The FPC anchored map of maize will be useful for map-based cloning, for genome sequencing efforts in maize, and for comparative genomics in grasses. The amplification primers for all mapped InDel and SNP loci, the diversity information for SNPs and InDels, and the corresponding overgoes to anchor bacterial artificial chromosome (BAC) contigs are provided as genetic resources. © Crop Science Society of America.
• Bruggmann, R., Bharti, A. K., Gundlach, H., Lai, J., Young, S., Pontaroli, A. C., Wei, F., Haberer, G., Fuks, G., Chunguang, D. u., Raymond, C., Estep, M. C., Liu, R., Bennetzen, J. L., Chan, A. P., Rabinowicz, P. D., Quackenbush, J., Barbazuk, W. B., Wing, R. A., , Birren, B., et al. (2006). Uneven chromosome contraction and expansion in the maize genome. Genome Research, 16(10), 1241-1251.
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  PMID: 16902087;PMCID: PMC1581433;Abstract: Maize (Zea mays or corn), both a major food source and an important cytogenetic model, evolved from a tetraploid that arose about 4.8 million years ago (Mya). As a result, maize has extensive duplicated regions within its genome. We have sequenced the two copies of one such region, generating 7.8 Mb of sequence spanning 17.4 cM of the short arm of chromosome 1 and 6.6 Mb (25.6 cM) from the long arm of chromosome 9. Rice, which did not undergo a similar whole genome duplication event, has only one orthologous region (4.9 Mb) on the short arm of chromosome 3, and can be used as reference for the maize homoeologous regions. Alignment of the three regions allowed identification of syntenic blocks, and indicated that the maize regions have undergone differential contraction in genic and intergenic regions and expansion by the insertion of retrotransposable elements. Approximately 9% of the predicted genes in each duplicated region are completely missing in the rice genome, and almost 20% have moved to other genomic locations. Predicted genes within these regions tend to be larger in maize than in rice, primarily because of the presence of predicted genes in maize with larger introns. Interestingly, the general gene methylation patterns in the maize homoeologous regions do not appear to have changed with contraction or expansion of their chromosomes. In addition, no differences in methylation of single genes and tandemly repeated gene copies have been detected. These results, therefore, provide new insights into the diploidization of polyploid species. ©2006 by Cold Spring Harbor Laboratory Press.
• Bruggmann, R., Bharti, A. K., Gundlach, H., Lai, J., Young, S., Pontaroli, A. C., Wei, F., Haberer, G., Fuks, G., Du, C., Raymond, C., Estep, M. C., Liu, R., Bennetzen, J. L., Chan, A. P., Rabinowicz, P. D., Quackenbush, J., Barbazuk, W. B., Wing, R. A., , Birren, B., et al. (2006). Uneven chromosome contraction and expansion in the maize genome. GENOME RESEARCH, 16(10), 1241-1251.
• Cunningham, C., Hikima, J., Jenny, M. J., Chapman, R. W., Fang, G., Saski, C., Lundqvist, M. L., Wing, R. A., Cupit, P. M., Gross, P. S., Warr, G. W., & Tomkins, J. P. (2006). New resources for marine genomics: Bacterial artificial chromosome libraries for the eastern and pacific oysters (Crassostrea virginica and C. gigas). Marine Biotechnology, 8(5), 521-533.
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  PMID: 16896533;Abstract: Large-insert genomic bacterial artificial chromosome (BAC) libraries of two culturally and economically important oyster species, Crassostrea virginica and C. gigas, have been developed as part of an international effort to develop tools and reagents that will advance our ability to conduct genetic and genomic research. A total of 73,728 C. gigas clones with an average insert size of 152 kb were picked and arrayed representing an 11.8-fold genome coverage. A total of 55,296 clones with an average insert size of 150 kb were picked and arrayed for C. virginica, also representing an 11.8-fold genome coverage. The C. gigas and C. virginica libraries were screened with probes derived from selected oyster genes using high-density BAC colony filter arrays. The probes identified 4 to 25 clones per gene for C. virginica and 5 to 50 clones per gene for C. gigas. We conducted a preliminary analysis of genetic polymorphism represented in the C. gigas library. The results suggest that the degree of divergence among similar sequences is highly variable and concentrated in intronic regions. Evidence supporting allelic polymorphism is reported for two genes and allelic and/or locus specific polymorphism for several others. Classical inheritance studies are needed to confirm the nature of these polymorphisms. The oyster BAC libraries are publicly available to the research community on a cost-recovery basis at www.genome.clemson.edu. © Springer Science+Business Media, Inc. 2006.
• Gowda, M., Venu, R., Raghupathy, M. B., Nobuta, K., Huameng, L. i., Wing, R., Stahlberg, E., Couglan, S., Haudenschild, C. D., Dean, R., Nahm, B., Meyers, B. C., & Wang, G. (2006). Deep and comparative analysis of the mycelium and appressorium transcriptomes of Magnaporthe grisea using MPSS, RL-SAGE, and oligoarray methods. BMC Genomics, 7.
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  PMID: 17156450;PMCID: PMC1764740;Abstract: Background: Rice blast, caused by the fungal pathogen Magnaporthe grisea, is a devastating disease causing tremendous yield loss in rice production. The public availability of the complete genome sequence of M. grisea provides ample opportunities to understand the molecular mechanism of its pathogenesis on rice plants at the transcriptome level. To identify all the expressed genes encoded in the fungal genome, we have analyzed the mycelium and appressorium transcriptomes using massively parallel signature sequencing (MPSS), robust-long serial analysis of gene expression (RL-SAGE) and oligoarray methods. Results: The MPSS analyses identified 12,531 and 12,927 distinct significant tags from mycelia and appressoria, respectively, while the RL-SAGE analysis identified 16,580 distinct significant tags from the mycelial library. When matching these 12,531 mycelial and 12,927 appressorial significant tags to the annotated CDS, 500 bp upstream and 500 bp downstream of CDS, 6,735 unique genes in mycelia and 7,686 unique genes in appressoria were identified. A total of 7,135 mycelium-specific and 7,531 appressorium-specific significant MPSS tags were identified, which correspond to 2,088 and 1,784 annotated genes, respectively, when matching to the same set of reference sequences. Nearly 85% of the significant MPSS tags from mycelia and appressoria and 65% of the significant tags from the RL-SAGE mycelium library matched to the M. grisea genome. MPSS and RL-SAGE methods supported the expression of more than 9,000 genes, representing over 80% of the predicted genes in M. grisea. About 40% of the MPSS tags and 55% of the RL-SAGE tags represent novel transcripts since they had no matches in the existing M. grisea EST collections. Over 19% of the annotated genes were found to produce both sense and antisense tags in the protein-coding region. The oligoarray analysis identified the expression of 3,793 mycelium-specific and 4,652 appressorium-specific genes. A total of 2,430 mycelial genes and 1,886 appressorial genes were identified by both MPSS and oligoarray. Conclusion: The comprehensive and deep transcriptome analys is by MPSS and RL-SAGE methods identified many novel sense and antisense transcripts in the M. grisea genome at two important growth stages. The differentially expressed transcripts that were identified, especially those specifically expressed in appressoria, represent a genomic resource useful for gaining a better understanding of the molecular basis of M. grisea pathogenicity. Further analysis of the novel antisense transcripts will provide new insights into the regulation and function of these genes in fungal growth, development and pathogenesis in the host plants. © 2006 Gowda et al; licensee BioMed Central Ltd.
• Hass-Jacobus, B. L., Futrell-Griggs, M., Abernathy, B., Westerman, R., Goicoechea, J., Stein, J., Klein, P., Hurwitz, B., Zhou, B., Rakhshan, F., Sanyal, A., Gill, N., Lin, J., Walling, J. G., Luo, M. Z., Siva, J., Kudrna, D., Kim, H. R., Ware, D., , Wing, R. A., et al. (2006). Integration of hybridization-based markers (overgos) into physical maps for comparative and evolutionary explorations in the genus Oryza and in Sorghum. BMC Genomics, 7.
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  PMID: 16895597;PMCID: PMC1590032;Abstract: Background: With the completion of the genome sequence for rice (Oryza sativa L.), the focus of rice genomics research has shifted to the comparison of the rice genome with genomes of other species for gene cloning, breeding, and evolutionary studies. The genus Oryza includes 23 species that shared a common ancestor 8-10 million years ago making this an ideal model for investigations into the processes underlying domestication, as many of the Oryza species are still undergoing domestication. This study integrates high-throughput, hybridization-based markers with BAC end sequence and fingerprint data to construct physical maps of rice chromosome I orthologues in two wild Oryza species. Similar studies were undertaken in Sorghum bicolor, a species which diverged from cultivated rice 40-50 million years ago. Results: Overgo markers, in conjunction with fingerprint and BAC end sequence data, were used to build sequence-ready BAC contigs for two wild Oryza species. The markers drove contig merges to construct physical maps syntenic to rice chromosome I in the wild species and provided evidence for at least one rearrangement on chromosome I of the O. sativa versus Oryza officinalis comparative map. When rice overgos were aligned to available S. bicolor sequence, 29% of the overgos aligned with three or fewer mismatches; of these, 41% gave positive hybridization signals. Overgo hybridization patterns supported colinearity of loci in regions of sorghum chromosome 3 and rice chromosome I and suggested that a possible genomic inversion occurred in this syntenic region in one of the two genomes after the divergence of S. bicolor and O. sativa. Conclusion: The results of this study emphasize the importance of identifying conserved sequences in the reference sequence when designing overgo probes in order for those probes to hybridize successfully in distantly related species. As interspecific markers, overgos can be used successfully to construct physical maps in species which diverged less than 8 million years ago, and can be used in a more limited fashion to examine colinearity among species which diverged as much as 40 million years ago. Additionally, overgos are able to provide evidence of genomic rearrangements in comparative physical mapping studies. © 2006 Hass-Jacobus et al; licensee BioMed Central Ltd.
• Hawkins, J. S., Kim, H., Nason, J. D., Wing, R. A., & Wendel, J. F. (2006). Differential lineage-specific amplification of transposable elements is responsible for genome size variation in Gossypium. GENOME RESEARCH, 16(10), 1252-1261.
• Hawkins, J. S., Kim, H., Nason, J. D., Wing, R. A., & Wendel, J. F. (2006). Differential lineage-specific amplification of transposable elements is responsible for genome size variation in Gossypium. Genome Research, 16(10), 1252-1261.
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  PMID: 16954538;PMCID: PMC1581434;Abstract: The DNA content of eukaryotic nuclei (C-value) varies ~200,000-fold, but there is only a ∼20-fold variation in the number of protein-coding genes. Hence, most C-value variation is ascribed to the repetitive fraction, although little is known about the evolutionary dynamics of the specific components that lead to genome size variation. To understand the modes and mechanisms that underlie variation in genome composition, we generated sequence data from whole genome shotgun (WGS) libraries for three representative diploid (n = 13) members of Gossypium that vary in genome size from 880 to 2460 Mb (1C) and from a phylogenetic outgroup, Gossypioides kirkii, with an estimated genome size of 588 Mb. Copy number estimates including all dispersed repetitive sequences indicate that 40%-65% of each genome is composed of transposable elements. Inspection of individual sequence types revealed differential, lineage-specific expansion of various families of transposable elements among the different plant lineages. Copia-like retrotransposable element sequences have differentially accumulated in the Gossypium species with the smallest genome, G. raimondii, while gypsy-like sequences have proliferated in the lineages with larger genomes. Phylogenetic analyses demonstrated a pattern of lineage-specific amplification of particular subfamilies of retrotransposons within each species studied. One particular group of gypsy-like retrotransposon sequences, Gorge3 (Gossypium retrotransposable gypsy-like element), appears to have undergone a massive proliferation in two plant lineages, accounting for a major fraction of genome-size change. Like maize, Gossypium has undergone a threefold increase in genome size due to the accumulation of LTR retrotransposons over the 5-10 Myr since its origin. ©2006 by Cold Spring Harbor Laboratory Press.
• Lin, Y., Chow, T., Luo, M., Kudrna, D., Lin, C., Wing, R. A., & Hsing, Y. C. (2006). Two highly representative rice BAC libraries of japonica cv Tainung 67 suitable for rice structural and functional genomic research. Plant Science, 170(4), 889-896.
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  Abstract: Two deep-coverage bacterial artificial chromosome (BAC) libraries of Oryza sativa japonica cv. Tainung 67 (TNG 67), a popular genetic stock in breeding programs and scientific research in Taiwan, have been constructed to facilitate positional cloning of rice genes and to analyze variety-specific genome composition, toward rice structural and functional genomic studies. Good high-molecular-weight DNA was produced well by nuclei preparation from two- to three-week-old seedlings, partially digested by either HindIII or EcoRI, and two cycles of size selection by pulsed-field gel electrophoresis (PFGE). The HindIII library consists of 45,312 clones, near lack of false positive clones, with an estimated average insert size of 138.4 kb and coverage of 15.1× haploid genome equivalents. The EcoRI library consists of 9984 clones, 2% of false positive clones, with an estimated average insert size of 137.8 kb and coverage of 3.2× haploid genome equivalents. Nine single-copy sequence tagged site (STS) markers, located on different chromosomes, were used to screen the two libraries, which a single BAC clone double-spotted on three filters, and hybridized 16-29, with an average of 21.2 BACs. As a result, these two libraries can complement each other and cover nearly 100% of the rice genome, consequently providing efficient tools to isolate any genes of interest. Resources, high-density filters, individual clones, and whole libraries, are available for public distribution and may be accessed at the Institute of Plant and Microbial Biology, Academia Sinica, or Arizona Genomic Institute (AGI).
• Luo, M., Kim, H., Kudrna, D., Sisneros, N. B., Lee, S., Mueller, C., Collura, K., Zuccolo, A., Buckingham, B., Grim, S. M., Yanagiya, K., Inoko, H., Shiina, T., Flajnik, M. F., Wing, R. A., & Ohta, Y. (2006). Construction of a nurse shark (Ginglymostoma cirratum) bacterial artificial chromosome (BAC) library and a preliminary genome survey. BMC Genomics, 7.
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  PMID: 16672057;PMCID: PMC1513397;Abstract: Background: Sharks are members of the taxonomic class Chondrichthyes, the oldest living jawed vertebrates. Genomic studies of this group, in comparison to representative species in other vertebrate taxa, will allow us to theorize about the fundamental genetic, developmental, and functional characteristics in the common ancestor of all jawed vertebrates. Aims: In order to obtain mapping and sequencing data for comparative genomics, we constructed a bacterial artificial chromosome (BAC) library for the nurse shark, Ginglymostoma cirratum. Results: The BAC library consists of 313,344 clones with an average insert size of 144 kb, covering -4.5 × 1010 bp and thus providing an II-fold coverage of the haploid genome. BAC end sequence analyses revealed, in addition to LINEs and SINEs commonly found in other animal and plant genomes, two new groups of nurse shark-specific repetitive elements, NSRE1 and NSRE2 that seem to be major components of the nurse shark genome. Screening the library with single-copy or multi-copy gene probes showed 6-28 primary positive clones per probe of which 50-90% were true positives, demonstrating that the BAC library is representative of the different regions of the nurse shark genome. Furthermore, some BAC clones contained multiple genes, making physical mapping feasible. Conclusion: We have constructed a deep-coverage, high -quality, large insert, and publicly available BAC library for a cartilaginous fish. It will be very useful to the scientific community interested in shark genomic structure, comparative genomics, and functional studies. We found two new groups of repetitive elements specific to the nurse shark genome, which may contribute to the architecture and evolution of the nurse shark genome. © 2006 Luo et al; licensee BioMed Central Ltd.
• Luo, M., Yeisoo, Y. u., Kim, H., Kudrna, D., Itoh, Y., Agate, R. J., Melamed, E., Goicoechea, J. L., Talag, J., Mueller, C., Wang, W., Currie, J., Sisneros, N. B., Wing, R. A., & Arnold, A. P. (2006). Erratum to "Utilization of a zebra finch BAC library to determine the structure of an avian androgen receptor genomic region" [Genomics 87 (2006) 181-190] (DOI:10.1016/j.ygeno.2005.09.005). Genomics, 87(5), 678-679.
• Luo, M., Yeisoo, Y. u., Kim, H., Kudrna, D., Itoh, Y., Agate, R. J., Melamed, E., Goicoechea, J. L., Talag, J., Mueller, C., Wang, W., Currie, J., Sisneros, N. B., Wing, R. A., & Arnold, A. P. (2006). Utilization of a zebra finch BAC library to determine the structure of an avian androgen receptor genomic region. Genomics, 87(1), 181-190.
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  PMID: 16321505;Abstract: The zebra finch (Taeniopygia guttata) is an important model organism for studying behavior, neuroscience, avian biology, and evolution. To support the study of its genome, we constructed a BAC library (TG__Ba) using DNA from livers of females. The BAC library consists of 147,456 clones with 98% containing inserts of an average size of 134 kb and represents 15.5 haploid genome equivalents. By sequencing a whole BAC, a full-length androgen receptor open reading frame was identified, the first in an avian species. Comparison of BAC end sequences and the whole BAC sequence with the chicken genome draft sequence showed a high degree of conserved synteny between the zebra finch and the chicken genome. © 2005 Elsevier Inc. All rights reserved.
• Piegu, B., Guyot, R., Picault, N., Roulin, A., Saniyal, A., Kim, H., Collura, K., Brar, D. S., Jackson, S., Wing, R. A., & Panaud, O. (2006). Doubling genome size without polyploidization: Dynamics of retrotransposition-driven genomic expansions in Oryza australiensis, a wild relative of rice. GENOME RESEARCH, 16(10), 1262-1269.
• Udall, J. A., Swanson, J. M., Haller, K., Rapp, R. A., Sparks, M. E., Hatfield, J., Yeisoo, Y. u., Yingru, W. u., Dowd, C., Arpat, A. B., Sickler, B. A., Wilkins, T. A., Jin, Y. G., Xiao, Y. C., Scheffler, J., Taliercio, E., Turley, R., McFadden, H., Payton, P., , Klueva, N., et al. (2006). A global assembly of cotton ESTs. Genome Research, 16(3), 441-450.
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  PMID: 16478941;PMCID: PMC1415220;Abstract: Approximately 185,000 Gossypium EST sequences comprising >94,800,000 nucleotides were amassed from 30 cDNA libraries constructed from a variety of tissues and organs under a range of conditions, including drought stress and pathogen challenges. These libraries were derived from allopolyploid cotton (Gossypium hirsutum; AT and DT genomes) as well as its two diploid progenitors, Gossypium arboreum (A genome) and Gossypium raimondii (D genome). ESTs were assembled using the Program for Assembling and Viewing ESTs (PAVE), resulting in 22,030 contigs and 29,077 singletons (51,107 unigenes). Further comparisons among the singletons and contigs led to recognition of 33,665 exemplar sequences that represent a nonredundant set of putative Gossypium genes containing partial or full-length coding regions and usually one or two UTRs. The assembly, along with their UniProt BLASTX hits, GO annotation, and Pfam analysis results, are freely accessible as a public resource for cotton genomics. Because ESTs from diploid and allotetraploid Gossypium were combined in a single assembly, we were in many cases able to bioinformatically distinguish duplicated genes in allotetraploid cotton and assign them to either the A or D genome. The assembly and associated information provide a framework for future investigation of cotton functional and evolutionary genomics. ©2006 by Cold Spring Harbor Laboratory Press.
• Wing, R., Ammiraju, J. S., Luo, M., Goicoechea, J. L., Wang, W., Kudrna, D., Mueller, C., Talag, J., Kim, H., Sisneros, N. B., Blackmon, B., Fang, E., Tomkins, J. B., Brar, D., MacKill, D., McCouch, S., Kurata, N., Lambert, G., Galbraith, D. W., , Arumuganathan, K., et al. (2006). The Oryza bacterial artificial chromosome library resource: construction and analysis of 12 deep-coverage large-insert BAC libraries that represent the 10 genome types of the genus Oryza. Genome research, 16(1).
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  Rice (Oryza sativa L.) is the most important food crop in the world and a model system for plant biology. With the completion of a finished genome sequence we must now functionally characterize the rice genome by a variety of methods, including comparative genomic analysis between cereal species and within the genus Oryza. Oryza contains two cultivated and 22 wild species that represent 10 distinct genome types. The wild species contain an essentially untapped reservoir of agriculturally important genes that must be harnessed if we are to maintain a safe and secure food supply for the 21st century. As a first step to functionally characterize the rice genome from a comparative standpoint, we report the construction and analysis of a comprehensive set of 12 BAC libraries that represent the 10 genome types of Oryza. To estimate the number of clones required to generate 10 genome equivalent BAC libraries we determined the genome sizes of nine of the 12 species using flow cytometry. Each library represents a minimum of 10 genome equivalents, has an average insert size range between 123 and 161 kb, an average organellar content of 0.4%-4.1% and nonrecombinant content between 0% and 5%. Genome coverage was estimated mathematically and empirically by hybridization and extensive contig and BAC end sequence analysis. A preliminary analysis of BAC end sequences of clones from these libraries indicated that LTR retrotransposons are the predominant class of repeat elements in Oryza and a roughly linear relationship of these elements with genome size was observed.
• Yan, H., Ito, H., Nobuta, K., Ouyang, S., Jin, W., Tian, S., Lu, C., Venu, R. C., Wang, G., Green, P. J., Wing, R. A., Buell, C. R., Meyers, B. C., & Jiang, J. (2006). Genomic and genetic characterization of rice Cen3 reveals extensive transcription and evolutionary implications of a complex centromere. PLANT CELL, 18(9), 2123-2133.
• Bowers, J. E., Arias, M. A., Asher, R., Avise, J. A., Ball, R. T., Brewer, G. A., Buss, R. W., Chen, A. H., Edwards, T. M., Estill, J. C., Exum, H. E., Goff, V. H., Herrick, K. L., L., C., Karunakaran, S., Lafayette, G. K., Lemke, C., Marler, B. S., Masters, S. L., , McMillan, J. M., et al. (2005). Comparative physical mapping links conservation of microsynteny to chromosome structure and recombination in grasses. Proceedings of the National Academy of Sciences of the United States of America, 102(37), 13206-13211.
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  PMID: 16141333;PMCID: PMC1201573;Abstract: Nearly finished sequences for model organisms provide a foundation from which to explore genomic diversity among other taxonomic groups. We explore genome-wide microsynteny patterns between the rice sequence and two sorghum physical maps that integrate genetic markers, bacterial artificial chromosome (BAC) fingerprints, and BAC hybridization data. The sorghum maps largely tile a genomic component containing 41% of BACs but 80% of single-copy genes that shows conserved microsynteny with rice and partially tile a nonsyntenic component containing 46% of BACs but only 13% of single-copy genes. The remaining BACs are centromeric (4%) or unassigned (8%). The two genomic components correspond to cytologically discernible "euchromatin" and "heterochromatin. " Gene and repetitive DNA distributions support this classification. Greater microcolinearity in recombinogenic (euchromatic) than nonrecombinogenic (heterochromatic) regions is consistent with the hypothesis that genomic rearrangements are usually deleterious, thus more likely to persist in nonrecombinogenic regions by virtue of Muller's ratchet. Interchromosomal centromeric rearrangements may have fostered diploidization of a polyploid cereal progenitor. Model plant sequences better guide studies of related genomes in recombinogenic than nonrecombinogenic regions. Bridging of 35 physical gaps in the rice sequence by sorghum BAC contigs illustrates reciprocal benefits of comparative approaches that extend at least across the cereals and perhaps beyond. © 2005 by The National Academy of Sciences of the USA.
• Buell, C. R., McCombie, W. R., Wing, R. A., Yuan, Q., Ouyang, S., Liu, J., Zhu, W., Wang, A., Maiti, R., Haas, B., Wortman, J., Pertea, M., Jones, K. M., Kim, M., Overton, L., Tsitrin, T., Fadrosh, D., Bera, J., Weaver, J., , Jin, S., et al. (2005). Sequence, annotation, and analysis of synteny between rice chromosome 3 and diverged grass species. The Rice Chromosome 3 Sequencing Consortium. Genome Research, 15(9), 1284-1291.
