Sequencing a Reference-grade Genome of the Domesticated Allotetraploid Cotton: the World's Largest Source of Renewable Textile Fiber

棉花是世界上最大的天然纺织纤维来源，也是主要的油料作物。仅在美国，每年棉花生产产生的收入估计就有1000亿美元。棉花是研究纤维细胞分化、细胞扩增、纤维素生物合成、多倍体基因组进化和作物驯化的优良模型系统。陆地棉或美洲棉（Gossypium hirsutum L.）和皮马棉或埃及棉（Gossypium barbadense L.）都是由异源四倍体种独立驯化的。提高棉纤维产量和质量将为石油基合成纤维提供一种可持续的替代品。获得驯化异源四倍体棉花的参考级基因组序列将促进基础和应用研究，为纤维发育、纤维素和壁生物合成提供新的见解，对生物质作物和生物燃料生产具有广泛影响。由于大多数作物都是多倍体起源，该项目开发的方法和原理也将推动其他具有复杂和多倍体基因组的作