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  PMID: 16109971;PMCID: PMC1199543;Abstract: Rice (Oryza sativa L.) chromosome 3 is evolutionarily conserved across the cultivated cereals and shares large blocks of synteny with maize and sorghum, which diverged from rice more than 50 million years ago. To begin to completely understand this chromosome, we sequenced, finished, and annotated 36.1 Mb (∼97%) from O. sativa subsp. japonica cv Nipponbare. Annotation features of the chromosome include 5915 genes, of which 913 are related to transposable elements. A putative function could be assigned to 3064 genes, with another 757 genes annotated as expressed, leaving 2094 that encode hypothetical proteins. Similarity searches against the proteome of Arabidopsis thaliana revealed putative homologs for 67% of the chromosome 3 proteins. Further searches of a nonredundant amino acid database, the Pfam domain database, plant Expressed Sequence Tags, and genomic assemblies from sorghum and maize revealed only 853 nontransposable element related proteins from chromosome 3 that lacked similarity to other known sequences. Interestingly, 426 of these have a paralog within the rice genome. A comparative physical map of the wild progenitor species, Oryza nivara, with japonica chromosome 3 revealed a high degree of sequence identity and synteny between these two species, which diverged ∼10,000 years ago. Although no major rearrangements were detected, the deduced size of the O. nivara chromosome 3 was 21% smaller than that of japonica. Synteny between rice and other cereals using an integrated maize physical map and wheat genetic map was strikingly high, further supporting the use of rice and, in particular, chromosome 3, as a model for comparative studies among the cereals. ©2005 by Cold Spring Harbor Laboratory Press.
• Choisne, N., Demange, N., Orjeda, G., Samain, S., D'Hont, A., Cattolico, L., Pelletier, E., Couloux, A., Segurens, B., Wincker, P., Scarpelli, C., Weissenbach, J., Salanoubat, M., Singh, N. K., Mohapatra, T., Sharma, T. R., Gaikwad, K., Singh, A., Dalal, V., , Srivastava, S. K., et al. (2005). The sequence of rice chromosomes 11 and 12, rich in disease resistance genes and recent gene duplications. BMC Biology, 3.
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  Abstract: Background: Rice is an important staple food and, with the smallest cereal genome, serves as a reference species for studies on the evolution of cereals and other grasses. Therefore, decoding its entire genome will be a prerequisite for applied and basic research on this species and all other cereals. Results: We have determined and analyzed the complete sequences of two of its chromosomes, 11 and 12, which total 55.9 Mb (14.3% of the entire genome length), based on a set of overlapping clones. A total of 5,993 non-transposable element related genes are present on these chromosomes. Among them are 289 disease resistance-like and 28 defense-response genes, a higher proportion of these categories than on any other rice chromosome. A three-Mb segment on both chromosomes resulted from a duplication 7.7 million years ago (mya), the most recent large-scale duplication in the rice genome. Paralogous gene copies within this segmental duplication can be aligned with genomic assemblies from sorghum and maize. Although these gene copies are preserved on both chromosomes, their expression patterns have diverged. When the gene order of rice chromosomes 11 and 12 was compared to wheat gene loci, significant synteny between these orthologous regions was detected, illustrating the presence of conserved genes alternating with recently evolved genes. Conclusion: Because the resistance and defense response genes, enriched on these chromosomes relative to the whole genome, also occur in clusters, they provide a preferred target for breeding durable disease resistance in rice and the isolation of their allelic variants. The recent duplication of a large chromosomal segment coupled with the high density of disease resistance gene clusters makes this the most recently envolved part of the rice genome. Based on syntenic alignments of these chromosomes, rice chromosome 11 and 12 do not appear to have resulted from a single whole-genome duplication event as peviously suggested. © 2005 Rice Chromosomes 11 and 12 Sequencing Consortia and Messing, licensee BioMed Central Ltd.
• Haberer, G., Young, S., Bharti, A. K., Gundlach, H., Raymond, C., Fuks, G., Butler, E., Wing, R. A., Rounsley, S., Birren, B., Nusbaum, C., Mayer, K. F., & Messing, J. (2005). Structure and architecture of the maize genome. Plant Physiology, 139(4), 1612-1624.
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  PMID: 16339807;PMCID: PMC1310546;Abstract: Maize (Zea mays or corn) plays many varied and important roles in society. It is not only an important experimental model plant, but also a major livestock feed crop and a significant source of industrial products such as sweeteners and ethanol. In this study we report the systematic analysis of contiguous sequences of the maize genome. We selected 100 random regions averaging 144 kb in size, representing about 0.6% of the genome, and generated a high-quality dataset for sequence analysis. This sampling contains 330 annotated genes, 91% of which are supported by expressed sequence tag data from maize and other cereal species. Genes averaged 4 kb in size with five exons, although the largest was over 59 kb with 31 exons. Gene density varied over a wide range from 0.5 to 10.7 genes per 100 kb and genes did not appear to cluster significantly. The total repetitive element content we observed (66%) was slightly higher than previous whole-genome estimates (58%-63%) and consisted almost exclusively of retroelements. The vast majority of genes can be aligned to at least one sequence read derived from gene-enrichment procedures, but only about 30% are fully covered. Our results indicate that much of the increase in genome size of maize relative to rice (Oryza sativa) and Arabidopsis (Arabidopsis thaliana) is attributable to an increase in number of both repetitive elements and genes. © 2005 American Society of Plant Biologists.
• Horn, R., Lecouls, A., Callahan, A., Dandekar, A., Garay, L., McCord, P., Howad, W., Chan, H., Verde, I., Main, D., Jung, S., Georgi, L., Forrest, S., Mook, J., Zhebentyayeva, T., Yeisoo, Y. u., Hye, R. K., Jesudurai, C., Sosinski, B., , Arús, P., et al. (2005). Candidate gene database and transcript map for peach, a model species for fruit trees. Theoretical and Applied Genetics, 110(8), 1419-1428.
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  PMID: 15846479;Abstract: Peach (Prunus persica) is a model species for the Rosaceae, which includes a number of economically important fruit tree species. To develop an extensive Prunus expressed sequence tag (EST) database for identifying and cloning the genes important to fruit and tree development, we generated 9,984 high-quality ESTs from a peach cDNA library of developing fruit mesocarp. After assembly and annotation, a putative peach unigene set consisting of 3,842 ESTs was defined. Gene ontology (GO) classification was assigned based on the annotation of the single "best hit" match against the Swiss-Prot database. No significant homology could be found in the GenBank nr databases for 24.3% of the sequences. Using core markers from the general Prunus genetic map, we anchored bacterial artificial chromosome (BAC) clones on the genetic map, thereby providing a framework for the construction of a physical and transcript map. A transcript map was developed by hybridizing 1,236 ESTs from the putative peach unigene set and an additional 68 peach cDNA clones against the peach BAC library. Hybridizing ESTs to genetically anchored BACs immediately localized 11.2% of the ESTs on the genetic map. ESTs showed a clustering of expressed genes in defined regions of the linkage groups. [The data were built into a regularly updated Genome Database for Rosaceae (GDR), available at ( http://www.genome.clemson.edu/gdr/ ).]. © Springer-Verlag 2005.
• Jantasuriyarat, C., Gowda, M., Haller, K., Hatfield, J., Guodong, L. u., Stahlberg, E., Zhou, B., Huameng, L. i., Kim, H., Yeisoo, Y. u., Dean, R. A., Wing, R. A., Soderlund, C., & Wang, G. (2005). Large-scale identification of expressed sequence tags involved in rice and rice blast fungus interaction. Plant Physiology, 138(1), 105-115.
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  PMID: 15888683;PMCID: PMC1104166;Abstract: To better understand the molecular basis of the defense response against the rice blast fungus (Magnaporthe grisea), a large-scale expressed sequence tag (EST) sequencing approach was used to identify genes involved in the early infection stages in rice (Oryza sativa). Six cDNA libraries were constructed using infected leaf tissues harvested from 6 conditions: resistant, partially resistant, and susceptible reactions at both 6 and 24 h after inoculation. Two additional libraries were constructed using uninoculated leaves and leaves from the lesion mimic mutant spl11. A total of 68,920 ESTs were generated from 8 libraries. Clustering and assembly analyses resulted in 13,570 unique sequences from 10,934 contigs and 2,636 singletons. Gene function classification showed that 42% of the ESTs were predicted to have putative gene function. Comparison of the pathogen-challenged libraries with the uninoculated control library revealed an increase in the percentage of genes in the functional categories of defense and signal transduction mechanisms and cell cycle control, cell division, and chromosome partitioning. In addition, hierarchical clustering analysis grouped the eight libraries based on their disease reactions. A total of 7,748 new and unique ESTs were identified from our collection compared with the KOME full-length cDNA collection. Interestingly, we found that rice ESTs are more closely related to sorghum (Sorghum bicolor) ESTs than to barley (Hordeum vulgare), wheat (Triticum aestivum), and maize (Zea mays) ESTs. The large cataloged collection of rice ESTs in this study provides a solid foundation for further characterization of the rice defense response and is a useful public genomic resource for rice functional genomics studies. © 2005 American Society of Plant Biologists.
• Kumar, C. S., Wing, R. A., & Sundaresan, V. (2005). Efficient insertional mutagenesis in rice using the maize En/Spm elements. Plant Journal, 44(5), 879-892.
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  PMID: 16297077;Abstract: We have developed a novel system for insertional mutagenesis in rice (Oryza sativa) based on the maize (Zea mays) enhancer/suppressor mutator (En/Spm) element. In this system, a single T-DNA construct with Spm-transposase and the non-autonomous defective suppressor mutator (dSpm) element is used in conjunction with green fluorescent protein (GFP) and Discosoma sp. Red Fluorescence Protein (DsRed) fluorescent markers to select unlinked stable transpositions of dSpm. Using this system, we could demonstrate high frequencies of unlinked germinal transposition of dSpm in rice. Analysis of dSpm flanking sequences from 353 stable insertion lines revealed that the dSpm insertions appear to be widely distributed on rice chromosomes with a preference for genic regions (70%). The dSpm insertions appear to differ from Activator-Dissociation (Ac-Ds) elements in genomic distribution and exhibit a greater fraction of unlinked transpositions when compared with Ds elements. The results obtained in this study demonstrate that the maize En/Spm element can be used as an effective tool for functional genomics in rice and can complement efforts using other insertional mutagens. Further, the efficacy of the non-invasive fluorescence-based selection system is promising for its application to other crops. © 2005 Blackwell Publishing Ltd.
• Nelson, W. M., Bharti, A. K., Butler, E., Wei, F., Fuks, G., Kim, H., Wing, R. A., Messing, J., & Soderlund, C. (2005). Whole-genome validation of high-information-content fingerprinting. Plant Physiology, 139(1), 27-38.
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  PMID: 16166258;PMCID: PMC1203355;Abstract: Fluorescent-based high-information-content fingerprinting (HICF) techniques have recently been developed for physical mapping. These techniques make use of automated capillary DNA sequencing instruments to enable both high-resolution and high-throughput fingerprinting. In this article, we report the construction of a whole-genome HICF FPC map for maize (Zea mays subsp. mays cv B73), using a variant of HICF in which a type IIS restriction enzyme is used to generate the fluorescently labeled fragments. The HICF maize map was constructed from the same three maize bacterial artificial chromosome libraries as previously used for the whole-genome agarose FPC map, providing a unique opportunity for direct comparison of the agarose and HICF methods; as a result, it was found that HICF has substantially greater sensitivity in forming contigs. An improved assembly procedure is also described that uses automatic end-merging of contigs to reduce the effects of contamination and repetitive bands. Several new features in FPC v7.2 are presented, including shared-memory multiprocessing, which allows dramatically faster assemblies, and automatic end-merging, which permits more accurate assemblies. It is further shown that sequenced clones may be digested in silico and located accurately on the HICF assembly despite size deviations that prevent the precise prediction of experimental fingerprints. Finally repetitive bands are isolated, and their effect on the assembly is studied. © 2005 American Society of Plant Biologists.
• Pratt, L. H., Liang, C., Shah, M., Sun, F., Wang, H., Reid, S. P., Gingle, A. R., Paterson, A. H., Wing, R., Dean, R., Klein, R., Nguyen, H. T., Ma, H., Zhao, X., Morishige, D. T., Mullet, J. E., & Cordonnier-Pratt, M. (2005). Sorghum expressed sequence tags identify signature genes for drought, pathogenesis, and skotomorphogenesis from a milestone set of 16,801 unique transcripts. Plant Physiology, 139(2), 869-884.
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  PMID: 16169961;PMCID: PMC1256002;Abstract: Improved knowledge of the sorghum transcriptome will enhance basic understanding of how plants respond to stresses and serve as a source of genes of value to agriculture. Toward this goal, Sorghum bicolor L. Moench cDNA libraries were prepared from light- and dark-grown seedlings, drought-stressed plants, Colletotrichum-infected seedlings and plants, ovaries, embryos, and immature panicles. Other libraries were prepared with meristems from Sorghum propinquum (Kunth) Hitchc. that had been photoperiodically induced to flower, and with rhizomes from S. propinquum and johnsongrass (Sorghum halepense L. Pers.). A total of 117,682 expressed sequence tags (ESTs) were obtained representing both 3′ and 5′ sequences from about half that number of cDNA clones. A total of 16,801 unique transcripts, representing tentative UniScripts (TUs), were identified from 55,783 3′ ESTs. Of these TUs, 9,032 are represented by two or more ESTs. Collectively, these libraries were predicted to contain a total of approximately 31,000 TUs. Individual libraries, however, were predicted to contain no more than about 6,000 to 9,000, with the exception of light-grown seedlings, which yielded an estimate of close to 13,000. In addition, each library exhibits about the same level of complexity with respect to both the number of TUs preferentially expressed in that library and the frequency with which two or more ESTs is found in only that library. These results indicate that the sorghum genome is expressed in highly selective fashion in the individual organs and in response to the environmental conditions surveyed here. Close to 2,000 differentially expressed TUs were identified among the cDNA libraries examined, of which 775 were differentially expressed at a confidence level of 98%. From these 775 TUs, signature genes were identified defining drought, Colletotrichum infection, skotomorphogenesis (etiolation), ovary, immature panicle, and embryo. © 2005 American Society of Plant Biologists.
• Wang, W., Tanurdzic, M., Luo, M., Sisneros, N., Hye, R. K., Weng, J., Kudrna, D., Mueller, C., Arumuganathan, K., Carlson, J., Chapple, C., Pamphilis, C. D., Mandoli, D., Tomkins, J., Wing, R. A., & Banks, J. A. (2005). Construction of a bacterial artificial chromosome library from the spikemoss Selaginella moellendorffii: A new resource for plant comparative genomics. BMC Plant Biology, 5.
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  PMID: 15955246;PMCID: PMC1177970;Abstract: Background: The lycophytes are an ancient lineage of vascular plants that diverged from the seed plant lineage about 400 Myr ago. Although the lycophytes occupy an important phylogenetic position for understanding the evolution of plants and their genomes, no genomic resources exist for this group of plants. Results: Here we describe the construction of a large-insert bacterial artificial chromosome (BAC) library from the lycophyte Selaginella moellendorffii. Based on cell flow cytometry, this species has the smallest genome size among the different lycophytes tested, including Huperzia lucidula, Diphaiastrum digita, Isoetes engelmanii and S. kraussiana. The arrayed BAC library consists of 9126 clones; the average insert size is estimated to be 122 kb. Inserts of chloroplast origin account for 2.3% of the clones. The BAC library contains an estimated ten genome-equivalents based on DNA hybridizations using five single-copy and two duplicated S. moellendorffii genes as probes. Conclusion: The S. moellenforffii BAC library, the first to be constructed from a lycophyte, will be useful to the scientific community as a resource for comparative plant genomics and evolution. © 2005 Wang et al; licensee BioMed Central Ltd.
• Wing, R. (2005). Plant Molecular Biology: Editorial. Plant Molecular Biology, 59(1), vii.
• Wing, R. A., Ammiraju, J. S., Luo, M., Kim, H., Yeisoo, Y. u., Kudrna, D., Goicoechea, J. L., Wang, W., Nelson, W., Rao, K., Brar, D., Mackill, D. J., Han, B., Soderlund, C., Stein, L., SanMiguel, P., & Jackson, S. (2005). The Oryza map alignment project: The golden path to unlocking the genetic potential of wild rice species. Plant Molecular Biology, 59(1), 53-62.
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  PMID: 16217601;Abstract: The wild species of the genus Oryza offer enormous potential to make a significant impact on agricultural productivity of the cultivated rice species Oryza sativa and Oryza glaberrima. To unlock the genetic potential of wild rice we have initiated a project entitled the 'Oryza Map Alignment Project' (OMAP) with the ultimate goal of constructing and aligning BAC/STC based physical maps of 11 wild and one cultivated rice species to the International Rice Genome Sequencing Project's finished reference genome - O. sativa ssp. japonica c. v. Nipponbare. The 11 wild rice species comprise nine different genome types and include six diploid genomes (AA, BB, CC, EE, FF and GG) and four tetrapliod genomes (BBCC, CCDD, HHKK and HHJJ) with broad geographical distribution and ecological adaptation. In this paper we describe our strategy to construct robust physical maps of all 12 rice species with an emphasis on the AA diploid O. nivara - thought to be the progenitor of modern cultivated rice. © Springer 2005.
• Wing, R., Ammiraju, J. S., Yu, Y., Luo, M., Kudrna, D., Kim, H., Goicoechea, J. L., Katayose, Y., Matsumoto, T., Wu, J., Sasaki, T., & Wing, R. A. (2005). Random sheared fosmid library as a new genomic tool to accelerate complete finishing of rice (Oryza sativa spp. Nipponbare) genome sequence: sequencing of gap-specific fosmid clones uncovers new euchromatic portions of the genome. TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik, 111(8).
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  The International Rice Genome Sequencing Project has recently announced the high-quality finished sequence that covers nearly 95% of the japonica rice genome representing 370 Mbp. Nevertheless, the current physical map of japonica rice contains 62 physical gaps corresponding to approximately 5% of the genome, that have not been identified/represented in the comprehensive array of publicly available BAC, PAC and other genomic library resources. Without finishing these gaps, it is impossible to identify the complete complement of genes encoded by rice genome and will also leave us ignorant of some 5% of the genome and its unknown functions. In this article, we report the construction and characterization of a tenfold redundant, 40 kbp insert fosmid library generated by random mechanical shearing. We demonstrated its utility in refining the physical map of rice by identifying and in silico mapping 22 gap-specific fosmid clones with particular emphasis on chromosomes 1, 2, 6, 7, 8, 9 and 10. Further sequencing of 12 of the gap-specific fosmid clones uncovered unique rice genome sequence that was not previously reported in the finished IRGSP sequence and emphasizes the need to complete finishing of the rice genome.
• Yang, T., Lee, S., Chang, S., Yeisoo, Y. u., Jong, H. d., & Wing, R. A. (2005). In-depth sequence analysis of the tomato chromosome 12 centromeric region: Identification of a large CAA block and characterization of pericentromere retrotranposons. Chromosoma, 114(2), 103-117.
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  PMID: 15965704;Abstract: We sequenced a continuous 326-kb DNA stretch of a microscopically defined centromeric region of tomato chromosome 12. A total of 84% of the sequence (270 kb) was composed of a nested complex of repeat sequences including 27 retrotransposons, two transposable elements, three MITEs, two terminal repeat retrotransposons in miniature (TRIMs), ten unclassified repeats and three chloroplast DNA insertions. The retrotransposons were grouped into three families of Ty3-Gypsy type long terminal repeat (LTR) retrotransposons (PCRT1-PCRT3) and one LINE-like retrotransposon (PCRT4). High-resolution fluorescence in situ hybridization analyses on pachytene complements revealed that PCRT1a occurs on the pericentromere heterochromatin blocks. PCRT1 was the prevalent retrotransposon family occupying more than 60% of the 326-kb sequence with 19 members grouped into eight subfamilies (PCRT1-PCRT1h) based on LTR sequence. The PCRT1a subfamily is a rapidly amplified element occupying tens of megabases. The other PCRT1 subfamilies (PCRT1b-PCRT1h) were highly degenerated and interrupted by insertions of other elements. The PCRT1 family shows identity with a previously identified tomato-specific repeat TGR2 and a CENP-B like sequence. A second previously described genomic repeat, TGR3, was identified as a part of the LTR sequence of an Athila-like PCRT2 element of which four copies were found in the 326-kb stretch. A large block of trinucleotide microsatellite (CAA)n occupies the centromere and large portions of the flanking pericentromere heterochromatin blocks of chromosome 12 and most of the other chromosomes. Five putative genes in the remaining 14% of the centromere region were identified, of which one is similar to a transcription regulator (ToCPL1) and a candidate jointless-2 gene. © Springer-Verlag 2005.
• Yang, T., Yeisoo, Y. u., Chang, S., Jong, H. D., Oh, C., Ahn, S., Fang, E., & Wing, R. A. (2005). Toward closing rice telomere gaps: Mapping and sequence characterization of rice subtelomere regions. Theoretical and Applied Genetics, 111(3), 467-478.
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  PMID: 15965650;Abstract: Despite the collective efforts of the international community to sequence the complete rice genome, telomeric regions of most chromosome arms remain uncharacterized. In this report we present sequence data from subtelomere regions obtained by analyzing telomeric clones from two 8.8 x genome equivalent 10-kb libraries derived from partial restriction digestion with HaeIII or Sau3AI (OSJNPb HaeIII and OSJNPc Sau3AI). Seven telomere clones were identified and contain 25-100 copies of the telomere repeat (CCCTAAA)n on one end and unique sequences on the opposite end. Polymorphic sequence-tagged site markers from five clones and one additional PCR product were genetically mapped on the ends of chromosome arms 2S, 5L, 10S, 10L, 7L, and 7S. We found distinct chromosome-specific telomere-associated tandem repeats (TATR) on chromosome 7 (TATR7) and on the short arm of chromosome 10 (TATR10s) that showed no significant homology to any International Rice Genome Sequencing Project (IRGSP) genomic sequence. The TATR7, a degenerate tandem repeat which is interrupted by transposable elements, appeared on both ends of chromosome 7. The TATR10s was found to contain an inverted array of three tandem repeats displaying an interesting secondary folding pattern that resembles a telomere loop (t-loop) and which may be involved in a protective function against chromosomal end degradation. © Springer-Verlag 2005.
• Arpat, A. B., Waugh, M., Sullivan, J. P., Gonzales, M., Frisch, D., Main, D., Wood, T., Leslie, A., Wing, R. A., & Wilkins, T. A. (2004). Functional genomics of cell elongation in developing cotton fibers. Plant Molecular Biology, 54(6), 911-929.
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  PMID: 15604659;Abstract: Cotton fibers are single-celled seed trichomes of major economic importance. Factors that regulate the rate and duration of cell expansion control fiber morphology and important agronomic traits. For genetic characterization of rapid cell elongation in cotton fibers, ∼ 14,000 unique genes were assembled from 46,603 expressed sequence tags (ESTs) from developmentally staged fiber cDNAs of a cultivated diploid species (Gossypium arboreum L.). Conservatively, the fiber transcriptome represents 35-40% of the genes in the cotton genome. In silico expression analysis revealed that rapidly elongating fiber cells exhibit significant metabolic activity, with the bulk of gene transcripts, represented by three major functional groups - cell wall structure and biogenesis, the cytoskeleton and energy/carbohydrate metabolism. Oligonucleotide microarrays revealed dynamic changes in gene expression between primary and secondary cell wall biogenesis showing that fiber genes in the dbEST are highly stage-specific for cell expansion - a conclusion supported by the absence of known secondary cell wall-specific genes from our fiber dbEST. During the developmental switch from primary to secondary cell wall syntheses, 2553 "expansion-associated" fiber genes are significantly down regulated. Genes (81) significantly up-regulated during secondary cell wall synthesis are involved in cell wall biogenesis and energy/carbohydrate metabolism, which is consistent with the stage of cellulose synthesis during secondary cell wall modification in developing fibers. This work provides the first in-depth view of the genetic complexity of the transcriptome of an expanding cell, and lays the groundwork for studying fundamental biological processes in plant biology with applications in agricultural biotechnology.
• Budiman, M. A., Chang, S. -., Lee, S., Yang, T. J., Zhang, H. -., Jong, H. D., & Wing, R. A. (2004). Localization of jointless-2 gene in the centromeric region of tomato chromosome 12 based on high resolution genetic and physical mapping. Theoretical and Applied Genetics, 108(2), 190-196.
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  PMID: 14504748;Abstract: Abscission is a universal process whereby plants shed their organs, such as flowers, fruit and leaves. In tomato, the non-allelic mutations jointless and jointless-2 have been discovered as recessive mutations that completely suppress the formation of pedicel abscission zones. A high resolution genetic map of jointless-2 was constructed using 1,122 jointless F2 plants. Restriction fragment length polymorphism (RFLP) marker RPD140 completely co-segregated with the jointless-2 locus and mapped in a 2.4 cM interval between RFLP markers CD22 and TG618. To chromosome walk to jointless-2, all three markers were used to screen a bacterial artificial chromosome (BAC) library and contigs were developed. Intensive efforts to expand and merge the BAC contigs were unsuccessful because of the highly repetitive sequence content on the distal ends of each contig. To determine the physical distance between and the orientation of the three contigs, we used high resolution pachytene fluorescence in situ hybridization (FISH) mapping. The RPD140 contig was positioned in the centromeric region of chromosome 12 between two large pericentric heterochromatin blocks, about 50 Mb from the TG618 contig on the short arm and 10 Mb from the CD22 contig on the long arm, respectively. Based on high resolution genetic and physical mapping, we conclude that the jointless-2 gene is located within or near the chromosome 12 centromere where 1 cM is approximately 25 Mb in length.
• Close, T. J., Wanamaker, S. I., Caldo, R. A., Turner, S. M., Ashlock, D. A., Dickerson, J. A., Wing, R. A., Muehlbauer, G. J., Kleinhofs, A., & Wise, R. P. (2004). A new resource for cereal genomics: 22K Barley GeneChip comes of age. Plant Physiology, 134(3), 960-968.
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  PMID: 15020760;PMCID: PMC389919;Abstract: In recent years, access to complete genomic sequences, coupled with rapidly accumulating data related to RNA and protein expression patterns, has made it possible to determine comprehensively how genes contribute to complex phenotypes. However, for major crop plants, publicly available, standard platforms for parallel expression analysis have been limited. We report the conception and design of the new publicly available, 22K Barley1 GeneChip probe array, a model for plants without a fully sequenced genome. Array content was derived from worldwide contribution of 350,000 high-quality ESTs from 84 cDNA libraries, in addition to 1,145 barley (Hordeum vulgare) gene sequences from the National Center for Biotechnology Information nonredundant database. Conserved sequences expressed in seedlings of wheat (Triticum aestivum), oat (Avena strigosa), rice (Oryza sativa), sorghum (Sorghum bicolor), and maize (Zea mays) were identified that will be valuable in the design of arrays across grasses. To enhance the usability of the data, BarleyBase, a MIAME-compliant, MySQL relational database, serves as a public repository for raw and normalized expression data from the Barleyl GeneChip probe array. Interconnecting links with PlantGDB and Gramene allow BarleyBase users to perform gene predictions using the 21,439 non-redundant Barleyl exemplar sequences or cross-species comparison at the genome level, respectively. We expect that this first generation array will accelerate hypothesis generation and gene discovery in disease defense pathways, responses to abiotic stresses, development, and evolutionary diversity in monocot plants.
• Dietrich, F. S., Voegeli, S., Brachat, S., Lerch, A., Gates, K., Steiner, S., Mohr, C., Pöhlmann, R., Luedi, P., Choi, S., Wing, R. A., Flavier, A., Gaffney, T. D., & Philippsen, P. (2004). The Ashbya gossypii Genome as a Tool for Mapping the Ancient Saccharomyces cerevisiae Genome. Science, 304(5668), 304-307.
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  PMID: 15001715;Abstract: We have sequenced and annotated the genome of the filamentous ascomycete Ashbya gossypii. With a size of only 9.2 megabases, encoding 4718 protein-coding genes, it is the smallest genome of a free-living eukaryote yet characterized. More than 90% of A. gossypii genes show both homology and a particular pattern of synteny with Saccharomyces cerevisiae. Analysis of this pattern revealed 300 inversions and translocations that have occurred since divergence of these two species. It also provided compelling evidence that the evolution of S. cerevisiae included a whole genome duplication or fusion of two related species and showed, through inferred ancient gene orders, which of the duplicated genes lost one copy and which retained both copies.
• Gardiner, J., Schroeder, S., Polacco, M. L., Sanchez-Villeda, H., Fang, Z., Morgante, M., Landewe, T., Fengler, K., Useche, F., Hanafey, M., Tingey, S., Chou, H., Wing, R., Soderlund, C., & Coe Jr., E. H. (2004). Anchoring 9,371 maize expressed sequence tagged unigenes to the bacterial artificial chromosome contig map by two-dimensional overgo hybridization. Plant Physiology, 134(4), 1317-1326.
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  PMID: 15020742;PMCID: PMC419808;Abstract: Our goal is to construct a robust physical map for maize (Zea mays) comprehensively integrated with the genetic map. We have used a two-dimensional 24 X 24 overgo pooling strategy to anchor maize expressed sequence tagged (EST) unigenes to 165,888 bacterial artificial chromosomes (BACs) on high-density filters. A set of 70,716 public maize ESTs seeded derivation of 10,723 EST unigene assemblies. From these assemblies, 10,642 overgo sequences of 40 bp were applied as hybridization probes. BAC addresses were obtained for 9,371 overgo probes, representing an 88% success rate. More than 96% of the successful overgo probes identified two or more BACs, while 5% identified more than 50 BACs. The majority of BACs identified (79%) were hybridized with one or two overgos. A small number of BACs hybridized with eight or more overgos, suggesting that these BACs must be gene rich. Approximately 5,670 overgos identified BACs assembled within one contig, indicating that these probes are highly locus specific. A total of 1,795 megabases (Mb; 87%) of the total 2,050 Mb in BAC contigs were associated with one or more overgos, which are serving as sequence-tagged sites for single nucleotide polymorphism development. Overgo density ranged from less than one overgo per megabase to greater than 20 overgos per megabase. The majority of contigs (52%) hit by overgos contained three to nine overgos per megabase. Analysis of approximately 1,022 Mb of genetically anchored BAC contigs indicates that 9,003 of the total 13,900 overgo-contig sites are genetically anchored. Our results indicate overgos are a powerful approach for generating gene-specific hybridization probes that are facilitating the assembly of an integrated genetic and physical map for maize.
• Grover, C. E., Kim, H., Wing, R. A., Paterson, A. H., & Wendel, J. F. (2004). Incongruent patterns of local and global genome size evolution in cotton. Genome Research, 14(8), 1474-1482.
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  PMID: 15256507;PMCID: PMC509256;Abstract: Genome sizes in plants vary over several orders of magnitude, reflecting a combination of differentially acting local and global forces such as biases in indel accumulation and transposable element proliferation or removal. To gain insight into the relative role of these and other forces, ∼105 kb of contiguous sequence surrounding the cellulose synthase gene CesAl was compared for the two coresident genomes (AT and DT) of the allopolyploid cotton species, Gossypium hirsutum. These two genomes differ approximately twofold in size, having diverged from a common ancestor ∼5-10 million years ago (Mya) and been reunited in the same nucleus at the time of polyploid formation, ∼1-2 Mya. Gene content, order, and spacing are largely conserved between the two genomes, although a few transposable elements and a single cpDNA fragment distinguish the two homoeologs. Sequence conservation is high in both intergenic and genic regions, with 14 conserved genes detected in both genomes yielding a density of 1 gene every 7.5 kb. In contrast to the twofold overall difference in DNA content, no disparity in size was observed for this 105-kb region, and 555 indels were detected that distinguish the two homoeologous BACs, approximately equally distributed between AT and DT in number and aggregate size. The data demonstrate that genome size evolution at this phylogenetic scale is not primarily caused by mechanisms that operate uniformly across different genomic regions and components; instead, the twofold overall difference in DNA content must reflect locally operating forces between gene islands or in largely gene-free regions. © by Cold Spring Harbor Laboratory Press.
• Hamblin, M. T., Mitchell, S. E., White, G. M., Gallego, J., Kukatla, R., Wing, R. A., Paterson, A. H., & Kresovich, S. (2004). Comparative population genetics of the panicoid grasses: Sequence polymorphism, linkage disequilibrium and selection in a diverse sample of Sorghum bicolor. Genetics, 167(1), 471-483.
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  PMID: 15166170;PMCID: PMC1470838;Abstract: Levels of genetic variation and linkage disequilibrium (LD) are critical factors in association mapping methods as well as in identification of loci that have been targets of selection. Maize, an outcrosser, has a high level of sequence variation and a limited extent of LD. Sorghum, a closely related but largely self-pollinating panicoid grass, is expected to have higher levels of LD. As a first step in estimation of population genetic parameters in sorghum, we surveyed 27 diverse S. bicolor accessions for sequence variation at a total of 29,186 bp in 95 short regions derived from genetically mapped RFLPs located throughout the genome. Consistent with its higher level of inbreeding, the extent of LD is at least severalfold greater in sorghum than in maize. Total sequence variation in sorghum is about fourfold lower than that in maize, while synonymous variation is fivefold lower, suggesting a smaller effective population size in sorghum. Because we surveyed a species-wide sample, the mating system, which primarily affects population-level diversity, may not be primarily responsible for this difference. Comparisons of polymorphism and divergence suggest that both directional and diversifying selection have played important roles in shaping variation in the sorghum genome.
• Messing, J., Bharti, A. K., Karlowski, W. M., Gundlach, H., Hye, R. K., Yeisoo, Y. u., Wei, F., Fuks, G., Soderlund, C. A., F., K., & Wing, R. A. (2004). Sequence composition and genome organization of maize. Proceedings of the National Academy of Sciences of the United States of America, 101(40), 14349-14354.
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  PMID: 15388850;PMCID: PMC521949;Abstract: Zea mays L. ssp. mays, or corn, one of the most important crops and a model for plant genetics, has a genome ≈80% the size of the human genome. To gain global insight into the organization of its genome, we have sequenced the ends of large insert clones, yielding a cumulative length of one-eighth of the genome with a DNA sequence read every 6.2 kb, thereby describing a large percentage of the genes and transposable elements of maize in an unbiased approach. Based on the accumulative 307 Mb of sequence, repeat sequences occupy 58% and genic regions occupy 7.5%. A conservative estimate predicts ≈59,000 genes, which is higher than in any other organism sequenced so far. Because the sequences are derived from bacterial artificial chromosome clones, which are ordered in overlapping bins, tagged genes are also ordered along continuous chromosomal segments. Based on this positional information, roughly one-third of the genes appear to consist of tandemly arrayed gene families. Although the ancestor of maize arose by tetraploidization, fewer than half of the genes appear to be present in two orthologous copies, indicating that the maize genome has undergone significant gene loss since the duplication event.
• Rong, J., Abbey, C., Bowers, J. E., Brubaker, C. L., Chang, C., Chee, P. W., Delmonte, T. A., Ding, X., Garza, J. J., Marler, B. S., Park, C., Pierce, G. J., Rainey, K. M., Rastogi, V. K., Schulze, S. R., Trolinder, N. L., Wendel, J. F., Wilkins, T. A., Williams-Coplin, T. D., , Wing, R. A., et al. (2004). A 3347-Locus Genetic Recombination Map of Sequence-Tagged Sites Reveals Features of Genome Organization, Transmission and Evolution of Cotton (Gossypium). Genetics, 166(1), 389-417.
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  PMID: 15020432;PMCID: PMC1470701;Abstract: We report genetic maps for diploid (D) and tetraploid (AtDt) Gossypium genomes composed of sequence-tagged sites (STS) that foster structural, functional, and evolutionary genomic studies. The maps include, respectively, 2584 loci at 1,72-cM (∼600 kb) intervals based on 2007 probes (AtDt) and 763 loci at 1.96-cM (∼500 kb) intervals detected by 662 probes (D). Both diploid and tetraploid cottons exhibit negative crossover interference; i.e., double recombinants are unexpectedly abundant. We found no major structural changes between Dt and D chromosomes, but confirmed two reciprocal translocations between At chromosomes and several inversions, Concentrations of probes in corresponding regions of the various genomes may represent centromeres, while genome-specific concentrations may represent heterochromatin, Locus duplication patterns reveal all 13 expected homeologous chromosome sets and lend new support to the possibility that a more ancient polyploidization event may have predated the A-D divergence of 6-11 million years ago. Identification of SSRs within 312 RFLP sequences plus direct mapping of 124 SSRs and exploration for CAPS and SNPs illustrate the "portability" of these STS loci across populations and detection systems useful for marker-assisted improvement of the world's leading fiber crop. These data provide new insights into polyploid evolution and represent a foundation for assembly of a finished sequence of the cotton genome.
• Singh, N. K., Raghuvanshi, S., Srivastava, S. K., Gaur, A., Pal, A. K., Dalal, V., Singh, A., Ghazi, I. A., Bhargav, A., Yadav, M., Dixit, A., Batra, K., Gaikwad, K., Sharma, T. R., Mohanty, A., Bharti, A. K., Kapur, A., Gupta, V., Kumar, D., , Vij, S., et al. (2004). Sequence analysis of the long arm of rice chromosome 11 for rice-wheat synteny. Functional and Integrative Genomics, 4(2), 102-117.
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  PMID: 15085449;Abstract: The DNA sequence of 106 BAC/PAC clones in the minimum tiling path (MTP) of the long arm of rice chromosome 11, between map positions 57.3 and 116.2× cM, has been assembled to phase 2 or PLN level. This region has been sequenced to 10× redundancy by the Indian Initiative for Rice Genome Sequencing (IIRGS) and is now publicly available in GenBank. The region, excluding overlaps, has been predicted to contain 2,932 genes using different software. A gene-by-gene BLASTN search of the NCBI wheat EST database of over 420,000 cDNA sequences revealed that 1,143 of the predicted rice genes (38.9%) have significant homology to wheat ESTs (bit score ≥ 100). Further BLASTN search of these 1,143 rice genes with the GrainGenes database of sequence contigs containing bin-mapped wheat ESTs allowed 113 of the genes to be placed in bins located on wheat chromosomes of different homoeologous groups. The largest number of genes, about one-third, mapped to the homoeologous group 4 chromosomes of wheat, suggesting a common evolutionary origin. The remaining genes were located on wheat chromosomes of different groups with significantly higher numbers for groups 3 and 5. Location of bin-mapped wheat contigs to chromosomes of all the seven homoeologous groups can be ascribed to movement of genes (transpositions) or chromosome segments (translocations) within rice or the hexaploid wheat genomes. Alternatively, it could be due to ancient duplications in the common ancestral genome of wheat and rice followed by selective elimination of genes in the wheat and rice genomes. While there exists definite conservation of gene sequences and the ancestral chromosomal identity between rice and wheat, there is no obvious conservation of the gene order at this level of resolution. Lack of extensive colinearity between rice and wheat genomes suggests that there have been many insertions, deletions, duplications and translocations that make the synteny comparisons much more complicated than earlier thought. However, enhanced resolution of comparative sequence analysis may reveal smaller conserved regions of colinearity, which will facilitate selection of markers for saturation mapping and sequencing of the gene-rich regions of the wheat genome. © Springer-Verlag 2004.
• Thon, M. R., Martin, S. L., Goff, S., Wing, R. A., & Dean, R. A. (2004). BAC end sequences and a physical map reveal transposable element content and clustering patterns in the genome of Magnaporthe grisea. Fungal Genetics and Biology, 41(7), 657-666.
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  PMID: 15275661;Abstract: Transposable elements (TEs) are viewed as major contributors to the evolution of fungal genomes. Genomic resources such as BAC libraries are an underutilized resource for studying genome-wide TE distribution. Using the BAC end sequences and physical map that are available for the rice blast fungus, Magnaporthe grisea, we describe a likelihood ratio test designed to identify clustering of TEs in the genome. A significant variation in the distribution of three TEs, MAGGY, MGL, and Pot2 was observed among the fingerprint contigs of the physical map. We utilized a draft sequence of M. grisea chromosome 7 to validate our results and found a similar pattern of clustering. By examining individual BAC end sequences, we found evidence for 11 unique integrations of MAGGY or MGL into Pot2 but no evidence for the reciprocal integration of Pot2 into another TE. This suggests that: (a) the presence of Pot2 in the genome predates that of the other TEs, (b) Pot2 was less transpositionally active than other TEs, or (c) that MAGGY and MGL have integration site preference for Pot2. High transition/transversion mutation ratios as well as bias in transition site context was observed in MAGGY and MGL elements, but not in Pot2 elements. These features are consistent with the effects of a Repeat-Induced Point (RIP) mutation-like process occurring in MAGGY and MGL elements. This study illustrates the general utility of a physical map and BAC end sequences for the study of genome-wide repetitive DNA content and organization. Index Descriptors: Transposon; Transposable element; Rice blast; Magnaporthe grisea; Pyricularia grisea; BAC library; Physical map © 2004 Elsevier Inc. All rights reserved.
• Tomkins, J., Fregene, M., Main, D., Kim, H., Wing, R., & Tohme, J. (2004). Bacterial artificial chromosome (BAC) library resource for positional cloning of pest and disease resistance genes in cassava (Manihot esculenta Crantz). Plant Molecular Biology, 56(4), 555-561.
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  PMID: 15630619;Abstract: Pest and disease problems are important constraints of cassava production and host plant resistance is the most efficient method of combating them. Breeding for host plant resistance is considerably slowed down by the crop's biological constraints of a long growth cycle, high levels of heterozygosity and a large genetic load. More efficient methods such as gene cloning and transgenesis are required to deploy resistance genes. To facilitate the cloning of resistance genes, bacterial artificial chromosome (BAC) library resources have been developed for cassava. Two libraries were constructed from the cassava clones, TMS 30001, resistant to the cassava mosaic disease (CMD) and the cassava bacterial blight (CBB), and MECU72, resistant to cassava white fly. The TMS30001 library has 55 296 clones with an insert size range of 40-150 kb with an average of 80 kb, while the MECU72 library consists of 92 160 clones and an insert size range of 25-250 kb average of 93 kb. Based on a genome size of 772 Mb, the TMS30001 and MECU72 libraries have a 5 and 11.3 haploid genome equivalents and a 95% and 99% chance of finding any sequence, respectively. To demonstrate the potential of the libraries, the TMS30001 library was screened by southern hybridization using a cassava analog (CBB1) of the Xa21 gene from rice that maps to a region containing a QTL for resistance to CBB as probe. Five BAC clones that hybridized to CBB1 were isolated and a Hind III fingerprint revealed 2-3 copies of the gene in individual BAC clones. A larger scale analysis of resistance gene analogs (RGAs) in cassava has also been conducted in order to understand the number and organization of RGAs. To scan for gene and repeat DNA content in the libraries, end-sequencing was performed on 2301 clones from the MECU72 library. A total of 1705 unique sequences were obtained with an average size of 715 bp. Database homology searches using BLAST revealed that 458 sequences had significant homology with known proteins and 321 with transposable elements. The use of the library in positional cloning of pest and disease resistance genes is discussed.
• Zhang, Y., Huang, Y., Zhang, L., Ying, L. i., Tingting, L. u., Yiqi, L. u., Feng, Q., Zhao, Q., Cheng, Z., Xue, Y., Wing, R. A., & Han, B. (2004). Structural features of the rice chromosome 4 centromere. Nucleic Acids Research, 32(6), 2023-2030.
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  PMID: 15064362;PMCID: PMC390372;Abstract: A complete sequence of a chromosome centromere is necessary for fully understanding centromere function. We reported the sequence structures of the first complete rice chromosome centromere through sequencing a large insert bacterial artificial chromosome clone-based contig, which covered the rice chromosome 4 centromere. Complete sequencing of the 124-kb rice chromosome 4 centromere revealed that it consisted of 18 tracts of 379 tandemly arrayed repeats known as CentO and a total of 19 centromeric retroelements (CRs) but no unique sequences were detected. Four tracts, composed of 65 CentO repeats, were located in the opposite orientation, and 18 CentO tracts were flanked by 19 retroelements. The CRs were classified into four types, and the type I retroelements appeared to be more specific to rice centromeres. The preferential insert of the CRs among CentO repeats indicated that the centromere-specific retroelements may contribute to centromere expansion during evolution. The presence of three intact retrotransposons in the centromere suggests that they may be responsible for functional centromere initiation through a transcription-mediated mechanism. © Oxford University Press 2004; all rights reserved.
• Georgi, L. L., Wang, Y., Reighard, G. L., Mao, L., Wing, R. A., & Abbott, A. G. (2003). Comparison of peach and Arabidopsis genomic sequences: Fragmentary conservation of gene neighborhoods. Genome, 46(2), 268-276.
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  PMID: 12723043;Abstract: We examined the degree of conservation of gene order in two plant species, Prunus persica (peach) and Arabidopsis thaliana (thale cress), whose lineages diverged more than 90 million years ago. In the three peach genomic regions studied, segments with a gene order congruent with A. thaliana were short (two to three genes in length); and for any peach region, corresponding segments were found in diverse locations in the A. thaliana genome. At the gene level and lower, the A. thaliana sequence was enormously useful for identifying likely coding regions in peach sequences and in determining their intron-exon structure. The peach BAC sequence data reported here contained a BLAST-detectable putative coding sequence an average of every 7 kb, and the peach introns identified in this study were, on average, almost twice the length of the corresponding introns in A. thaliana.
• Senchina, D. S., Alvarez, I., Cronn, R. C., Liu, B., Rong, J., Noyes, R. D., Paterson, A. H., Wing, R. A., Wilkins, T. A., & Wendel, J. F. (2003). Rate variation among nuclear genes and the age of polyploidy in Gossypium. Molecular Biology and Evolution, 20(4), 633-643.
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  PMID: 12679546;Abstract: Molecular evolutionary rate variation in Gossypium (cotton) was characterized using sequence data for 48 nuclear genes from both genomes of allotetraploid cotton, models of its diploid progenitors, and an outgroup. Substitution rates varied widely among the 48 genes, with silent and replacement substitution levels varying from 0.018 to 0.162 and from 0.000 to 0.073, respectively, in comparisons between orthologous Gossypium and outgroup sequences. However, about 90% of the genes had silent substitution rates spanning a more narrow threefold range. Because there was no evidence of rate heterogeneity among lineages for any gene and because rates were highly correlated in independent tests, evolutionary rate is inferred to be a property of each gene or its genetic milieu rather than the clade to which it belongs. Evidence from approximately 200,000 nucleotides (40,000 per genome) suggests that polyploidy in Gossypium led to a modest enhancement in rates of nucleotide substitution. Phylogenetic analysis for each gene yielded the topology expected from organismal history, indicating an absence of gene conversion or recombination among homoeologs subsequent to allopolyploid formation. Using the mean synonymous substitution rate calculated across the 48 genes, allopolyploid cotton is estimated to have formed circa 1.5 million years ago (MYA), after divergence of the diploid progenitors about 6.7 MYA.
• Wing, R., Luo, M., & Wing, R. A. (2003). An improved method for plant BAC library construction. Methods in molecular biology (Clifton, N.J.), 236.
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  Large genomic DNA insert-containing libraries are required as critical tools for physical mapping, positional cloning, and genome sequencing of complex genomes. The bacterial artificial chromosome (BAC) cloning system has become a dominant system over others to clone large genomic DNA inserts. As the costs of positional cloning, physical mapping, and genome sequencing continuously decrease, there is an increasing demand for high-quality deep-coverage large insert BAC libraries. In our laboratory, we have constructed many high-quality deep-coverage large insert BAC libraries including arabidopsis, manocot and dicot crop plants, and plant pathogens. Here, we present the protocol used in our laboratory to construct BAC libraries.
• Wing, R., Yang, T., Yu, Y., Nah, G., Atkins, M., Lee, S., Frisch, D. A., & Wing, R. A. (2003). Construction and utility of 10-kb libraries for efficient clone-gap closure for rice genome sequencing. TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik, 107(4).
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  Rice is an important crop and a model system for monocot genomics, and is a target for whole genome sequencing by the International Rice Genome Sequencing Project (IRGSP). The IRGSP is using a clone by clone approach to sequence rice based on minimum tiles of BAC or PAC clones. For chromosomes 10 and 3 we are using an integrated physical map based on two fingerprinted and end-sequenced BAC libraries to identifying a minimum tiling path of clones. In this study we constructed and tested two rice genomic libraries with an average insert size of 10 kb (10-kb library) to support the gap closure and finishing phases of the rice genome sequencing project. The HaeIII library contains 166,752 clones covering approximately 4.6x rice genome equivalents with an average insert size of 10.5 kb. The Sau3AI library contains 138,960 clones covering 4.2x genome equivalents with an average insert size of 11.6 kb. Both libraries were gridded in duplicate onto 11 high-density filters in a 5 x 5 pattern to facilitate screening by hybridization. The libraries contain an unbiased coverage of the rice genome with less than 5% contamination by clones containing organelle DNA or no insert. An efficient method was developed, consisting of pooled overgo hybridization, the selection of 10-kb gap spanning clones using end sequences, transposon sequencing and utilization of in silico draft sequence, to close relatively small gaps between sequenced BAC clones. Using this method we were able to close a majority of the gaps (up to approximately 50 kb) identified during the finishing phase of chromosome-10 sequencing. This method represents a useful way to close clone gaps and thus to complete the entire rice genome.
• Yu, Y., Rambo, T., Currie, J., Saski, C., Kim, H. R., Collura, K., Thompson, S., Simmons, J., Yang, T. -., Nah, G., Patel, A. J., Thurmond, S., Henry, D., Oates, R., Palmer, M., Pries, G., Gibson, J., Anderson, H., Paradkar, M., , Crane, L., et al. (2003). In-depth view of structure, activity, and evolution of rice chromosome 10. Science, 300(5625), 1566-1569.
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  PMID: 12791992;Abstract: Rice is the world's most important food crop and a model for cereal research. At 430 megabases in size, its genome is the most compact of the cereals. We report the sequence of chromosome 10, the smallest of the 12 rice chromosomes (22.4 megabases), which contains 3471 genes. Chromosome 10 contains considerable heterochromatin with an enrichment of repetitive elements on 10S and an enrichment of expressed genes on 10L. Multiple insertions from organellar genomes were detected. Collinearity was apparent between rice chromosome 10 and sorghum and maize. Comparison between the draft and finished sequence demonstrates the importance of finished sequence.
• Chen, M., Presting, G., Barbazuk, W. B., Goicoechea, J. L., Blackmon, B., Fang, G., Kim, H., Frisch, D., Yeisoo, Y. u., Sun, S., Higingbottom, S., Phimphilai, J., Phimphilai, D., Thurmond, S., Gaudette, B., Ping, L. i., Liu, J., Hatfield, J., Main, D., , Farrar, K., et al. (2002). An integrated physical and genetic map of the rice genome. Plant Cell, 14(3), 537-545.
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  PMID: 11910002;PMCID: PMC150577;Abstract: Rice was chosen as a model organism for genome sequencing because of its economic importance, small genome size, and syntenic relationship with other cereal species. We have constructed a bacterial artificial chromosome fingerprint-based physical map of the rice genome to facilitate the whole-genome sequencing of rice. Most of the rice genome (∼90.6%) was anchored genetically by overgo hybridization, DNA gel blot hybridization, and in silico anchoring. Genome sequencing data also were integrated into the rice physical map. Comparison of the genetic and physical maps reveals that recombination is suppressed severely in centromeric regions as well as on the short arms of chromosomes 4 and 10. This integrated high-resolution physical map of the rice genome will greatly facilitate whole-genome sequencing by helping to identify a minimum tiling path of clones to sequence. Furthermore, the physical map will aid map-based cloning of agronomically important genes and will provide an important tool for the comparative analysis of grass genomes.
• Cheng, Z., Buell, C. R., Wing, R. A., & Jiang, J. (2002). Resolution of fluorescence in-situ hybridization mapping on rice mitotic prometaphase chromosomes, meiotic pachytene chromosomes and extended DNA fibers. Chromosome Research, 10(5), 379-387.
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  PMID: 12296520;Abstract: Fluorescence in-situ hybridization (FISH) is a quick and affordable approach to map DNA sequences to specific chromosomal regions. Although FISH is one of the most important physical mapping techniques, research on the resolution of FISH on different cytological targets is scarce in plants. In this study, we report the resolution of FISH mapping on mitotic prometaphase chromosomes, meiotic pachytene chromosomes and extended DNA fibers in rice. A majority of the FISH signals derived from bacterial artificial chromosome (BAC) clones separated by approximately 1 Mb of DNA cannot be resolved on mitotic prometaphase chromosomes. In contrast, the relative positions of closely linked or even partially overlapping BAC clones can be resolved on a euchromatic region of rice chromosome 10 at the early pachytene stage. The resolution of pachytene FISH is dependent on early or late pachytene stages and also on the location of the DNA probes in the euchromatic or heterochromatic regions. We calibrated the fiber-FISH technique in rice using seven sequenced BAC clones. The average DNA extension was 3.21 kb/μm among the seven BAC clones. Fiber-FISH results derived from a BAC contig that spanned 1 Mb DNA matched remarkably to the sequencing data, demonstrating the high resolution of this technique in cytological mapping.
• Coe, E., Cone, K., McMullen, M., Chen, S., Davis, G., Gardiner, J., Liscum, E., Polacco, M., Paterson, A., Sanchez-Villeda, H., Soderlund, C., & Wing, R. (2002). Access to the maize genome: An integrated physical and genetic map. Plant Physiology, 128(1), 9-12.
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  PMID: 11788746;PMCID: PMC1540197;
• Cone, K. C., McMullen, M. D., Bi, I. V., Davis, G. L., Yim, Y., Gardiner, J. M., Polacco, M. L., Sanchez-Villeda, H., Fang, Z., Schroeder, S. G., Havermann, S. A., Bowers, J. E., Paterson, A. H., Soderlund, C. A., Engler, F. W., Wing, R. A., & Coe Jr., E. H. (2002). Genetic, physical, and informatics resources for maize. On the road to an integrated map. Plant Physiology, 130(4), 1598-1605.
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  PMID: 12481043;PMCID: PMC1540265;
• Gemmill, R. M., Bolin, R., Albertsen, H., Tomkins, J. P., & Wing, R. A. (2002). Pulsed-field gel electrophoresis for long-range restriction mapping.. Current protocols in human genetics / editorial board, Jonathan L. Haines ... [et al.], Chapter 5, Unit5.1.
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  PMID: 18428330;Abstract: This unit describes procedures for generating long-range restriction maps of genomic DNA and for analysis of large insert clones. The basic protocol details restriction digestion of agarose-embedded DNA, PFGE separation, Southern transfer, and hybridization. Support protocols describe the preparation of high-molecular-weight genomic DNA samples in agarose blocks and in agarose microbeads, respectively. Additional support protocols describe the preparation of DNA size standards from l phage and two yeast species, Saccharomyces cerevisiae and Schizosaccharomyces pombe. An alternative method of preparing S. cerevisiae size standards using lithium dodecyl sulfate (LiDS) solubilization is provided. The final protocol details the preparation of BAC DNA suitable for digestion, mapping, and sequencing.
• Goff, S. A., Ricke, D., Lan, T., Presting, G., Wang, R., Dunn, M., Glazebrook, J., Sessions, A., Oeller, P., Varma, H., Hadley, D., Hutchison, D., Martin, C., Katagiri, F., Lange, B. M., Moughamer, T., Xia, Y., Budworth, P., Zhong, J., , Miguel, T., et al. (2002). A draft sequence of the rice genome (Oryza sativa L. ssp. japonica). Science, 296(5565), 92-100.
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  PMID: 11935018;Abstract: The genome of the japonica subspecies of rice, an important cereal and model monocot, was sequenced and assembled by whole-genome shotgun sequencing. The assembled sequence covers 93% of the 420-megabase genome. Gene predictions on the assembled sequence suggest that the genome contains 32,000 to 50,000 genes. Homologs of 98% of the known maize, wheat, and barley proteins are found in rice. Synteny and gene homology between rice and the other cereal genomes are extensive, whereas synteny with Arabidopsis is limited. Assignment of candidate rice orthologs to Arabidopsis genes is possible in many cases. The rice genome sequence provides a foundation for the improvement of cereals, our most important crops.
• Jiang, N., Bao, Z., Temnykh, S., Cheng, Z., Jiang, J., Wing, R. A., McCouch, S. R., & Wessier, S. R. (2002). Dasheng: A recently amplified nonautonomous long terminal repeat element that is a major component of pericentromeric regions in rice. Genetics, 161(3), 1293-1305.
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  PMID: 12136031;PMCID: PMC1462185;Abstract: A new and unusual family of LTR elements, Dasheng, has been discovered in the genome of Oryza sativa following database searches of ∼100 Mb of rice genomic sequence and 78 Mb of BAC-end sequence information. With all of the cis-elements but none of the coding domains normally associated with retrotransposons (e.g., gag, pol), Dasheng is a novel nonautonomous LTR element with high copy number. Over half of the ∼1000 Dasheng elements in the rice genome are full length (5.6-8.6 kb), and 60% are estimated to have amplified in the past 500,000 years. Using a modified AFLP technique called transposon display, 215 elements were mapped to all 12 rice chromosomes. Interestingly, more than half of the mapped elements are clustered in the heterochromatic regions around centromeres. The distribution pattern was further confirmed by FISH analysis. Despite clustering in heterochromatin, Dasheng elements are not nested, suggesting their potential value as molecular markers for these marker-poor regions. Taken together, Dasheng is one of the highest-copy-number LTR elements and one of the most recent elements to amplify in the rice genome.
• Martin, S. L., Blackmon, B. P., Rajagopalan, R., Houfek, T. D., Sceeles, R. G., Denn, S. O., Mitchell, T. K., Brown, D. E., Wing, R. A., & Dean, R. A. (2002). MagnaportheDB: A federated solution for integrating physical and genetic map data with BAC end derived sequences for the rice blast fungus Magnaporthe grisea. Nucleic Acids Research, 30(1), 121-124.
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  PMID: 11752272;PMCID: PMC99159;Abstract: We have created a federated database for genome studies of Magnaporthe grisea, the causal agent of rice blast disease, by integrating end sequence data from BAC clones, genetic marker data and BAC contig assembly data. A library of 9216 BAC clones providing >25-fold coverage of the entire genome was end sequenced and fingerprinted by HindIII digestion. The Image/FPC software package was then used to generate an assembly of 188 contigs covering >95% of the genome. The database contains the results of this assembly integrated with hybridization data of genetic markers to the BAC library. AceDB was used for the core database engine and a MySQL relational database, populated with numerical representations of BAC clones within FPC contigs, was used to create appropriately scaled images. The database is being used to facilitate sequencing efforts. The database also allows researchers mapping known genes or other sequences of interest, rapid and easy access to the fundamental organization of the M.grisea genome. This database, MagnaportheDB, can be accessed on the web at http://www.cals.ncsu.edu/fungalnomics/mgdatabase/int.htm.
• Roche, D., Conner, J. A., Budiman, M. A., Frisch, D., Wing, R., Hanna, W. W., & Ozias-Akins, P. (2002). Construction of BAC libraries from two apomictic grasses to study the microcolinearity of their apospory-specific genomic regions. Theoretical and Applied Genetics, 104(5), 804-812.
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  Abstract: We have constructed bacterial artificial chromosome (BAC) libraries from two grass species that reproduce by apospory, a form of gametophync apomixis. The library of an apomictic polyhaploid genotype (line MS228-20, with a 2C genome size of approximately 4,500 Mbp) derived from a cross between the obligate apomict, Pennisetum squamulatum, and pearl millet (P. glaucum) comprises 118,272 clones with an average insert size of 82 kb. The library of buffelgrass (Cenchrus ciliaris, apomictic line B-12-9, with a 2C genome size of approximately 3,000 Mbp) contains 68,736 clones with an average insert size of 109 kb. Based on the genome sizes of these two lines and correcting for the number for false-positive and organellar clones, library coverages were found to be 3.7 and 4.8 haploid genome equivalents for MS 228-20 and B12-9, respectively. Both libraries were screened by hybridization with six SCARs (sequence-characterized amplified regions), whose tight linkage in a single apospory-specific genomic region had been previously demonstrated in both species. Analysis of these BAC clones indicated that some of the SCAR markers are actually amplifying duplicated regions linked in coupling in both genomes and that restriction enzyme mapping will be necessary to sort out the duplications.
• Tomkins, J. P., Davis, G., Main, D., Yim, Y., Duru, N., Musket, T., Goicoechea, J. L., Frisch, D. A., Coe Jr., E. H., & Wing, R. A. (2002). Construction and characterization of a deep-coverage bacterial artificial chromosome library for maize. Crop Science, 42(3), 928-933.
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  Abstract: Modern cultivated maize (Zea mays L.)is one of the primary agronomic crops in the USA with an estimated genome size of 2500 megabases (Mb). To develop the resources for positional cloning and structural genomics in maize, we constructed a bacterial artificial chromosome (BAC) library for the inbred line B73 using the cloning enzyme Hind III. The library contains 247 680 clones (645 384-well plates). A random sampling of 697 clones indicated an average insert size of 136 kilobase (kb) (range = 42 to 379 kb) and 0.4% empty vectors. Screening the colony filters for chloroplast DNA content indicated an exceptionally low 0.18% contamination with chloroplast DNA. Thus, the library provides 13.5 haploid genome equivalents allowing >99% probability of recovering any specific sequence of interest. High-density filters were gridded robotically using a Genetix Q-BOT (Hampshire, UK) in a 4 by 4 double-spotted array on 22.5cm2 filters. Partial screening (6x coverage) of the library with 20 single copy probes identified an average 7.1 positive signals per probe, with a range of 3 to 15 positive signals per probe. To evaluate the utility of the library for sequence tagged connector (STC) analysis, 768 BAC clones were end sequenced in both forward and reverse directions giving a total of 1415 successful reads. End sequences were queried against SWISS-PROT, Genbank NR, MIPS Arabidopsis, maize genomic sequence dbGSS, and maize cDNA database dbEST. Results in spreadsheet format from these searches is publicly available at the CUGI website (www.genome.clemson.edu/projects/stc/maize/ZMMBBb/).
• Tomkins, J. P., Luo, M., Fang, G. C., Main, D., Goicoechea, J. L., Atkins, M., Frisch, D. A., Page, R. E., Guzmán-Novoa, E., Yu, Y., Hunt, G., & Wing, R. A. (2002). New genomic resources for the honey bee (Apis mellifera L.): Development of a deep-coverage BAC library and a preliminary STC database. Genetics and Molecular Research, 1(4), 306-316.
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  PMID: 14963821;Abstract: We have constructed a bacterial artificial chromosome (BAC) library for a European honey bee strain using the cloning enzyme HindIII in order to develop resources for structural genomics research. The library contains 36,864 clones (ninety-six 384-well plates). A random sampling of 247 clones indicated an average insert size of 113 kb (range = 27 to 213 kb) and 2% empty vectors. Based on an estimated genome size of 270 Mb, this library provides approximately 15 haploid genome equivalents, allowing >99% probability of recovering any specific sequence of interest. High-density colony filters were gridded robotically using a Genetix Q-BOT in a 4 × 4 double-spotted array on 22.5-cm2 filters. Screening of the library with four mapped honey bee genomic clones and two bee cDNA probes identified an average of 21 positive signals per probe, with a range of 7-38 positive signals per probe. An additional screening was performed with nine aphid gene fragments and one Drosophila gene fragment resulting in seven of the nine aphid probes and the Drosophila probe producing positive signals with a range of 1 to 122 positive signals per probe (average of 45). To evaluate the utility of the library for sequence tagged connector analysis, 1152 BAC clones were end sequenced in both forward and reverse directions, giving a total of 2061 successful reads of high quality. End sequences were queried against SWISS-PROT, insect genomic sequence GSS, insect EST, and insect transposable element databases. Results in spreadsheet format from these searches are publicly available at the Clemson University Genomics Institute (CUGI) website in a searchable format (http://www.genome.clemson.edu/projects/stc/bee/AM_Ba/).
• Wechter, W. P., Begum, D., Presting, G., Kim, J. J., Wing, R. A., & Kluepfel, D. A. (2002). Physical mapping, BAC-end sequence analysis, and marker tagging of the soilborne nematicidal bacterium, Pseudomonas synxantha BG33R. OMICS A Journal of Integrative Biology, 6(1), 11-21.
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  PMID: 11881828;Abstract: A bacterial artificial chromosome (BAC) library was constructed for the genome of the rhizosphere-inhabiting fluorescent pseudomonad Pseudomonas synxantha BG33R. Three thousand BAC clones with an average insert size of 140 kbp and representing a 70-fold genomic coverage were generated and arrayed onto nylon membranes. EcoRI fingerprint analysis of 986 BAC clones generated 23 contigs and 75 singletons. Hybridization analysis allowed us to order the 23 contigs and condense them into a single contig, yielding an estimated genome size of 5.1 Mb for P. synxantha BG33R. A minimum-tile path of 47 BACs was generated and end-sequenced. The genetic loci involved in ring nematode egg-kill factor production in BG33R Tn5 mutants, 246 (vgrG homolog), 1122 (sensor kinase homolog), 1233 (UDP-galactose epimerase homolog), 1397 (ferrisiderophore receptor homolog), and 1917 (ribosomal subunit protein homolog), have been mapped onto the minimum-tile BAC library. Two of the genetic regions that flank Tn5 insertions in BG33R egg-kill-negative mutants 1233 and 1397 are separated by a single BAC clone. Fragments isolated by ligation-mediated PCR of the Tn5 mutagenized regions of 29 randomly selected, non-egg-kill-related, insertion mutants have been anchored onto the ordered physical map of P. synxantha.
• Wei, F., Wing, R. A., & Wise, R. P. (2002). Genome dynamics and evolution of the Mla (powdery mildew) resistance locus in barley. Plant Cell, 14(8), 1903-1917.
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  PMID: 12172030;PMCID: PMC151473;Abstract: Genes that confer defense against pathogens often are clustered in the genome and evolve via diverse mechanisms. To evaluate the organization and content of a major defense gene complex in cereals, we determined the complete sequence of a 261-kb BAC contig from barley cv Morex that spans the Mla (powdery mildew) resistance locus. Among the 32 predicted genes on this contig, 15 are associated with plant defense responses; 6 of these are associated with defense responses to powdery mildew disease but function in different signaling pathways. The Mla region is organized as three gene-rich islands separated by two nested complexes of transposable elements and a 45-kb gene-poor region. A heterochromatic-like region is positioned directly proximal to Mla and is composed of a gene-poor core with 17 families of diverse tandem repeats that overlap a hypermethylated, but transcriptionally active, gene-dense island. Paleontology analysis of long terminal repeat retrotransposons indicates that the present Mla region evolved over a period of >7 million years through a variety of duplication, inversion, and transposon-insertion events. Sequence-based recombination estimates indicate that R genes positioned adjacent to nested long terminal repeat retrotransposons, such as Mla, do not favor recombination as a means of diversification. We present a model for the evolution of the Mla region that encompasses several emerging features of large cereal genomes.
• Zhao, Q., Zhang, Y., Cheng, Z., Chen, M., Wang, S., Feng, Q., Huang, Y., Ying, L. i., Tang, Y., Zhou, B., Chen, Z., Shuliang, Y. u., Zhu, J., Xin, H. u., Jie, M. u., Ying, K., Hao, P., Zhang, L., Yiqi, L. u., , Zhang, L. S., et al. (2002). A fine physical map of the rice chromosome 4. Genome Research, 12(5), 817-823.
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  PMID: 11997348;PMCID: PMC186569;Abstract: As part of an international effort to completely sequence the rice genome, we have produced a fine bacterial artificial chromosome (BAC)-based physical map of the Oryza sativa japonica Nipponbare chromosome 4 through an integration of 114 sequenced BAC clones from a taxonomically related subspecies O. sativa indica Guangluai 4 and 182 RFLP and 407 expressed sequence tag (EST) markers with the fingerprinted data of the Nipponbare genome. The map consists of II contigs with a total length of 34.5 Mb covering 94% of the estimated chromosome size (36.8 Mb). BAC clones corresponding to telomeres, as well as to the centromere position, were determined by BAC-pachytene chromosome fluorescence in situ hybridization (FISH). This gave rise to an estimated length ratio of 5.13 for the long arm and 2.9 for the short arm (on the basis of the physical map), which indicates that the short arm is a highly condensed one. The FISH analysis and physical mapping also showed that the short arm and the pericentromeric region of the long arm are rich in heterochromatin, which occupied 45% of the chromosome, indicating that this chromosome is likely very difficult to sequence. To our knowledge, this map provides the first example of a rapid and reliable physical mapping on the basis of the integration of the data from two taxonomically related subspecies.
• Cheng, Z., Buell, C. R., Wing, R. A., Minghong, G. u., & Jiang, J. (2001). Toward a cytological characterization of the rice genome. Genome Research, 11(12), 2133-2141.
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  PMID: 11731505;PMCID: PMC311230;Abstract: Rice (Oryza sativa L.) will be the first major crop, as well as the first monocot plant species, to be completely sequenced. Integration of DNA sequence-based maps with cytological maps will be essential to fully characterize the rice genome. We have isolated a set of 24 chromosomal arm-specific bacterial artificial chromosomes to facilitate rice chromosome identification. A standardized rice karyotype was constructed using meiotic pachytene chromosomes of O. sativa spp. japonica rice var. Nipponbare. This karyotype is anchored by centromere-specific and chromosomal arm-specific cytological landmarks and is fully integrated with the most saturated rice genetic linkage maps in which Nipponbare was used as one of the mapping parents. An ideogram depicting the distribution of heterochromatin in the rice genome was developed based on the patterns of 4′,6-diamidino-2-phenylindole staining of the Nipponbare pachytene chromosomes. The majority of the heterochromatin is distributed in the pericentric regions with some rice chromosomes containing a significantly higher proportion of heterochromatin than other chromosomes. We showed that pachytene chromosome-based fluorescence in situ hybridization analysis is the most effective approach to integrate DNA sequences with euchromatic and heterochromatic features.
• Cheng, Z., Presting, G. G., Buell, C. R., Wing, R. A., & Jiang, J. (2001). High-resolution pachytene chromosome mapping of bacterial artificial chromosomes anchored by genetic markers reveals the centromere location and the distribution of genetic recombination along chromosome 10 of rice. Genetics, 157(4), 1749-1757.
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  PMID: 11290728;PMCID: PMC1461616;Abstract: Large-scale physical mapping has been a major challenge for plant geneticists due to the lack of techniques that are widely affordable and can be applied to different species. Here we present a physical map of rice chromosome 10 developed by fluorescence in situ hybridization (FISH) mapping of bacterial artificial chromosome (BAC) clones on meiotic pachytene chromosomes. This physical map is fully integrated with a genetic linkage map of rice chromosome 10 because each BAC clone is anchored by a genetically mapped restriction fragment length polymorphism marker. The pachytene chromosome-based FISH mapping shows a superior resolving power compared to the somatic metaphase chromosome-based methods. The telomere-centromere orientation of DNA clones separated by 40 kb can be resolved on early pachytene chromosomes. Genetic recombination is generally evenly distributed along rice chromosome 10. However, the highly heterochromatic short arm shows a lower recombination frequency than the largely euchromatic long arm. Suppression of recombination was found in the centromeric region, but the affected region is far smaller than those reported in wheat and barley. Our FISH mapping effort also revealed the precise genetic position of the centromere on chromosome 10.
• Draye, X., Lin, Y. -., Qian, X. -., Bowers, J. E., Burow, G. B., Morrell, P. L., Peterson, D. G., Presting, G. G., Ren, S. -., Wing, R. A., & Paterson, A. H. (2001). Toward integration of comparative genetic, physical, diversity, and cytomolecular maps for grasses and grains, using the sorghum genome as a foundation. Plant Physiology, 125(3), 1325-1341.
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  PMID: 11244113;PMCID: PMC65612;Abstract: The small genome of sorghum (Sorghum bicolor L. Moench.) provides an important template for study of closely related large-genome crops such as maize (Zea mays) and sugarcane (Saccharum spp.), and is a logical complement to distantly related rice (Oryza sativa) as a "grass genome model." Using a high-density RFLP map as a framework, a robust physical map of sorghum is being assembled by integrating hybridization and fingerprint data with comparative data from related taxa such as rice and using new methods to resolve genomic duplications into locus-specific groups. By taking advantage of allelic variation revealed by heterologous probes, the positions of corresponding loci on the wheat (Triticum aestivum), rice, maize, sugarcane, and Arabidopsis genomes are being interpolated on the sorghum physical map. Bacterial artificial chromosomes for the small genome of rice are shown to close several gaps in the sorghum contigs; the emerging rice physical map and assembled sequence will further accelerate progress. An important motivation for developing genomic tools is to relate molecular level variation to phenotypic diversity. "Diversity maps," which depict the levels and patterns of variation in different gene pools, shed light on relationships of allelic diversity with chromosome organization, and suggest possible locations of genomic regions that are under selection due to major gene effects (some of which may be revealed by quantitative trait locus mapping). Both physical maps and diversity maps suggest interesting features that may be integrally related to the chromosomal context of DNA - progress in cytology promises to provide a means to elucidate such relationships. We seek to provide a detailed picture of the structure, function, and evolution of the genome of sorghum and its relatives, together with molecular tools such as locus-specific sequence-tagged site DNA markers and bacterial artificial chromosome contigs that will have enduring value for many aspects of genome analysis.
• Luo, M., Wang, Y., Frisch, D., Joobeur, T., Wing, R. A., & Dean, R. A. (2001). Melon bacterial artificial chromosome (BAC) library construction using improved methods and identification of clones linked to the locus conferring resistance to melon Fusarium wilt (Fom-2). Genome, 44(2), 154-162.
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  PMID: 11341724;Abstract: Utilizing improved methods, two bacterial artificial chromosome (BAC) libraries were constructed for the multidisease-resistant line of melon MR-1. The HindIII library consists of 177 microtiter plates in a 384-well format, while the EcoRI library consists of 222 microtiter plates. Approximately 95.6% of the HindIII library clones contain nuclear DNA inserts with an average size of 118 kb, providing a coverage of 15.4 genome equivalents. Similarly, 96% of the EcoRI library clones contain nuclear DNA inserts with an average size of 114 kb, providing a coverage of 18.7 genome equivalents. Both libraries were evaluated for contamination with high-copy vector, empty pIndigoBac536 vector, and organellar DNA sequences. High-density filters were screened with two genetic markers FM and AM that co-segregate with Fom-2, a gene conferring resistance to races 0 and 1 of Fusarium wilt. Fourteen and 18 candidate BAC clones were identified for the FM and AM probes, respectively, from the HindIII library, while 34 were identified for the AM probe from filters A, B, and C of the EcoRI library.
• Mao, L., Begum, D., Goff, S. A., & Wing, R. A. (2001). Sequence and analysis of the tomato JOINTLESS locus. Plant Physiology, 126(3), 1331-1340.
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  PMID: 11457984;PMCID: PMC116490;Abstract: A 119-kb bacterial artificial chromosome from the JOINTLESS locus on the tomato (Lycopersicon esculentum) chromosome 11 contained 15 putative genes. Repetitive sequences in this region include one copia-like LTR retrotransposon, 13 simple sequence repeats, three copies of a novel type III foldback transposon, and four putative short DNA repeats. Database searches showed that the foldback transposon and the short DNA repeats seemed to be associated preferably with genes. The predicted tomato genes were compared with the complete Arabidopsis genome. Eleven out of 15 tomato open reading frames were found to be colinear with segments on five Arabidopsis bacterial artificial chromosome/P1-derived artificial chromosome clones. The synteny patterns, however, did not reveal duplicated segments in Arabidopsis, where over half of the genome is duplicated. Our analysis indicated that the microsynteny between the tomato and Arabidopsis genomes was still conserved at a very small scale but was complicated by the large number of gene families in the Arabidopsis genome.
• Martinez, B., Tomkins, J., Wackett, L. P., Wing, R., & Sadowsky, M. J. (2001). Complete nucleotide sequence and organization of the atrazine catabolic plasmid pADP-1 from Pseudomonas sp. strain ADP. Journal of Bacteriology, 183(19), 5684-5697.
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  PMID: 11544232;PMCID: PMC95461;Abstract: The complete 108,845-nucleotide sequence of catabolic plasmid pADP-1 from Pseudomonas sp. strain ADP was determined. Plasmid pADP-1 was previously shown to encode AtzA, AtzB, and AtzC, which catalyze the sequential hydrolytic removal of s-triazine ring substituents from the herbicide atrazine to yield cyanuric acid. Computational analyses indicated that pADP-1 encodes 104 putative open reading frames (ORFs), which are predicted to function in catabolism, transposition, and plasmid maintenance, transfer, and replication. Regions encoding transfer and replication functions of pADP-1 had 80 to 100% amino acid sequence identity to pR751, an IncPβ plasmid previously isolated from Enterobacter aerogenes. pADP-1 was shown to contain a functional mercury resistance operon with 99% identity to Tn5053. Complete copies of transposases with 99% amino acid sequence identity to TnpA from IS1071 and TnpA from Pseudomonas pseudoalcaligenes were identified and flank each of the atzA, atzB, and atzC genes, forming structures resembling nested catabolic transposons. Functional analyses identified three new catabolic genes, atzD, atzE, and atzF, which participate in atrazine catabolism. Crude extracts from Escherichia coli expressing AtzD hydrolyzed cyanuric acid to biuret. AtzD showed 58% amino acid sequence identity to TrzD, a cyanuric acid amidohydrolase, from Pseudomonas sp. strain NRRLB-12227. Two other genes encoding the further catabolism of cyanuric acid, atzE and atzF, reside in a contiguous cluster adjacent to a potential LysR-type transcriptional regulator. E. coli strains bearing atzE and atzF were shown to encode a biuret hydrolase and allophanate hydrolase, respectively, atzDEF are cotranscribed. AtzE and AtzF are members of a common amidase protein family. These data reveal the complete structure of a catabolic plasmid and show that the atrazine catabolic genes are dispersed on three disparate regions of the plasmid. These results begin to provide insight into how plasmids are structured, and thus evolve, to encode the catabolism of compounds recently added to the biosphere.
• Presting, G. G., Budiman, M. A., Wood, T., Yeisoo, Y. u., Kim, H., Goicoechea, J. L., Fang, E., Blackman, B., Jiang, J., Woo, S., Dean, R. A., Frisch, D., & Wing, R. A. (2001). A framework for sequencing the rice genome. Novartis Foundation Symposium, 236, 13-27.
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  PMID: 11387975;Abstract: Rice is an important food crop and a model plant for other cereal genomes. The Clemson University Genomics Institute framework project, begun two years ago in anticipation of the now ongoing international effort to sequence the rice genome, is nearing completion. Two bacterial artificial chromosome (BAC) libraries have been constructed from the Oryza sativa cultivar Nipponbare. Over 100 000 BAC end sequences have been generated from these libraries and, at a current total of 28 Mbp, represent 6.5% of the total rice genome sequence. This sequence information has allowed us to draw first conclusions about unique and redundant rice genomic sequences. In addition, more than 60 000 clones (19 genome equivalents) have been successfully fingerprinted and assembled into contigs using FPC software. Many of these contigs have been anchored to the rice chromosomes using a variety of techniques. Hybridization experiments have shown these contigs to be very robust. Contig assembly and hybridization experiments have revealed some surprising insights into the organization of the rice genome, which will have significant repercussions for the sequencing effort. Integration of BAC end sequence data with anchored contig information has provided unexpected revelations on sequence organization at the chromosomal level.
• Tomkins, J. P., Peterson, D. G., Yang, T. J., Main, D., Ablett, E. F., Henry, R. J., Lee, L. S., Holton, T. A., Waters, D., & Wing, R. A. (2001). Grape (Vitis vinifera L.) BAC library construction, preliminary STC analysis, and identification of clones associated with flavonoid and stilbene biosynthesis. American Journal of Enology and Viticulture, 52(4), 287-291.
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  Abstract: We have constructed a grape BAC library using the cultivar Syrah. The library contains 55,296 clones stored in 144 384-well microtiter plates. A random sampling of 381 BACs indicated an average insert size of 144 kb with a range of 30 to 355 kb, and less than 4% of the clones do not contain inserts. Eighty-nine percent of BAC clones in the library have an average insert size greater than 100 kb. Based on a genome size of 483 Mb, library coverage is 16.5 haploid genome equivalents. Screening the BAC library colony filters with cpDNA sequences showed that contamination of the genomic library with chloroplast clones was low (1.5%). Library screening of an 11X coverage (2 BAC colony filters) with 12 cDNA probes corresponding to flavonoid and stilbene biosynthesis genes resulted in an average of 13 hits per probe (range = 1 to 27). To gain a glimpse into the grape genome and evaluate the library for sequence-tagged connector (STC) development, 768 BAC clones were end sequenced in both forward and reverse directions. The STCs were queried against the SWISS-PROT database and significant hits were sorted according to putative function.
• Tomkins, J. P., Peterson, D. G., Yang, T. J., Main, D., Wilkins, T. A., Paterson, A. H., & Wing, R. A. (2001). Development of genomic resources for cotton (Gossypium hirsutum L.): BAC library construction, preliminary STC analysis, and identification of clones associated with fiber development. Molecular Breeding, 8(3), 255-261.
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  Abstract: We have constructed a cotton (Gossypium hirsutum L.) BAC library using the elite Acala-type cultivar Maxxa. The BAC library contains 129 024 clones comprising 8.3 haploid genome equivalents based on a AD genome size of 2118 Mb. A random sampling of 435 BACs indicated an average insert length of 137 kb with a range of 80 to 275 kb, and 1.4% of the BACs do not contain inserts. Of the BAC clones in the sample 99% had an average insert length equal to or greater than 100 kb. Contamination of the genomic library with chloroplast clones was low (1.5%). To evaluate the library for genome representation and to identify clones associated with cotton fiber development, six BAC colony filters (7.2x representation) were screened with PCR-generated gene-specific probes for the GhMYB class of transcription factor genes giving an average of 12 positive signals per probe (range 6-20). To gain a glimpse into the cotton genome and evaluate the library for sequence-tagged connector (STC) development, 1536 BAC clones were end sequenced in both forward and reverse direction. The STCs were queried against the SwissProt and MIPS Arabidopsis databases and significant hits were sorted according to putative function.
• Tomkins, J. P., Wood, T. C., Barnes, L. S., Westman, A., & Wing, R. A. (2001). Evaluation of genetic variation in the daylily (Hemerocallis spp.) using AFLP markers. Theoretical and Applied Genetics, 102(4), 489-496.
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  Abstract: The daylily (Hemerocallis spp.) is one of the most economically important ornamental plant species in commerce. Interestingly, it is also one of the most heavily bred crops during the past 60 years. Since the American Hemerocallis Society began acting as the official registry of daylily cultivars in 1947, more than 40 000 registrations have been processed. In order to determine the effects of intensive breeding on cultivar development, and to study relationships among different species, genetic variation in the daylily was estimated using AFLP markers. Nineteen primary genotypes (species and early cultivars) and 100 modern cultivars from different time periods were evaluated using 152 unambiguous bands (average 79% polymorphism rate) derived from three AFLP primer combinations. Overall, pairwise similarity estimates between entries ranged between 0.618 and 0.926 (average=0.800). When comparing cultivar groups from different time periods (1940-1998), genetic similarity was initially increased, compared to the primary diploid genotypes, remained constant from 1940 to 1980, and then steadily increased as breeding efforts intensified and hybridizers began focusing on a limited tetraploid germplasm pool derived by colchicine conversion. Among modern (1991-1998) daylily cultivars, genetic similarity has increased by approximately 10% compared to the primary genotypes. These data were also used to evaluate recent taxonomic classifications among daylily species which, with a few minor exceptions, were generally supported by the AFLP data.
• Tomkins, J. P., Wood, T. C., Stacey, M. G., Loh, J. T., Judd, A., Goicoechea, J. L., Stacey, G., Sadowsky, M. J., & Wing, R. A. (2001). A marker-dense physical map of the Bradyrhizobium japonicum genome. Genome Research, 11(8), 1434-1440.
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  PMID: 11483585;PMCID: PMC311109;Abstract: Bacterial artificial chromosome (BAC) clones are effective mapping and sequencing reagents for use with a wide variety of small and large genomes. This report describes the development of a physical framework for the genome of Bradyrhizobium japonicum, the nitrogen-fixing symbiont of soybean. A BAC library for B. japonicum was constructed that provides a 77-fold genome coverage based on an estimated genome size of 8.7 Mb. The library contains 4608 clones with an average insert size of 146 kb. To generate a physical map, the entire library was fingerprinted with HindIII, and the fingerprinted clones were assembled into contigs using the Fingerprint Contig software (FPC; Sanger Centre, UK). The FPC analysis placed 3410 clones in six large contigs. The ends of 1152 BAC inserts were sequenced to generate a sequence-tagged connector (STC) framework. To join and orient the contigs, high-density BAC colony filters were probed with 41 known gene probes and 17 end sequences from contig boundaries. STC sequences were searched against the public databases using FASTA and BLASTX algorithms. Query results allowed the identification of 113 high probability matches with putative functional identities that were placed on the physical map. Combined with the hybridization data, a high-resolution physical map with 194 positioned markers represented in two large contigs was developed, providing a marker every 45 kb. Of these markers, 177 are known or putative B. japonicum genes. Additionally, 1338 significant BLASTX results (E < 10-4) were manually sorted by function to produce a functionally categorized database of relevant B. japonicum STC sequences that can also be traced to specific locations in the physical map.
• Wang, W., Zhai, W., Luo, M., Jiang, G., Chen, X., Li, X., Wing, R. A., & Zhu, L. (2001). Chromosome landing at the bacterial blight resistance gene Xa4 locus using a deep coverage rice BAC library. Molecular and General Genetics, 265(1), 118-125.
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  PMID: 11370857;Abstract: Xa4 is a dominantly inherited rice gene that confers resistance to Philippine race 1 of the bacterial blight pathogen Xanthomonas oryzae pv. oryzae in rice. In order to isolate the gene by positional cloning, a bacterial artificial chromosome (BAC) library was constructed from genomic DNA isolated from an Xa4-harboring accession, IRBB56. The library contains 55,296 clones with an average insert size of 132 kb, providing 14 rice genome equivalents. Three DNA markers closely linked to Xa4 were used to screen the library. The marker RS13, a resistance gene analogue that co-segregates with Xa4, identified 18 clones, of which four and six, respectively, were simultaneously detected by the other two markers, G181 and L1044. Fingerprinting and Southern analysis indicated that these clones overlapped and define an interval spanning 420 kb. In an F2 population derived from an indica variety, IR24, and its Xa4-containing near isogenic line (NIL), IRBB4, the susceptible plants were screened in order to map the Xa4 gene genetically and physically. Out of 24 insert ends isolated from the BACs in the contig, three revealed polymorphisms between IR24 and IRBB4. Two insert ends, 56M22F and 26D24R, flanked Xa4 on each side. Based on the overlap of the BACs, six overlapping clones were considered to include the Xa4 allele, one of which, 106P13, was chosen for further investigation.
• Yang, Z. -., Ye, X. -., Choi, S., Molina, J., Moonan, F., Wing, R. A., Roose, M. L., & Mirkov, T. E. (2001). Construction of a 1.2-Mb contig including the citrus tristeza virus resistance gene locus using a bacterial artificial chromosome library of Poncirus trifoliata (L.) Raf.. Genome, 44(3), 382-393.
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  PMID: 11444697;Abstract: The citrus tristeza virus resistance gene (Ctv) is a single dominant gene in Poncirus trifoliata, a sexually compatible relative of citrus. To clone this gene, a bacterial artificial chromosome (BAC) library has been constructed from an individual plant that was homozygous for Ctv. This library contains 45 696 clones with an average insert size of 80 kb, corresponding to 9.6 genome equivalents. Screening of the BAC library with five chloroplast DNA probes indicated that 0.58% of the BAC clones contained chloroplast-derived inserts. The chromosome walk across the Ctv locus was initiated using three closely linked genetic markers: C19, AD8, and Z16. The walk has been completed and a contig of ca. 1.2 Mb was constructed. Based on new data, the genetic map in the Ctv region was revised, with Ctv being located between AD8-Z16 and C19 at distances of 1.2 and 0.6 cM, respectively. Utilizing DNA fragments isolated from the contig as RFLP markers, the Ctv locus was further mapped to a region of ca. 300 kb. This contig contains several putative disease-resistance genes similar to the rice Xa21 gene, the tomato Cf-2 gene, and the Arabidopsis thaliana RPS2 gene. This library will therefore allow cloning of Ctv and other putative disease-resistance genes.
• Budiman, M. A., Mao, L., Wood, T. C., & Wing, R. A. (2000). A deep-coverage tomato BAC library and prospects toward development of an STC framework for genome sequencing. Genome Research, 10(1), 129-136.
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  PMID: 10645957;PMCID: PMC310507;Abstract: Recently a new strategy using BAC end sequences as sequence-tagged connectors (STCs] was proposed for whole-genome sequencing projects. In this study, we present the construction and detailed characterization of a 15.0 haploid genome equivalent BAC library for the cultivated tomato, Lycopersicon esculentum cv. Heinz 1706. The library contains 129,024 clones with an average insert size of 117.5 kb and a chloroplast content of 1.11%. BAC end sequences from 1490 ends were generated and analyzed as a preliminary evaluation for using this library to develop an STC framework to sequence the tomato genome. A total of 1205 BAC end sequences (80.9%) were obtained, with an average length of 360 high-quality bases, and were searched against the GenBank database. Using a cutoff expectation value of < 10-6, and combining the results from BLASTN, BLASTX, and TBLASTX searches, 24.3% of the BAC end sequences were similar to known sequences, of which almost half (48.7%) share sequence similarities to retrotransposons and 7% to known genes. Some of the transposable element sequences were the first reported in tomato, such as sequences similar to maize transposon Activator (Ac) ORF and tobacco pararetrovirus-like sequences. Interestingly, there were no BAC end sequences similar to the highly repeated TGRI and TGRII elements. However, the majority (70.3%) of STCs did not share significant sequence similarities to any sequences in GenBank at either the DNA or predicted protein levels, indicating that a large portion of the tomato genome is still unknown. Our data demonstrate that this BAC library is suitable for developing an STC database to sequence the tomato genome. The advantages of developing an STC framework for whole-genome sequencing of tomato are discussed.
• Choi, S., Begum, D., Koshinsky, H., Ow, D. W., & Wing, R. A. (2000). A new approach for the identification and cloning of genes: the pBACwich system using Cre/lox site-specific recombination.. Nucleic acids research, 28(7), E19.
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  PMID: 10710436;PMCID: PMC102802;Abstract: With current plant transformation methods ( Agrobacterium, biolistics and protoplast fusion), insertion of DNA into the genome occurs randomly and in many instances at multiple sites. Associated position effects, copy number differences and multigene interactions can make gene expression experiments difficult to interpret and plant phenotypes less predictable. An alternative approach to random integration of large DNA fragments into plants is to utilize one of several site-specific recombination (SSR) systems, such as Cre/ lox. Cre has been shown in numerous instances to mediate lox site-specific recombination in animal and plant cells. By incorporating the Cre/ lox SSR system into a bacterial artificial chromosome (BAC) vector, a more precise evaluation of large DNA inserts for genetic complementation should be possible. Site-specific insertion of DNA into predefined sites in the genome may eliminate unwanted 'position effects' caused by the random integration of exogenously introduced DNA. In an effort to make the Cre/ lox system an effective tool for site-directed integration of large DNAs, we constructed and tested a new vector potentially capable of integrating large DNA inserts into plant and fungal genomes. In this study, we present the construction of a new BAC vector, pBACwich, for the system and the use of this vector to demonstrate SSR of large DNA inserts (up to 230 kb) into plant and fungal genomes.
• Druka, A., Kudrna, D., Han, F., Kilian, A., Steffenson, B., Frisch, D., Tomkins, J., Wing, R., & Kleinhofs, A. (2000). Physical mapping of the barley stem rust resistance genie rpg4. Molecular and General Genetics, 264(3), 283-290.
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  PMID: 11085268;Abstract: The barley stem rust resistance gene rpg4 was physically and genetically localized on two overlapping BAC clones covering an estimated 300-kb region of the long arm of barley chromosome 7(5H). Initially, our target was mapped within a 6.0-cM region between the previously described flanking markers MWG740 and ABG391. This region was then saturated by integrating new markers from several existing barley and rice maps and by using BAC libraries of barley cv. Morex and rice cv. Nipponbare. Physical/genetic distances in the vicinity of rpg4 were found to be 1.0 Mb/cM, which is lower than the average for barley (4 Mb/cM) and lower than that determined by translocation breakpoint mapping (1.8 Mb/cM). Synteny at high resolution levels has been established between the region of barley chromosome 7(5H) containing the rpg4 locus and the subtelomeric region of rice chromosome 3 between markers S16474 and E10757. This 1.7-cM segment of the rice genome was covered by two overlapping BAC clones, about 250 kb of total length. In barley the markers S16474 and E10757 genetically delimit rpg4, lying 0.6 cM distal and 0.4 cM proximal to the locus, respectively.
• Lin, Y. R., Draye, X., Qian, X., Ren, S., Zhu, L. H., Tomkins, J., Wing, R. A., Li, Z., & Paterson, A. H. (2000). Locus-specific contig assembly in highly-duplicated genomes, using the BAC-RF method.. Nucleic acids research, 28(7), E23.
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  PMID: 10710440;PMCID: PMC102806;Abstract: Polyploidy, the presence of multiple sets of chromosomes that are similar but not identical, complicates both chromosome walking and assembly of sequence-ready contigs for many plant taxa including a large number of economically-significant crops. Traditional 'dot-blot hybridization' or PCR-based assays for identifying BAC clones corresponding to a mapped DNA landmark usually do not provide sufficient information to distinguish between allelic and non-allelic loci. A restriction fragment matching method using pools of BAC DNA in combination with dot-blots reveals the locus specificity of individual BACs that correspond to multi-locus DNA probes, in a manner that can efficiently be applied on a large scale. This approach also provides an alternative means of mapping DNA loci that exploits many advantages of 'radiation hybrid' mapping in taxa for which such hybrids are not available. The BAC-RF method is a practical and reliable approach for using high-density RFLP maps to anchor sequence-ready BAC contigs in highly-duplicated genomes, provides an alternative to high-density robotic gridding for screening BAC libraries when the necessary equipment is not available, and permits the expedient isolation of individual members of multigene or repetitive DNA families for a wide range of genetic and evolutionary investigations.
• Mao, L., Begum, D., Chuang, H., Budiman, M. A., Szymkowiak, E. J., Irish, E. E., & Wing, R. A. (2000). JOINTLESS is a MADS-box gene controlling tomato flower abscission zone development. Nature, 406(6798), 910-913.
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  PMID: 10972295;Abstract: Abscission is a universal and dynamic process in plants whereby organs such as leaves, flowers and fruit are shed, both during normal development, and in response to tissue damage and stress. Shedding occurs by separation of cells in anatomically distinct regions of the plant, called abscission zones (AZs). During abscission, the plant hormone ethylene stimulates cells to produce enzymes that degrade the middle lamella between cells in the AZ. The physiology and regulation of abscission at fully developed AZs is well known, but the molecular biology underlying their development is not. Here we report the first isolation of a gene directly involved in the development of a functional plant AZ. Tomato plants with the jointless mutation4 fail to develop AZs on their pedicels and so abscission of flowers or fruit does not occur normally. We identify JOINTLESS as a new MADS-box gene in a distinct phylogenetic clade separate from those functioning in floral organs. We propose that a deletion in JOINTLESS accounts for the failure of activation of pedicel AZ development in jointless tomato plants.
• Mao, L., Wood, T. C., Yeisoo, Y. u., Budiman, M. A., Tomkins, J., Woo, S., Sasinowski, M., Presting, G., Frisch, D., Goff, S., Dean, R. A., & Wing, R. A. (2000). Rice Transposable elements: A survey of 73,000 sequence-tagged-connectors. Genome Research, 10(7), 982-990.
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  PMID: 10899147;PMCID: PMC310901;Abstract: As part of an international effort to sequence the rice genome, the Clemson University Genomics Institute is developing a sequence-tagged-connector (STC) framework. This framework includes the generation of deep-coverage BAC libraries from O. sativa ssp. japonica c.v. Nipponbare and the sequencing of both ends of the genomic DNA insert of the BAC clones. Here, we report a survey of the transposable elements (TE) in >73,000 STCs. A total of 6848 STCs were found homologous to regions of known TE sequences (E
• Wei, F., Gobelman-Werner, K., Morroll, S. M., Kurth, J., Mao, L., Wing, R., Leister, D., Schulze-Lefert, P., & Wise, R. P. (2000). Erratum: The Mla (powdery mildew) resistance cluster is associated with three NBS-LRR gene families and suppressed recombination within a 240-kb DNA interval on chromosome 5S (1HS) of Barley (Genetics 153 (1929-1948)). Genetics, 154(2), 953-.
• Yu, Y., Tomkins, J. P., Waugh, R., Frisch, D. A., Kudrna, D., Kleinhofs, A., Brueggeman, R. S., Muehlbauer, G. J., Wise, R. P., & Wing, R. A. (2000). A bacterial artificial chromosome library for barley (Hordeum vulgare L.) and the identification of clones containing putative resistance genes. Theoretical and Applied Genetics, 101(7), 1093-1099.
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  Abstract: Modern cultivated barley is an important cereal crop with an estimated genome size of 5000 Mb. To develop the resources for positional cloning and structural genomic analyses in barley, we constructed a bacterial artificial chromosome (BAC) library for the cultivar Morex using the cloning enzyme HindIII. The library contains 313344 clones (816 384-well plates). A random sampling of 504 clones indicated an average insert size of 106 kbp (range=30-195 kbp) and 3.4% empty vectors. Screening the colony filters for chloroplast DNA content indicated an exceptionally low 1.5% contamination with chloroplast DNA. Thus, the library provides 6.3 haploid genome equivalents allowing a >99% probability of recovering any specific sequence of interest. High-density filters were gridded robotically using a Genetix Q-BOT in a 4x4 double-spotted array on 22.5-cm2 filters. Each set of 17 filters allows the entire library to be screened with 18432 clones represented per filter. Screening the library with 40 single copy probes identified an average 6.4 clones per probe, with a range of 1-13 clones per probe. A set of resistance-gene analog (RGA) sequences identified 121 RGA-containing BAC clones representing 20 different regions of the genome with an average of 6.1 clones per locus. Additional screening of the library with a P-loop disease resistance primer probe identified 459 positive BAC clones. These data indicate that this library is a valuable resource for structural genomic applications in barley.
• Yuan, Q., Liang, F., Hsiao, J., Zismann, V., Benito, M., Quackenbush, J., Wing, R., & Buell, R. (2000). Anchoring of rice BAC clones to the rice genetic map in silico. Nucleic Acids Research, 28(18), 3636-3641.
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  PMID: 10982886;PMCID: PMC110739;Abstract: A wealth of molecular resources have been developed for rice genomics, including dense genetic maps, expressed sequence tags (ESTs), yeast artificial chromosome maps, bacterial artificial chromosome (BAC) libraries and BAC end sequence databases. Integration of genetic and physical maps involves labor-intensive empirical experiments. To accelerate the integration of the bacterial clone resources with the genetic map for the International Rice Genome Sequencing Project, we cleaned and filtered the available EST and BAC end sequences for repetitive sequences and then searched all available rice genetic markers with our filtered databases. We identified 418 genetic markers that aligned with at least one BAC end sequence with >95% sequence identity, providing a set of large insert clones with an average separation of 1 Mb that can serve as nucleation points for the sequencing phase of the International Rice Genome Sequencing Project.
• Zhang, H. -., Budiman, M. A., & Wing, R. A. (2000). Genetic mapping of jointless-2 to tomato chromosome 12 using RFLP and RAPD markers. Theoretical and Applied Genetics, 100(8), 1183-1189.
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  Abstract: Abscission zones are specialized regions in plants, usually located at the base of most plant parts, such as flowers, fruit and leaves, where organs are shed. Although a great deal of information is known about the physiological and biochemical events that lead to organ shedding, very little is known of the molecular events that lead to the formation of the abscission zone itself. In tomato, two recessive mutations have been discovered that completely suppress the formation of flower and fruit pedicel abscission zones, i.e., jointless (j) and jointless-2 (j-2), both tentatively localized to chromosome 11 about 30 cM apart. Because the study of the control of abscission zone development is important for both basic and applied research we are using a map-based cloning approach to identify the jointless genes. The first step in any positional cloning experiment is to establish segregating mapping populations for the target gene and identify closely linked molecular markers that flank the locus. In this study, bulked segregant analysis was used to identify a RAPD marker associated with the j-2 locus, RPD140. To determine the chromosome location of RPD140, we converted it to an RFLP marker that was then mapped on the Cornell reference tomato map in a marker-dense region of chromosome 12. To verify that the j-2 locus was located on tomato chromosome 12, we used nine chromosome 12 RFLP markers linked with RPD140 to map the j-2 gene in an interspecific F2 mapping population of 151 plants segregating for j-2. The j-2 gene was localized to a 3.0-cM interval between RPD140 and TG618 on tomato chromosome 12.
• Lijavetzky, D., Muzzi, G., Wicker, T., Keller, B., Wing, R., & Dubcovsky, J. (1999). Construction and characterization of a bacterial artificial chromosome (BAC) library for the A genome of wheat. Genome, 42(6), 1176-1182.
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  PMID: 10659785;Abstract: A genomic bacterial artificial chromosome (BAC) library of the A genome of wheat has been constructed. Triticum monococcum accession DV92 was selected for this purpose because it is a cultivated diploid wheat and one of the parental lines used in the construction of a saturated genetic map. Leaves from this accession were used to isolate high-molecular-weight DNA from nuclei. This DNA was partially digested with restriction enzyme Hind III, subjected to double size selection, electroeluted and cloned into the pINDIGO451 BAC vector. The library consists of 276 480 clones with an average insert size of 115 kb. Excluding the 1.33% of empty clones and 0.14% of clones with chloroplast DNA, the coverage of this library is 5.6 genome equivalents. With this genome coverage the probability of having any DNA sequence represented in this library is higher than 99.6%. Clones were sorted in 720 384-well plates and blotted onto 15 high-density filters. High-density filters were screened with several single or low-copy clones and five positive BAC clones were selected for further analysis. Since most of the T. monococcum BAC ends included repetitive sequences, a modification was introduced into the classical end-isolation procedure to select low copy sequences for chromosome walking.
• Tomkins, J. P., Mahalingam, R., Smith, H., Goicoechea, J. L., Knap, H. T., & Wing, R. A. (1999). A bacterial artificial chromosome library for soybean PI 437654 and identification of clones associated with cyst nematode resistance. Plant Molecular Biology, 41(1), 25-32.
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  PMID: 10561065;Abstract: We have constructed a soybean bacterial artificial chromosome (BAC) library using the plant introduction (PI) 437654. The library contains 73728 clones stored in 192 384-well microtiter plates. A random sampling of 230 BACs indicated an average insert size of 136 kb with a range of 20 to 325 kb, and less than 4% of the clones do not contain inserts. Ninety percent of BAC clones in the library have an average insert size greater than 100 kb. Based on a genome size of 1115 Mb, library coverage is 9 haploid genome equivalents. Screening the BAC library colony filters with cpDNA sequences showed that contamination of the genomic library with chloroplast clones was low (1.85%). Library screening with three genomic RFLP probes linked to soybean cyst nematode (SCN) resistance genes resulted in an average of 18 hits per probe (range 7 to 30). Two separate pools of forward and reverse suppression subtractive cDNAs obtained from SCN-infected and uninfected roots of PI 437654 were hybridized to the BAC library filters. The 488 BACs identified from positive signals were fingerprinted and analyzed using FPC software (version 4.0) resulting in 85 different contigs. Contigs were grouped and analyzed in three categories: (1) contigs of BAC clones which hybridized to forward subtracted cDNAs, (2) contigs of BAC clones which hybridized to reverse subtracted cDNAs, and (3) contigs of BAC clones which hybridized to both forward and reverse subtracted cDNAs. This protocol provides an estimate of the number of genomic regions involved in early resistance response to a pathogenic attack.
• Tomkins, J. P., Miller-Smith, H., Sasinowski, M., Choi, S., Sasinowska, H., Verce, M. F., Freedman, D. L., Dean, R. A., & Wing, R. A. (1999). Physical map and gene survey of the Ochrobactrum anthropi genome using bacterial artificial chromosome contigs. Microbial and Comparative Genomics, 4(3), 203-217.
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  PMID: 10587947;Abstract: Bacterial artificial chromosome (BAC) clones are effective mapping and sequencing reagents for use with a wide variety of small and large genomes. This report describes research aimed at determining the genome structure of Ochrobactrum anthropi, an opportunistic human pathogen that has potential applications in biodegradation of hazardous organic compounds. A BAC library for O. anthropi was constructed that provides a 70-fold genome coverage based on an estimated genome size of 4.8 Mb. The library contains 3072 clones with an average insert size of 112 kb. High-density colony filters of the library were made, and a physical map of the genome was constructed using a hybridization without replacement strategy. In addition, 1536 BAC clones were fingerprinted with HindIII and analyzed using IMAGE and Fingerprint Contig software (FPC, Sanger Centre, U.K.). The FPC results supported the hybridization data, resulting in the formation of two major contigs representing the two major replicons of the O. anthropi genome. After determining a reduced tiling path, 138 BAC ends from the reduced tile were sequenced for a preliminary gene survey. A search of the public databases with the BLASTX algorithm resulted in 77 strong hits (E-value < 0.001), of which 89% showed similarity to a wide variety of prokaryotic genes. These results provide a contig-based physical map to assist the cloning of important genomic regions and the potential sequencing of the O. anthropi genome.
• Tomkins, J. P., Yu, Y., Miller-Smith, H., Frisch, D. A., Woo, S. S., & Wing, R. A. (1999). A bacterial artificial chromosome library for sugarcane. Theoretical and Applied Genetics, 99(3-4), 419-424.
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  PMID: 22665173;Abstract: Modern cultivated sugarcane is a complex aneuploid polyploid with an estimated genome size of 3000 Mb. Although most traits in sugarcane show complex inheritance, a rest locus showing monogenic inheritance has been documented. In order to facilitate cloning of the rust locus, we have constructed a bacterial artificial chromosome (BAC) library for the cultivar R570. The library contains 103,296 clones providing 4.5 sugarcane genome equivalents A random sampling of 240 clones indicated an average insert size of 130 kb allowing a 98% probability of recovering any specific sequence of interest. High-density filters were gridded robotically using a Genetix Q-BOT in a 4 x 4 double-spotted array on 22.5 cm2 filters. Each set of five filters provides a genome coverage of 4x with 18,432 clones represented per filter. Screening of the library with three different barley chloroplast gene probes indicated an exceptionally low chloroplast DNA content of less than 1%. To demonstrate the library's potential for map-based cloning, single-copy RFLP sugarcane mapping probes anchored to nine different linkage groups and three different gene probes were used to screen the library. The number of positive hybridization signals resulting from each probe ranged from 8 to 60. After determining addresses of the signals, clones were evaluated for insert size and HindIII-fingerprinted. The fingerprints were then used to determine clone relationships and assemble contigs. For comparison with other monocot genomes, sugarcane RFLP probes were also used to screen a Sorghum bicolor BAC library and two rice BAC libraries. The rice and sorghum BAC clones were characterized for insert size and fingerprinted, and the results compared to sugarcane. The library was screened with a rust resistance RFLP marker and candidate BAC clones were subjected to RFLP fragment matching to identify those corresponding to the same genomic region as the rust gene.
• Wei, F., Gobelman-Werner, K., Morroll, S. M., Kurth, J., Mao, L., Wing, R., Leister, D., Schulze-Lefert, P., & Wise, R. P. (1999). The Mla (powdery mildew) resistance cluster is associated with three NBS-LRR gene families and suppressed recombination within a 240-kb DNA interval on chromosome 5S (1HS) of barley. Genetics, 153(4), 1929-1948.
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  PMID: 10581297;PMCID: PMC1460856;Abstract: Powdery mildew of barley, caused by Erysiphe graminis f. sp. hordei, is a model system for investigating the mechanism of gene-for-gene interaction between large-genome cereals and obligate-fungal pathogens. A large number of loci that confer resistance to this disease are located on the short arm of chromosome 5(1H). The Mla resistance-gene cluster is positioned near the telomeric end of this chromosome arm. AFLP-, RAPD-, and RFLP-derived markers were used to saturate the Mla region in a high-resolution recombinant population segregating for the (Mla6 + Mla14) and (Mla13 + Ml-Ru3) resistance specificities. These tightly linked genetic markers were used to identify and develop a physical contig of YAC and BAC clones spanning the Mla cluster. Three distinct NBS-LRR resistance-gene homologue (RGH) families were revealed via computational analysis of low-pass and BAC-end sequence data derived from Mla-spanning clones. Genetic and physical mapping delimited the Mla-associated, NBS-LRR gene families to a 240-kb interval. Recombination within the RGH families was at least 10-fold less frequent than between markers directly adjacent to the Mla cluster.
• Gómez, M., Islam-Faridi, M., Zwick, M. S., Czeschin Jr., D. G., Hart, G. E., Wing, R. A., Stelly, D. M., & Price, H. J. (1998). Tetraploid nature of Sorghum bicolor (L.) Moench. Journal of Heredity, 89(2), 188-190.
• Huang, Y., Jordan, W. R., Wing, R. A., & Morgan, P. W. (1998). Gene expression induced by physical impedance in maize roots. Plant Molecular Biology, 37(6), 921-930.
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  PMID: 9700065;Abstract: Two cDNA clones, pIIG1 and pIIG2, corresponding to mRNAs that accumulate in maize root tips subjected to 10 rain of physical impedance, were isolated by differential screening of a cDNA library. The deduced proteins, based on DNA sequence analysis, have molecular masses of 13 and 23 kDa for pIIG1 and pIIG2, respectively. pHG1 showed 97% similarity at the nucleic acid level to a maize root cortical cell delineating protein (pZRP3) and was also similar to some bimodular proteins that are developmentally or stress regulated in other plant species. In situ localization of pIIG1 showed some expression in cortical cells of control maize roots; however, after a 10 min physical impedance treatment, pIIG1 accumulation increased greatly in cortical cells and extended to include the procambial region. pIIG2 did not show sequence similarity with any identified gene of known function, but a bipartite nuclear targeting sequence occurs in its deduced amino acid sequence which indicates it may function in the nucleus. Thus, rapid accumulation of specific mRNAs occurs in maize roots in response to impedance stress, and these mRNAs may be responsible for some responses of the roots to physical impedance.
• Miller, J. T., Jackson, S. A., Nasuda, S., Gill, B. S., Wing, R. A., & Jiang, J. (1998). Cloning and characterization of a centromere-specific repetitive DNA element from Sorghum bicolor. Theoretical and Applied Genetics, 96(6-7), 832-839.
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  Abstract: A 823-bp Sau3AI fragment (pSau3A10) was subcloned from a sorghum bacterial artificial chromosome (BAC) clone, 13I16, that contains DNA sequences specific to the centromeres of grass species. Sequence analysis showed that pSau3A10 consists of six copies of an approximately 137-bp monomer. The six monomers were organized into three dimers. The monomers within the dimers shared 62-72% homology and the dimers were 79-82% homologous with each other. Fluorescence in situ hybridization (FISH) analysis indicated that the Sau3A10 family is present only in the centromeres of sorghum chromosomes. Sequencing, Southern hybridization, and Fiber-FISH analyses indicated that the Sau3A10 family is tandemly arranged and is present in uninterrupted stretches of up to at least 81 kb of DNA. Slot-blot analysis estimated that the Sau3A10 family constitutes 1.6-1.9% of the sorghum genome. The long stretches of Sau3A10 sequences were interrupted by other centromeric DNA elements. Southern analysis indicated that the Sau3A10 sequence is one of the most abundant DNA families located in sorghum centromeres and is conserved only in closely related sorghum species. Methylation experiments indicated that the cytosine of the CG sites in sorghum centromeric regions is generally methylated. The structure and organization of the Sau3A10 family shared similarities with centromeric DNA repeats in other eukaryotic species. It is suggested that the Sau3A10 family is probably an important part of sorghum centromeres.
• Zwick, M. S., Islam-Faridi, M. N., Czeschin Jr., D. G., Wing, R. A., Hart, G. E., Stelly, D. M., & Price, H. J. (1998). Physical mapping of the liguleless linkage group in sorghum bicolor using rice RFLP-selected sorghum BACs. Genetics, 148(4), 1983-1992.
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  PMID: 9560411;PMCID: PMC1460102;Abstract: Physical mapping of BACs by fluorescent in situ hybridization (FISH) was used to analyze the liguleless (lg-1) linkage group in sorghum and compare it to the conserved region in rice and maize. Six liguleless-associated rice restriction fragment length polymorphism (RFLP) markers were used to select 16 homeologous sorghum BAGs, which were in turn used to physically map the liguleless linkage group in sorghum. Results show a basic conservation of the liguleless region in sorghum relative to the linkage map of rice. One marker which is distal in rice is more medial in sorghum, and another marker which is found within the linkage group in rice is on a different chromosome in sorghum. BACs associated with linkage group I hybridize to chromosome I(t), which was identified by using FISH in a sorghum cytogenetic stock trisomic for chromosome I (denoted I(t)), and a BAC associated with linkage group E hybridized to an unidentified chromosome. Selected BAGs, representing RFLP loci, were end-cloned for RFLP mapping, and the relative linkage order of these clones was in full agreement with the physical data. Similarities in locus order and the association of RFLP-selected BAC markers with two different chromosomes were found to exist between the linkage map of the liguleless region in maize and the physical map of the liguleless region in sorghum.
• Chen, M., Sanmiguel, P., Oliveira, A. D., Woo, S. -., Zhang, H., Wing, R. A., & Bennetzen, J. L. (1997). Microcolinearity in sh2-homologous regions of the maize, rice, and sorghum genomes. Proceedings of the National Academy of Sciences of the United States of America, 94(7), 3431-3435.
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  PMID: 9096411;PMCID: PMC20387;Abstract: Large regions of genomic colinearity have been demonstrated among grass species by recombinational mapping, but the degree of chromosomal conservation at the sub-centimorgan level has not been extensively investigated. We cloned the rice and sorghum genes homologous to the sh2 locus of maize on bacterial artificial chromosomes (BACs), and observed that a homologue of the maize a1 gene was also present on each of these BACs. In sorghum, we found a direct duplication of a1 homologues separated by about 10 kb. In maize, sh2 and a1 are approximately 140 kb apart and transcribed in the same direction, with sh2 upstream of a1. In rice and sorghum, this arrangement is fully conserved. However, the sh2 and a1 homologues are separated by about 19 kb in both rice and sorghum. We found low-copy-number and repetitive DNAs between the sh2 and a1 homologues of sorghum and rice. The sh2 and a1 homologues cross-hybridized, but the repetitive DNA and most low-copy-number sequences between these genes did not. These results indicate that maize, sorghum, and rice have conserved gene order and composition in the sh2-a1 region, but have acquired extensive qualitative and quantitative differences in the sequences between these genes.
• Gómez, M. I., Islam-Faridi, M. N., Woo, S., Schertz, K. F., Czeschin Jr., D., Zwick, M. S., Wing, R. A., Stelly, D. M., & Price, H. J. (1997). FISH of a maize sh2-selected sorghum BAC to chromosomes of Sorghum bicolor. Genome, 40(4), 475-478.
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  PMID: 18464840;Abstract: Fluorescence in situ hybridization (FISH) of a 205 kb Sorghum bicolor bacterial artificial chromosome (BAC) containing a sequence complementary to maize sh2 cDNA produced a large pair of FISH signals at one end of a midsize metacentric chromosome of S. bicolor. Three pairs of signals were observed in metaphase spreads of chromosomes of a sorghum plant containing an extra copy of one arm of the sorghum chromosome arbitrarily designated with the letter D. Therefore, the sequence cloned in this BAC must reside in the arm of chromosome D represented by this monotelosome. This demonstrates a novel procedure for physically mapping cloned genes or other single-copy sequences by FISH, sh2 in this case, by using BACs containing their complementary sequences. The results reported herein suggest homology, at least in part, between one arm of chromosome D in sorghum and the long arm of chromosome 3 in maize.
• Zhang, H., & Wing, R. A. (1997). Physical mapping of the rice genome with BACs. Plant Molecular Biology, 35(1-2), 115-127.
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  PMID: 9291965;Abstract: The development of genetics in this century has been catapulted forward by several milestones: rediscovery of Mendel's laws, determination of DNA as the genetic material, discovery of the double-helix structure of DNA and its implications for genetic behavior, and most recently, analysis of restriction fragment length polymorphisms (RFLPs). Each of these milestones has generated a huge wave of progress in genetics. Consequently, our understanding of organismal genetics now extends from phenotypes to their molecular genetic basis. It is now clear that the next wave of progress in genetics will hinge on genome molecular physical mapping, since a genome physical map will provide an invaluable, readily accessible system for many detailed genetic studies and isolation of many genes of economic or biological importance. Recent development of large-DNA fragment (>100 kb) manipulation and cloning technologies, such as pulsed-field gel electrophoresis (PFGE), and yeast artificial chromosome (YAC) and bacterial artificial chromosome (BAC) cloning, has provided the powerful tools needed to generate molecular physical maps for higher-organism genomes. This chapter will discuss (1) an ideal physical map of plant genome and its applications in plant genetic and biological studies, (2) reviews on physical mapping of the genomes of Caenorhabditis elegans, Arabidopsis thaliana, and man, (3) large-insert DNA libraries: cosmid, YAC and BAC, and genome physical mapping, (4)physical mapping of the rice genome with BACs, and (5) perspectives on the physical mapping of the rice genome with BACs.
• Zhu, H., Choi, S., Johnston, A. K., Wing, R. A., & Dean, R. A. (1997). A large-insert (130 kbp) bacterial artificial chromosome library of the rice blast fungus Magnaporthe grisea: Genome analysis, contig assembly, and gene cloning. Fungal Genetics and Biology, 21(3), 337-347.
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  PMID: 9290247;Abstract: Magnaporthe grisea (Hebert) Barr causes rice blast, one of the most devastating diseases of rice (Oryza sativa) worldwide. This fungus is an ideal organism for studying a number of aspects of plant pathogen interactions, including infection-related morphogenesis, avirulence, and pathogen evolution. To facilitate M. grisea genome analysis, physical mapping, and positional cloning, we have constructed a bacterial artificial chromosome (BAC) library from the rice infecting strain 70-15. A new method was developed for separation of partially digested large-molecular-weight DNA fragments that facilitated library construction with large inserts. The library contains 9216 clones, with an average insert size of 130 kbp (>25 genome equivalents) stored in 384-well microtiter plates that can be double spotted robotically on to a single nylon membrane. Several unlinked single-copy DNA probes were used to screen 4608 clones in the library and an average of 13 (minimum of 6) overlapping BAC clones was found in each case. Hybridization of total genomic DNA to the library and analysis of individual clones indicated that ~26% of the clones contain single-copy DNA. Approximately 35% of BAC clones contained the retrotransposon MAGGY. The library was used to identify BAC clones containing a adenylate cyclase gene (mac1). In addition, a 550-kbp contig composed of 6 BAC clones was constructed that encompassed two adjacent RFLP markers on chromosome 2. These data show that the BAC library is suitable for genome analysis of M. grisea. Copies of colony hybridization membranes are available upon request.
• Zwick, M. S., Hanson, R. E., McKnight, T. D., Islam-Faridi, M. N., Stelly, D. M., Wing, R. A., & Price, H. J. (1997). A rapid procedure for the isolation of C₀t-1 DNA from plants. Genome, 40(1), 138-142.
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  PMID: 18464813;Abstract: In situ hybridization (ISH) for the detection of single- or low-copy sequences, particularly large DNA fragments cloned into YAC or BAC vectors, generally requires the suppression or 'blocking' of highly-repetitive DNAs. C0t-1 DNA is enriched for repetitive DNA elements, high or moderate in copy number, and can therefore be used more effectively than total genomic DNA to prehybridize and competitively hybridize repetitive elements that would otherwise cause nonspecific hybridization. C0t-1 DNAs from several mammalian species are commercially available, however, none is currently available for plants to the best of our knowledge. We have developed a simple 1-day procedure to generate C0t-1 DNA without the use of specialized equipment.
• Avramova, Z., Tikhonov, A., SanMiguel, P., Jin, Y., Liu, C., Woo, S., Wing, R. A., & Bennetzen, J. L. (1996). Gene identification in a complex chromosomal continuum by local genomic cross-referencing. Plant Journal, 10(6), 1163-1168.
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  PMID: 9011097;Abstract: Most higher plants have complex genomes containing large quantities of repetitive DNA interspersed with low-copy-number sequences. Many of these repetitive DNAs are mobile and have homology to RNAs in various cell types. This can make it difficult to identify the genes in a long chromosomal continuum. It was decided to use genic sequence conservation and grass genome co-linearity as tools for gens identification. A bacterial artificial chromosome (BAC) clone containing sorghum genomic DNA was selected using a maize Adh1 probe. The 165 kb sorghum BAC was tested for hybridization to a set of clones representing the contiguous 280 kb of DNA flanking maize Adh1. None of the repetitive maize DNAs hybridized, but most of the low-copy-number sequences did. A low-copy-number sequence that did cross-hybridize was found to be a gene, while one that did not was found to be a low-copy-number retrotransposon that was named Reina. Regions of cross-hybridization were co-linear between the two genomes, but closer together in the smaller sorghum genome. These results indicate that local genomic cross-referencing by hybridization of orthologous clones can be an efficient and rapid technique for gene identification and studies of genome organization.
• Bennetzen, J. L., SanMiguel, P., Liu, C. N., Chen, M., Tikhonov, A., Costa, A., Jin, Y. K., Avramova, Z., Woo, S. S., Zhang, H., & Wing, R. A. (1996). The Hybaid Lecture. Microcollinearity and segmental duplication in the evolution of grass nuclear genomes.. Symposia of the Society for Experimental Biology, 50, 1-3.
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  PMID: 9039427;Abstract: Recent studies have shown that grass genomes have very similar gene compositions and regions of conserved gene order, as exemplified by collinear genetic maps of DNA markers. We have begun the detailed study of sequence organization in large (100-500 kb) segments of the nuclear genomes of maize, sorghum and rice. Our results indicate collinearity of genes in the regions homoeologous to the maize adh1 and sh2-a1 genes. Comparable genes were found to be physically closer to each other in grasses with small genomes (rice and sorghum) than they are in maize. In several instances, we have found evidence of tandem and 'distantly tandem' duplications of segments containing maize and sorghum genes. These duplications complicate characterizations of microcollinearity and could also interfere with some map-based approaches to gene isolation.
• Jiang, J., Nasuda, S., Dong, F., Scherrer, C. W., Woo, S., Wing, R. A., Gill, B. S., & Ward, D. C. (1996). A conserved repetitive DNA element located in the centromeres of cereal chromosomes. Proceedings of the National Academy of Sciences of the United States of America, 93(24), 14210-14213.
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  PMID: 8943086;PMCID: PMC19519;Abstract: Repetitive DNA sequences have been demonstrated to play an important role for centromere function of eukaryotic chromosomes, including those from fission yeast, Drosophila melanogaster, and humans. Here we report on the isolation of a repetitive DNA element located in the centromeric regions of cereal chromosomes. A 745-bp repetitive DNA clone, pSau3A9, was isolated from sorghum (Sorghum bicolor). This DNA element is located in the centromeric regions of all sorghum chromosomes, as demonstrated by fluorescence in situ hybridization. Repetitive DNA sequences homologous to pSau3A9 also are present in the centromeric regions of chromosomes from other cereal species, including rice, maize, wheat, barley, rye, and oats. Probe pSau3A9 also hybridized to the centromeric region of B chromosomes from rye and maize. The repetitive nature and its conservation in distantly related plant species indicate that the pSau3A9 family may be associated with centromere function of cereal chromosomes. The absence of DNA sequences homologous to pSau3A9 in dicot species suggests a faster divergence of centromere-related sequences compared with the telomere-related sequences in plants.
• Vielle-Calzada, J., Nuccio, M. L., Budiman, M. A., Thomas, T. L., Burson, B. L., Hussey, M. A., & Wing, R. A. (1996). Comparative gene expression in sexual and apomictic ovaries of Pennisetum ciliare (L.) Link. Plant Molecular Biology, 32(6), 1085-1092.
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  PMID: 9002607;Abstract: Limited emphasis has been given to the molecular study of apomixis, an asexual method of reproduction where seeds are produced without fertilization. Most buffelgrass (Pennisetum ciliare (L.) Link syn = Cenchrus ciliaris L.) genotypes reproduce by obligate apomixis (apospory); however, rare sexual plants have been recovered. A modified differential display procedure was used to compare gene expression in unpollinated ovaries containing ovules with either sexual or apomictic female gametophytes. The modification incorporated end-labeled poly(A)+ anchored primers as the only isotopic source, and was a reliable and consistent approach for detecting differentially displayed transcripts. Using 20 different decamers and two anchor primers, 2268 cDNA fragments between 200 and 600 bp were displayed. From these, eight reproducible differentially displayed cDNAs were identified and cloned. Based on northern analysis, one cDNA was detected in only the sexual ovaries, two cDNAs in only apomictic ovaries and one cDNA was present in both types of ovaries. Three fragments could not be detected and one fragment was detected in ovaries, stems, and leaves. Comparison of gene expression during sexual and apomictic development in buffelgrass represents a new model system and a strategy for investigating female reproductive development in the angiosperms.
• Zhang, H., Choi, S., Woo, S., Zhikang, L. i., & Wing, R. A. (1996). Construction and characterization of two rice Bacterial Artificial Chromosome libraries from the parents of a permanent recombinant inbred mapping population. Molecular Breeding, 2(1), 11-24.
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  Abstract: Rice is a leading grain crop and the staple food for over half of the "world" population. Rice is also an ideal species for genetic and biological studies of cereal crops and other monocotyledonous plants because of its small genome and well developed genetic system. To facilitate rice genome analysis leading to physical mapping, the identification of molecular markers closely linked to economic traits, and map-based cloning, we have constructed two rice bacterial artificial chromosome (BAC) libraries from the parents of a permanent mapping population (Lemont and Teqing) consisting of 400 F9 recombinant inbred lines (RILs). Lemont (japonica) and Teqing (indica) represent the two major genomes of cultivated rice, both are leading commercial varieties and widely used germplasm in rice breeding programs. The Lemont library contains 7296 clones with an average insert size of 150 kb, which represents 2.6 rice haploid genome equivalents. The Teqing library contains 14 208 clones with an average insert size of 130 kb, which represents 4.4 rice haploid genome equivalents. Three single-copy DNA probes were used to screen the libraries and at least two overlapping BAC clones were isolated with each probe from each library, ranging from 45 to 260 kb in insert size. Hybridization of BAC clones with chloroplast DNA probes and fluorescent in situ hybridization using BAC DNA as probes demonstrated that both libraries contain very few clones of chloroplast DNA origin and are likely free of chimeric clones. These data indicate that both BAC libraries should be suitable for map-based cloning of rice genes and physical mapping of the rice genome. © 1996 Kluwer Academic Publishers.
• Cai, L., Taylor, J. F., Wing, R. A., Gallagher, D. S., Woo, S. -., & Davis, S. K. (1995). Construction and characterization of a bovine bacterial artificial chromosome library. Genomics, 29(2), 413-425.
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  PMID: 8666390;Abstract: A bacterial artificial chromosome (BAC) library has been constructed for use in bovine genome mapping using the pBeloBAC11 vector. Currently, the library consists of 23,040 clones, which achieves a 70% probability (P = 0.70) of the library containing a specific unique DNA sequence. Sixty thousand clones, or about three haploid bovine genomes, will be required to achieve a 95% probability (P = 0.95) of containing a unique sequence. An average insert size of 146 kb was estimated from the analysis of 77 randomly selected BAC clones produced by one or two rounds of size selection. The bovine DNA inserts proved to be very stable for at least 100 cell generations. No chimetic clones were detected among 11 large, size-selected BAC clones using fluorescence in situ hybridization (FISH) on metaphase bovine chromosomes. The polymerase chain reaction (PCR) was used to screen the library for single-copy nuclear sequences. Thirty-three of 46 (72%) sequences were present in the library in at least one copy, which is consistent with the estimated 70% probability of this library containing a unique DNA sequence. A BAC clone containing the 3β-hydroxy-5-ene steroid dehydrogenase (HSD3B) gene was physically mapped to bovine chromosome 3 by FISH. Two new microsatellite markers were isolated from the HSD3B-positive BAC clone as sequence-tagged sites for genetic mapping. These markers cosegregated, and no recombinants were detected in 193 informative meioses. Plasmid end rescue and the inverse polymerase chain reaction methods were used to rescue both ends of this BAC clone, and chromosome walking was performed using PCR primers designed within the end region sequences. Based on our experimental results, the BAC system provides a very useful tool for complex genome analysis.
• Choi, S., Creelman, R. A., Mullet, J. E., & Wing, R. A. (1995). Construction and characterization of a bacterial artificial chromosome library of Arabidopsis thaliana. Plant Molecular Biology Reporter, 13(2), 124-128.
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  Abstract: We constructed an ordered 3,948-clone arabidopsis BAC library. The library has a combined average insert size of 100 kb (n=54). Assuming a haploid genome size of 100,000 kb, the BAC library contains 3.95 haploid genome equivalents with a 98 percent probability of isolating a specific genomic region. The library was screened with five arabidopsis cDNA probes and one tomato probe; all probes hybridized to at least one (and in most cases three) BAC clones in the library. © 1995 Kluwer Academic Publishers.
• Hanson, R. E., Zwick, M. S., Sangdun, C., Islam-Faridi, M., McKnight, T. D., Wing, R. A., Price, H. J., & Stelly, D. M. (1995). Fluorescent in situ hybridization of a bacterial artificial chromosome. Genome, 38(4), 646-651.
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  PMID: 7672600;
• Woo, S., Rastogi, V. K., Zhang, H., Paterson, A. H., Schertz, K. F., & Wing, R. A. (1995). Isolation of megabase-size DNA from sorghum and applications for physical mapping and bacterial and yeast artificial chromosome library construction. Plant Molecular Biology Reporter, 13(1), 82-94.
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  Abstract: A method was developed for the isolation of megabase-size DNA from Sorghum bicolor. Sorghum protoplasts were isolated from young leaf tissue, embedded in an agarose matrix as microbeads or plugs, followed by cell lysis and protein degradation. The DNA prepared by this method was larger than 1 Mb in size and readily digestible with restriction enzymes. The DNA was shown to be suitable for physical mapping, and was successfully used for the construction of BAC and YAC libraries. © 1995 Kluwer Academic Publishers.
• Xinping, Z., Wing, R. A., & Paterson, A. H. (1995). Cloning and characterization of the majority of repetitive DNA in cotton (Gossypium L.). Genome, 38(6), 1177-1188.
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  PMID: 8654914;
• Zhang, H., Zhao, X., Ding, X., Paterson, A. H., & Wing, R. A. (1995). Preparation of megabase-size DNA from plant nuclei. Plant Journal, 7(1), 175-184.
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  Abstract: A novel technique has been developed for the preparation of high molecular weight (HMW) DNA from plant nuclei. This technique involves physical homogenization of plant tissues, nuclei isolation, embedding of the nuclei in low-melting-point agarose microbeads or plugs, and DNA purification in situ. This technique is simple, rapid, and economical, and the majority of the DNA prepared is over 5.7 Mb in size. The genomic DNA content of the HMW DNA prepared by this technique is enriched by at least threefold and the chloroplast DNA content is reduced by over twofold relative to that prepared from plant protoplasts by existing methods. The DNA is readily digestible with different restriction enzymes and partial digestions of the DNA could be reproducibly performed. This method has been successfully used for the preparation of HMW DNA from a wide range of plant taxa, including grasses, legumes, vegetables, and trees. These results demonstrate that the DNA prepared by this technique is suitable for plant genome analysis by pulsed-field gel electrophoresis and for the construction of yeast and bacterial artificial chromosomes.
• Chittenden, L. M., Schertz, K. F., Lin, Y. R., Wing, R. A., & Paterson, A. H. (1994). A detailed RFLP map of Sorghum bicolor X S.propinquum, suitable for high-density mapping, suggests ancestral duplication of Sorghum chromosomes or chromosomal segments. Theoretical and Applied Genetics, 87(8), 925-933.
• Wing, R. A., Zhang, H., & Tanksley, S. D. (1994). Map-based cloning in crop plants. Tomato as a model system: I. Genetic and physical mapping of jointless. MGG Molecular & General Genetics, 242(6), 681-688.
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  PMID: 7908716;Abstract: A map-based cloning scheme is being used to isolate the jointless (j) gene of tomato. The jointless locus is defined by a single recessive mutation that completely suppresses the formation of the fruit and flower pedicel and peduncle abscission zone. jointless was mapped in an F2 population of an interspecific cross between Lycopersicon esculentum and Lycopersicon pennellii to a 7.1 cM interval between two restriction fragment length polymorphism (RFLP) markers TG523 and TG194. Isogenic DNA pools were then constructed from a subset of the mapping population and screened with 800 random decamers for random amplification of polymorphic DNA (RAPD) polymorphisms. Five new RAPD markers were isolated and mapped to chromosome 11, two of which were mapped within the targeted interval. One marker, RPD158, was mapped 1.5 cM to the opposite side of jointless relative to TG523 and thus narrowed the interval between the closest flanking markers to 3.0 cM. Physical mapping by pulse-field gel electrophoresis using TG523 and RPD158 as probes demonstrated that both markers hybridize to a common 600 kb SmaI restriction fragment. This provided an estimate of 200 kb/cM for the relationship between physical and genetic distances in the region of chromosome 11 containing the j locus. The combined results provide evidence for the feasibility of the next step toward isolation of the jointless gene by map-based cloning - a chromosome walk or jump to jointless. © 1994 Springer-Verlag.
• Woo, S. -., Jiang, J., Gill, B. S., Paterson, A. H., & Wing, R. A. (1994). Construction and characterization of a bacterial artificial chromosome library of Sorghum bicolor. Nucleic Acids Research, 22(23), 4922-4931.
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  PMID: 7800481;PMCID: PMC523757;Abstract: The construction of representative large insert DNA libraries is critical for the analysis of complex genomes. The predominant vector system for such work is the yeast artificial chromosome (YAC) system. Despite the success of YACs, many problems have been described including: chimerism, tedious steps in library construction and low yields of YAC insert DNA. Recently a new E.coli based system has been developed, the bacterial artificial chromosome (BAC) system, which offers many potential advantages over YACs. We tested the BAC system in plants by constructing an ordered 13 440 clone sorghum BAC library. The library has a combined average insert size, from single and double size selections, of 157 kb. Sorghum inserts of up to 315 kb were isolated and shown to be stable when grown for over 100 generations in liquid media. No chimeric clones were detected as determined by fluorescence in situ hybridization of ten BAC clones to metaphase and interphase S.bicolor nuclei. The library was screened with six sorghum probes and three maize probes and all but one sorghum probe hybridized to at least one BAC clone in the library. To facilitate chromosome walking with the BAC system, methods were developed to isolate the proximal ends of restriction fragments inserted into the BAC vector and used to isolate both the left and right ends of six randomly selected BAC clones. These results demonstrate that the S.bicolor BAC library will be useful for several physical mapping and map-based cloning applications not only in sorghum but other related cereal genomes, such as maize. Furthermore, we conclude that the BAC system is suitable for most large genome applications, is more 'user friendly' than the YAC system, and will likely lead to rapid progress in cloning biologically significant genes from plants.
• Zhang, H., Martin, G. B., Tanksley, S. D., & Wing, R. A. (1994). Map-based cloning in crop plants: tomato as a model system II. Isolation and characterization of a set of overlapping yeast artificial chromosomes encompassing the jointless locus. MGG Molecular & General Genetics, 244(6), 613-621.
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  PMID: 7969030;Abstract: A map-based cloning technique for crop plants is being developed using tomato as a model system. The target gene jointless is a recessive mutation that completely suppresses the formation of flower and fruit pedicel abscission zones. Previously, the jointless locus was mapped to a 3 cM interval between the two molecular markers TG523 and RPD158. Physical mapping of the jointless region by pulsed-field gel electrophoresis demonstrated that TG523 and RPD158 reside on a 600 kb SmaI fragment. In this study, TG523 was used as a probe to screen a tomato yeast artificial chromosome (YAC) library. Six tomato YAC (TY) clones were isolated, ranging from 220 to 380 kb in size. Genetic mapping of YAC ends demonstrated that this set of overlapping YACs encompasses the jointless locus. Two YAC ends, TY159L (L indicates left end) and TY143R (R indicates right end), cosegregate with the jointless locus. Only one of the six YACs (TY142) contained single-copy DNA sequences at both ends that could be mapped. The two ends of TY142 were mapped to either side of the jointless locus, indicating that TY142 contains a contiguous 285 kb tomato DNA fragment that probably includes the jointless locus. Physical mapping of the TY142 clone revealed that TY159L and TY143R reside on a 55 kb SalI fragment. Southern blot hybridization analysis of the DNAs of tomato lines nearly isogenic for the jointless mutation has allowed localization of the target locus to a region of less than 50 kb within the TY142 clone. © 1994 Springer-Verlag.
• Zhao, X., Zhang, H., Wing, R. A., & Paterson, A. H. (1994). A simple method for isolation of megabase DNA from cotton. Plant Molecular Biology Reporter, 12(2), 110-115.
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  Abstract: A simple method for preparation of high-molecular-weight DNA from cotton was developed. This method includes two major steps, (i) isolating nuclei and (ii) embedding nuclei into agarose microbeads. DNA isolated by this procedure is larger than 5.7 Mb in size, and is suitable for physical mapping by PFGE and YAC/BAC cloning. © 1994 Kluwer Academic Publishers.
• Paterson, A. H., & Wing, R. A. (1993). Genome mapping in plants. Current Opinion in Biotechnology, 4(2), 142-147.
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  Abstract: Genome mapping permits the study of morphological, physiological, and developmental processes in which genetic variants exist, and requires minimal a priori information. Further exploitation of the polymerase chain reaction, yeast artificial chromosomes, and comparative analysis of distantly related taxa, will contribute greatly to the fundamental understanding of plant biology and crop production.
• Wang, G. -., Wing, R. A., & Paterson, A. H. (1993). PCR amplification from single seeds, facilitating DNA marker-assisted breeding. Nucleic Acids Research, 21(10), 2527-.
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  PMID: 8506153;PMCID: PMC309568;
• Wing, R. A., Rastogi, V. K., Zhang, H., Paterson, A. H., & Tanksley, S. D. (1993). An improved method of plant megabase DNA isolation in agarose microbeads suitable for physical mapping and YAC cloning. Plant Journal, 4(5), 893-898.
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  PMID: 8275106;Abstract: The isolation of high quality megabase DNA from plant cells that is susceptible to a variety of molecular reagents is a critical first step in the physical analysis of complex genomes. A method for the isolation of such DNA by encapsulating plant protoplasts in agarose microbeads is presented. In comparison with the conventional agarose plug method, microbeads provide a dramatic increase in the surface area yielding megabase DNA that can be treated essentially as an aqueous DNA solution. Examples of the utility of DNA prepared by this technique for physical mapping, partial restriction enzyme digestion and cloning of large inserts as YACs are presented.
• Tanksley, S. D., Ganal, M. W., Prince, J. P., Vicente, M. D., Bonierbale, M. W., Broun, P., Fulton, T. M., Giovannoni, J. J., Grandillo, S., Martin, G. B., Messeguer, R., Miller, J. C., Miller, L., Paterson, A. H., Pineda, O., Roder, M. S., Wing, R. A., Wu, W., & Young, N. D. (1992). High density molecular linkage maps of the tomato and potato genomes. Genetics, 132(4), 1141-1160.
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  PMID: 1360934;PMCID: PMC1205235;Abstract: High density molecular linkage maps, comprised of more than 1000 markers with an average spacing between markers of approximately 1.2 cM (ca. 900 kb), have been constructed for the tomato and potato genomes. As the two maps are based on a common set of probes, it was possible to determine, with a high degree of precision, the breakpoints corresponding to 5 chromosomal inversions that differentiate the tomato and potato genomes. All of the inversions appear to have resulted from single breakpoints at or near the centromeres of the affected chromosomes, the result being the inversion of entire chromosome arms. While the crossing over rate among chromosomes appears to be uniformly distributed with respect to chromosome size, there is tremendous heterogeneity of crossing over within chromosomes. Regions of the map corresponding to centromeres and centromeric heterochromatin, and in some instances telomeres, experience up to 10-fold less recombination than other areas of the genome. Overall, 28% of the mapped loci reside in areas of putatively suppressed recombination. This includes loci corresponding to both random, single copy genomic clones and transcribed genes (detected with cDNA probes). The extreme heterogeneity of crossing over within chromosomes has both practical and evolutionary implications. Currently tomato and potato are among the most thoroughly mapped eukaryotic species and the availability of high density molecular linkage maps should facilitate chromosome walking, quantitative trait mapping, marker-assisted breeding and evolutionary studies in these two important and well studied crop species.
• Giovannoni, J. J., Wing, R. A., Ganal, M. W., & Tanksley, S. D. (1991). Isolation of molecular markers from specific chromosomal intervals using DNA pools from existing mapping populations. Nucleic Acids Research, 19(23), 6553-6558.
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  PMID: 1684420;PMCID: PMC329217;Abstract: We present a general method for isolating molecular markers specific to any region of a chromosome using existing mapping populations. Two pools of DNA from individuals homozygous for opposing alleles for a targeted chromosomal interval, defined by two or more linked RFLP markers, are constructed from members of an existing mapping population. The DNA pools are then screened for polymorphism using random oligonuclotide primers and PCR (1). Polymorphic DNA bands should represent DNA sequences within or adjacent to the selected interval. We tested this method in tomato using two genomic intervals containing genes responsible for regulating pedicle abscission pointless) and fruit ripening (non-ripening). DNA pools containing 7 to 14 F2 individuals for each interval were screened with 200 random primers. Three polymorphic markers were thus identified, two of which were subsequently shown to be tightly linked to the selected intervals. The third marker mapped to the same chromosome (11) but 45 cM away from the selected interval. A particularly attractive attribute of this method is that a single mapping population can be used to target any interval in the genome. Although this method has been demonstrated in tomato, it should be applicable to any sexually reproducing organism for which segregating populations are being used to construct genetic linkage maps.
• Twell, D., Yamaguchi, J., Wing, R. A., Ushiba, J., & McCormick, S. (1991). Promoter analysis of genes that are coordinately expressed during pollen development reveals pollen-specific enhancer sequences and shared regulatory elements. Genes and Development, 5(3), 496-507.
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  PMID: 1840556;Abstract: We have investigated the functional organization and properties of cis regulatory elements in the promoter regions of two genes from tomato (LAT52 and LAT59) that are preferentially and coordinately expressed during pollen maturation. Promoter deletion analysis in transgenic plants demonstrated that only minimal (
• Wing, R. A., Yamaguchi, J., Larabell, S. K., Ursin, V. M., & McCormick, S. (1990). Molecular and genetic characterization of two pollen-expressed genes that have sequence similarity to pectate lyases of the plant pathogen Erwinia. Plant Molecular Biology, 14(1), 17-28.
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  PMID: 1983191;Abstract: A set of cDNAs that are expressed in tomato anthers were isolated [24]. We further characterized two of these cDNAs (LAT56 and LAT59) and their corresponding genomic clones. LAT56 and LAT59 show low levels of steady-state mRNA in immature anthers and maximal levels in mature anthers and pollen. The LAT56 and LAT59 genes are single-copy in the tomato genome, and are linked on chromosome 3, approximately 5 cM apart. Although these cDNAs did not cross-hybridize, their deduced protein sequences (P56 and P59) have 54% amino acid identity. The LAT56 and LAT59 genes each have two introns, but they are located in non-homologous positions. P56 and P59 show significant protein sequence similarity to pectate lyases of plant pathogenic bacteria. The similarity of P56 and P59 to the bacterial pectate lyases is equivalent to the homology described for different pectate lyase sequences of the genus Erwinia. We suggest that the pollen expression of LAT56 and LAT59 might relate to a requirement for pectin degradation during pollen tube growth. © 1989 Kluwer Academic Publishers.
• Twell, D., Wing, R., Yamaguchi, J., & McCormick, S. (1989). Isolation and expression of an anther-specific gene from tomato. MGG Molecular & General Genetics, 217(2-3), 240-245.
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  PMID: 2770694;Abstract: We have isolated and sequenced an anther-specific cDNA clone and a corresponding genomic clone from tomato. The gene (LAT52) encodes an 800-nucleotide-long transcript that is detectable in pollen, anthers and at 20-to 50-fold lower levels in petals. LAT52 mRNA is not detectable in pistils, sepals or non-reproductive tissues. Steady-state levels of LAT52 mRNA are detectable in immature anthers containing pollen at the tetrad stage and increase progressively throughout microsporogenesis until anthesis (pollen shed). The LAT52 gene contains 5′ and 3′ untranslated regions of 110 and approximately 150 nucleotides, respectively, and a single intron with a highly repetitive sequence. A TATA box motif is located 28 nucleotides upstream of the transcription start site. The gene encodes a putative protein of 18 kDa that is cysteine rich and has an N-terminal hydrophobic region with characteristics similar to eucaryotic secretory signal sequences. LAT52 is a single or low copy gene in tomato and shares homology with sequences in tobacco. © 1989 Springer-Verlag.
• Matoba, S., Fukayama, J., Wing, R. A., & Ogrydziak, D. M. (1988). Intracellular precursors and secretion of alkaline extracellular protease of Yarrowia lipolytica.. Molecular and cellular biology, 8(11), 4904-4916.
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  PMID: 3211132;PMCID: PMC365584;Abstract: Processing and secretion of the alkaline extracellular protease (AEP) from the yeast Yarrowia lipolytica was studied by pulse-chase and immunoprecipitation experiments. Over half of newly synthesized AEP was secreted by 6 min. Over 99% of AEP activity which was external to the cytoplasmic membrane was located in the supernatant medium. Polypeptides of 55, 52, 44, 36, and 32 kilodaltons (55K, 52K, 44K, 36K, and 32K polypeptides) were immunoprecipitated from [3H]leucine-labeled cell extracts by rabbit antibodies raised against mature, secreted AEP (32K polypeptide). Experiments with tunicamycin and endoglycosidase H indicated that the 55K, 52K, and 44K polypeptides contained about 2 kilodaltons of N-linked oligosaccharide and that the 36K and 32K polypeptides contained none. Results of pulse-chase experiments did not fit a simple precursor-product relationship of 55K----52K----44K----36K----32K. In fact, maximum labeling intensity of the 52K polypeptide occurred later than for the 44K and 36K polypeptides. Secretion of polypeptides of 19 and 20 kilodaltons derived from the proregion of AEP indicated that one major processing pathway was 55K----52K----32K. The gene coding for AEP (XPR2) was cloned and sequenced. The sequence and the immunoprecipitation results suggest that AEP is originally synthesized with an additional preproI-proII-proIII amino-terminal region. Processing definitely involves cleavage(s) after pairs of basic amino acids and the addition of one N-linked oligosaccharide. Signal peptidase cleavage, dipeptidyl aminopeptidase cleavages, and at least one additional proteolytic cleavage may also be involved.
• Davidow, L. S., Apostolakos, D., O'Donnell, M. M., Proctor, A. R., Ogrydziak, D. M., Wing, R. A., Stasko, I., & DeZeeuw, J. R. (1985). Integrative transformation of the yeast Yarrowia lipolytica. Current Genetics, 10(1), 39-48.
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  Abstract: We have derived a DNA-mediated transformation system for the yeast Yarrowia lipolytica based on the lithium acetate method Ito et al. (1983) developed for Saccharomyces cerevisiae. The first plasmid used, pLD25, contains the Y. lipolytica LEU2 gene (coding for the enzyme beta-isopropylmalate dehydrogenase, EC 1.1.1.85) on a 6.6 kb piece of DNA inserted into pBR322. The recipient strain ATCC 20688 contains the rarely reverting mutation leu2-35. The Y. lipolytica LEU2 gene complements leuB mutants in Escherichia coli and leu2 mutants in S. cerevisiae and it also hybridizes weakly to the S. cerevisiae LEU2 gene. Y. lipolytica transformation frequencies of up to 104 Leu+ cells per microgram of DNA, per 108 viable cells have been obtained from plasmds linearized with restriction enzymes. The more than 100-fold increase in transformation frequency obtained by using linearized DNA instead of intact plasmid resembles the situation seen in S. cerevisiae for site-directed integrative transformation (Orr-Weaver et al. 1981). The transformants were stable when grown in non-selective medium. We found that pLD25 integrated at the leu2 region when either linear or intact plasmid was used to transform Y. lipolytica. © 1985 Springer-Verlag.

Presentations

• Wing, R. A. (2022, December). The Center for Desert Agriculture: Finding ways to feed the world without destroying our planet!. KAUST Research Open Week. KAUST: KAUST.
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  Go to 8hr 30min to see my presentation
• Wing, R. A. (2022, February). Forgotten Food -Future Food Approaches for Food Security in the Region. IOFS Strategic Commodities & Food Safety Forum: Rice Development Block. Sheraton Grand Doha Resort & Convention Hotel: Islamic Organization for Food Security.
• Wing, R. A. (2022, February). Harvesting 15MY of Evolutionary History to Help Solved the 10-billion People Qustion. PGEC Weekly Seminar Series. Zoomland: USDA/ARS Plant Gene Expression Center.
• Wing, R. A. (2022, February). Harvesting 15MY of Evoutionary History to Help Solved the 10-billion People Question. KAUST/CIRAD Spring Lecture Series. Zoom: KAUST/CIRAD.
• Wing, R. A. (2022, February). What if we could feed the next billion people and support the planet. The Expo 2020 Dubai World Majlis - Farms of the Future. Dubai, UAE: Australian Pavilion.
• Wing, R. A. (2022, January). The Center for Desert Agriculture: Finding ways to feed the world without destroying our planet!. KAUST Gifted Student Program - Convocation. Zoomland: KAUST.
• Wing, R. A. (2021, February). The panOryza Genome Project: Harvesting 15MY of Oryza Evolutionary History to Help Solve the 10-billion People Question. KeyGene Webinar Building for a Pangenome - Part 1. Webinarr (the Netherlands): KeyGene.
• Wing, R. A. (2021, June). Towards a Digital Genebank of Cultivated Rice & its Wild Oryza Relatives to Accelerate Sustainable Crop Production. Transforming Rice Breeding The program of the first meeting of the Center of Excellence for rice. Zoomland: Islamic Organization for Food Security.
• Wing, R. A. (2021, November). Neo-Domestication: Developing New Crops from Native Plants. International Workshop on the Future of Seawater Farming & Saline Agriculture in the Kingdom of Saudi Arabia. Zoomland (MEWA headquarter, Riyadh, KSA): Ministry of the Environment, Water & Agriculture.
• Wing, R. A. (2021, November). The 10-billion people question: How do we feed our world without destroying our planet?. COP26 - Understanding and Employing Climate Change Science -Panel 2. Zoomland (Glasgow, Scotland, UK): AEON Collective/KAUST.
• Wing, R. A. (2021, Novemberr). Construction and Analysis of a Platinum Standard Reference Genome Panel for all 27 Species of the Genus Oryza. 18th International Symposium on Rice Functional Genomics. Barcelona, Spain: CRAG: Center for Research in Agricultural Genomics.
• Wing, R. A., & Wing, R. A. (2021, November). 15 Million Year of Evolutionary History in the Genus Oryza to Help Solve the 10-Billion People Question. 2021 International Cooperation Seminar on the Conservation and Utilization of Biological Resources. Zoomland (Guangxi University, China): State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources Guangxi University.
• Wing, R. A. (2019, April). The International Oryza Map Alignment Project (*IOMAP): Harvesting 15-MY of evolutionary history of rice and its wild relative to help solve the 10-billion people question. Invited Plant Science Workshop Speaker. Thuwal, Saudi Arabia: King Abdullah University of Science & Technology.
• Wing, R. A. (2019, August). Harvesting 15MY of Oryza Evolutionary History to Help Solve the 10-billion People Question. KAUST Marine Science Summer Course. Thuwal, Saudi Arabia: KAUST.
• Wing, R. A. (2019, February). The International Oryza Map Alignment Project (*IOMAP): Harvesting 15-MY of evolutionary history of rice and its wild relative to help solve the 10-billion people question. Invited Seminar Speaker. Cuernavaca, Mexico: Universidad Nacional Autónoma de México (UNAM – Genome Sciences Center).
• Wing, R. A. (2019, February). The International Oryza Map Alignment Project (*IOMAP): Harvesting 15-MY of evolutionary history of rice and its wild relative to help solve the 10-billion people question. Invited Seminar Speaker. León, Mexico: Universidad Nacional Autónoma de México (UNAM – School of Higher Studies).
• Wing, R. A. (2019, February). The International Oryza Map Alignment Project (*IOMAP): Harvesting 15-MY of evolutionary history of rice and its wild relative to help solve the 10-billion people question. Invited Seminar Speaker. Mexico City, Mexico.: Universidad Nacional Autónoma de México (UNAM – Main Campus).
• Wing, R. A. (2019, February). The International Oryza Map Alignment Project (*IOMAP): Harvesting 15-MY of evolutionary history of rice and its wild relative to help solve the 10-billion people question. Invited Seminar Speaker. Mexico City, Mexico: Universidad Nacional Autónoma de México (UNAM - Botanical Garden/Institute of Biology).
• Wing, R. A. (2019, February). The International Oryza Map Alignment Project (*IOMAP): Harvesting 15-MY of evolutionary history of rice and its wild relative to help solve the 10-billion people question. Invited Speaker. Irapuato, Guanajuato, Mexico: Laboratorio Nacional de Genómica para la Biodiversidad y Departamento de Ingeniería Genética de Plantas, Cinvestav.
• Wing, R. A. (2019, January). Havesting 15 Million Year of Oryza Genome Evolution to Help Solve the 10-Billion People Question - New Gene Evolution. International Plant & Animal Genome XXVII. San Diego: Comparative Genomics Workshop.
• Wing, R. A. (2019, January). Havesting 15 Million Year of Oryza Genome Evolution to Help Solve the 10-Billion People Question - The Oryza Pan Reference Genome. International Plant & Animal Genome XXVII. San Diego, CA: Gramene Workshop.
• Wing, R. A. (2019, January). The 10-billion people question. FFA Guest Speaker, Tanque Verde High School. Tanque Verde High School, Tucson, Arizona: Tanque Verde High School.
• Wing, R. A. (2019, June). Harvesting 15MY of Oryza Evolutionary History to Help Solve the 10-billion People Question. KEYS Symposium 2019 “Crop innovations in a climate change era”. Wageningen, Netherlands: KeyGene.
• Wing, R. A. (2019, May). Harvesting 15MY of Oryza Evolutionary History to Help Solve the 10-billion People Question. UK Rice Research Consortium Meeting. Nottingham, UK: University of Nottingham.
• Wing, R. A. (2019, November). Latest Advances in Rice Genetics and Genomics. 11th The Rice Trader World Rice Conference. Manila, Philippines: The Rice Trader World Rice Conference.
• Wing, R. A. (2019, October). Harvesting 15MY of Oryza Evolutionary History to Help Solve the 10-billion People Question. Frasergen Seminar Series. Wuhan, China: Frasergen.
• Wing, R. A. (2019, October). The International Oryza Map Alignment Project (IOMAP) from a Platinum Reference Genome Sequence Perspective. 17th International Symposium on Rice Functional Genomics. Taipei, Taiwan: Academia Sinica.
• Wing, R. A. (2019, September). Towards establishment of a set of 42 platinum standard reference genome sequences that span the genetic diversity of cultivated rice & and its wild relatives using PacBio long-read sequencing and BioNano validation technologies. AGI’s PacBio Sequel II Launch Party. Tucson, Arizona: University of Arizona.
• Wing, R. A. (2018, November). Harvesting 15MY of evolutionary history of the genus Oryza to help solve the 10-billion people question. The First "Tsung-lo Lo" Symposium. Shanghai, China: National Key Laboratory of Plant Molecular Genetics and the Shanghai Institute of Plant Physiology & Ecology, Chinese Academy of Sciences.
• Wing, R. A. (2018, September). Harvesting 15MY of Oryza Evolution to Help to Solve the 10 Billion People Question. Ecosystem Genomics Seminar Series , UA. University of Arizona, Keating BioReserach Building: Bio5.
• Wing, R. A. (2017, January). Harnessing 15 MY of Oryza Genome Evolution to Help Solve the 10-billion People Question. International Plant & Animal Genome Meeting XXV. San Diego: Rice Functional Genomics Workshop.
• Wing, R. A. (2017, January). Introducing 5 New High-Quality PacBio Genome Assemblies for Rice to Help Solve the 10-billion People Question. International Plant & Animal Genome Meeting XXV. San Diego: Pacific Biosciences Industry Workshop.
• Wing, R. A. (2017, October). Harnessing 15-MY of Oryza Genome Evolution to Help Solve the 10-billion People Question. International Workshop - Evolution of Biological Traits. Center for Advanced Studies, Seestraße 13, 80802 München, Germany: Ludwig Maximilian University, Center for Advanced Studies, Seestraße 13, 80802 München.
• Wing, R. A. (2017, October). Harnessisng 15MY of Oryza evolution to help solve the 10-billion people question. Agricultural Genomics 2017: Functional Genomics towards Green Crops fofr Sustainable Agriculture. Wuhan, China: Nature Conferences/Huazhong Agricultural University.
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  Opening Keynote Lecture
• Wing, R. A. (2016, October). Harnessing 15MY of Oryza Genome Evolution to Help Solve the 10-Billion People Question. Progress report and the Antle Endowed Chair. Tanamura & Antle Farm, Salinas, Ca: Tanimura & Antle Inc..
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  I was invited to update the Tanimura and Antle Farm on my research activities over the past 2 years.
• Wing, R. A. (2015, February). Genetically Modified Organisms – An Informal Discussion. Environmental Health Sciences Cafe at Gentle Ben's. Tucson, AZ: Environmental Health Sciences & Gentle Ben's.
• Wing, R. A. (2015, February). The International Oryza Map Alignment Project: Development of a genus-wide comparative genomics platform to help solve the 9-billion people question. New York University - Abu Dhabi. Abu Dhabi, UAE: NYU-AD.
• Wing, R. A. (2015, January). Evolution of the collective Oryza genome: Development and analysis of a genus-wide genome biology platform to help solve the 9-billion people question. International Plant & Anima Genome XXIII: Rice Functional Genomics Workshop. San Diego, CA: NA.
• Wing, R. A. (2015, January). Old School/New School Genome Sequencing: One Step Backwards–a Quantum Leap Forward. International Plant & Anima Genome XXIII: The Resurgence of Reference Quality Genome Sequence Workshop. San Diego, CA: NA.
• Wing, R. A. (2015, January). Top 20 BMAP Progress & Platinum Genome Sequencing Progress on 2 Rice Genomes. International Plant & Anima Genome XXIII: Brassicales Map Alignment Project Workshop. San Diego, CA: NA.
• Wing, R. A. (2015, July). BAC to the Future with PacBio’s Long Read Sequencing Technology. 3rd Asia PacBio User Group Meeting. Singapore: Pacific Biosciences.
• Wing, R. A. (2015, July). Natural Variation Across the Oryza Genus. Plant & Animal Genome - Asia. Singapore: PAG-Asia.
• Wing, R. A. (2015, March). Evolution of the collective Oryza genome: Development of a genus-wide comparative genomics platform to help solve the 9-billion people question. B-Debate: Evolution of Plant Phenotypes. From Genomes to Traits. Barcelona, Spain: B-Debate: International Center for Scientific Debate.
• Wing, R. A. (2015, March). Evolution of the collective Oryza genome: Development of a genus-wide comparative genomics platform to help solve the 9-billion people question. UC Riverside, Institute for Integrative Genome Biology's Seminar Series. Riverside, CA: IIGB, UC Riversity.
• Wing, R. A. (2015, March). Why African Rice Perseveres as a Globally Important Crop. Tucson Festival of Book - Science City. Tucson, AZ: Tucson Festival of Books.
• Wing, R. A. (2014, April). The International-Oryza Map Alignment Project: Establishment and Analysis of a Within Genus Comparative Genomics Platform to Help Solve the 9-Billion People Question. 10th Annual Soybean Biotechnology Symposium. Colombia, MO: University of Missouri.
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  Plenary Lecture
• Wing, R. A. (2014, December). The International Oryza Map Alignment Project: Development of a genus-wide comparative genomics platform to help solve the 9 billion-people question. World Policy Conference V. Seoul, South Korea: WPC.
• Wing, R. A. (2014, January). Brassicales Map Alignment Project Workshop - Boechera Whole Genome Profiling: Toward a Platinum Standard Genome. International Plant & Animal Genome Conference. San Diego, CA.
• Wing, R. A. (2014, January). Challenges and the Future of Genome Sciences. Tucson Winter Breeding Institute. Tucson, Arizona: Tucson Winter Breeding Institute.
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  1.5 hr lecture
• Wing, R. A. (2014, January). Plant Genomes and Genome Sequencing – History. Tucson Winter Breeding Institute. Tucson, Arizona: Tucson Winter Breeding Institute.
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  1.5 hour lecture
• Wing, R. A. (2014, June). BAC to the Future – From Gene Assembly Space to Platinum Standard Genomes. INRA CNRGV - Plant Genomes Day. Toulouse, France: INRA CNRGV.
• Wing, R. A. (2014, June). The genome of African rice (Oryza glaberrima): Evidence for independent domestication. UP - Plant Genomics & Evolutionary Biology Seminar Series. Perpignan, France: University of Perpignan.
• Wing, R. A. (2014, March). The genome of African rice (Oryza glaberrima): Evidence for independent domestication. Institute of Genetics Seminar Series. Gainesville, FL: University of Florida.
• Wing, R. A. (2014, March). The genome of African rice (Oryza glaberrima): Evidence for independent domestication. UNLV Biology Department Seminar Series. Las Vegas, NV: UNLV.
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  Seminar
• Wing, R. A. (2014, May). From Genome Biology to Functional Breeding: New Opportunities & Challenges for IRRI and the World. IRRI, Plant breeding and Genetics Seminar. Los Banos, Philippines: IRRI.
• Wing, R. A. (2014, May). From Genome Biology to Functional Breeding: New Opportunities & Challenges for IRRI and the World. Plant & Animal Genome - Asia. Singapore: International Rice Informatics Consortium.
• Wing, R. A. (2014, May). The Arizona Phenotyping Network – A Plan for Economic Development of Arizona Agriculture for the Future. 22nd Annual Convention Arizona Seed Trade Association Meeting. Tubac, Arizona: 22nd Annual Convention Arizona Seed Trade Association.
• Wing, R. A. (2014, May). Using Rice Genomics to Help Solve the 9-Billion People QuestionUsing Rice Genomics to Help Solve the 9-Billion People Question. Borderlands Brewery Science Cafe. Tucson, AZ: Borderland Brewery.
• Wing, R. A. (2014, November). Helping Solve the 9-Billion People Question. 5th Annual Plant Science Family Night. Tucson, AZ: Ventana Vista Elementary School/University of Arizona.
• Wing, R. A. (2014, November). The genome sequence of African rice (Oryza glaberrima)and evidence for independent domestication. 12th International Symposium on Rice Functional Genomics. Tucson, AZ: University of Arizona.
• Wing, R. A. (2014, October). Evolution of the collective Oryza genome: Development and analysis of a genus-wide genome biology platform to help solve the 9-billion people question. Ecology & Evolutionary Biology Seminar Series. Tucson, AZ: University of Arizona.
• Wing, R. A. (2014, October). The Arizona Phenotyping Network A Plan for Economic Development of Arizona Agriculture for the Future. YAC Special Semnar. Yuma, AZ: Yuma Ag Center.
  More info

  This talk was organized by YAC and the UA Development Foundation for Representatives of the Sam Wall Foundation.
• Wing, R. A. (2012). Rice 2020 Revised: An Urgent Call to Mobilize and Coordinate Rice Functional Genomics Worldwide. 10th International Symposium on Rice Functional Genomics. Chiangmei, Thailand.
• Wing, R. A. (2012). Sequencing the Collective Oryza Genome. 20th Plant & Animal Genome Meeting, Rice Functional Genomics Workshop. San Diego, CA.
• Wing, R. A. (2012). Sequencing the Collective Oryza genome to Help Solve the 9-Billion People Question. 10th International Symposium on Rice Functional Genomics. Chiangmei, Thailand.
• Wing, R. A. (2012). Sequencing the Collective Oryza genome to Help Solve the 9-Billion People Question. 31st IUBS General Assembly Conference. Suzhou, China.
• Wing, R. A. (2012). Sequencing the Collective Oryza genome to Help Solve the 9-Billion People Question. 3rd International Symposium on Genomics and Crop Genetic Improvement. Wuhan, China.
• Wing, R. A. (2012). Sequencing the Collective Oryza genome to Help Solve the 9-Billion People Question. Dialogue on Designer Rice for the future: Perceptions & Prospects. Hyderabad, India.
• Wing, R. A. (2012). Sequencing the Collective Oryza genome to Help Solve the 9-Billion People Question. Frontiers of life science and innovation of agricultural biotechnology - Doctoral Student Forum Of China. Wuhan, China.
• Wing, R. A. (2012). Sequencing the Collective Oryza genome to Help Solve the 9-Billion People Question. Plant Genetics and Breeding Seminar Series. Los Banos, The Philippines: International Rice Research Institute.
• Wing, R. A. (2012). Sequencing the Collective Oryza genome to Help Solve the 9-Billion People Question. Plant Genomics in China XIII. Tai an, Shandong Province, China.
• Wing, R. A. (2012). Sequencing the Collective Oryza genome to Help Solve the 9-Billion People Question. USDA Semi-Arid Research Center Seminar Series. Maricopa, AZ: USDA Semi-Arid Research Center.
• Wing, R. A. (2012). Sequencing the collective Oryza genome to help solve the 9-billion people question. Quebec City, Canada.
• Wing, R. A. (2011). Development and Analysis of Comparative Genomics Platforms from Closely Related Species: Oryza and Brassicaceae as Case Studies. PAG's Evolution of Genome Size Workshop. San Diego, CA.
• Wing, R. A. (2011). Sequencing the Collective Oryza Genome. Rice Functional Genomics Workshop. 20th Plant & Animal Genome Meeting. San Diego, CA.
• Wing, R. A. (2011). The International Oryza Map Alignment Project: Sequencing the collective Oryza genome - O. glaberrima as a model system. PAG's Keygene NV Workshop. San Diego, CA.
• Wing, R. A. (2011). The Oryza Map Alignment Project: Development and Analysis of a Genus-wide Comparative Genomics Platform to Help Solve the 9-Billion People Questio. 9th International Symposium on Rice Functional Genomics. Taipei, Taiwan.
• Wing, R. A. (2011). The Oryza Map Alignment Project: Development and Analysis of a Genus-wide Comparative Genomics Platform to Help Solve the 9-Billion People Question. Riverside, CA: UCR's Institute for Integrative Genome Biology.
• Wing, R. A. (2011). The Oryza Map Alignment Project: Development and Analysis of a within-Genus Comparative Genomics Platform for the World's Most Important Food Crop-Rice. 18th Penn State Plant Biology Symposium. State College, PA.
• Wing, R. A. (2011). The Oryza Map Alignment Project: Development and Analysis of a within-Genus Comparative Genomics Platform for the World's Most Important Food Crop-Rice. UGA's Plant Center Spring Symposium. Athens, GA.
• Wing, R. A. (2011). The Oryza Map Alignment Project: Development and analysis of a within-genus comparative genomics platform for the world's most important food crop- Rice. Corvallis, OR: OSU's Center for Genome Research and Biocomputing.

Poster Presentations

• Wing, R. A. (2018, July). The International Oryza Map Alignment Project (*IOMAP): Exploring 15-MY of evolutionary history of rice and its wild relative to help solve the 10-billion people question. American Association of Plant Biology Annual Meeting, Montreal, CA. Montreal, Canada: ASPB.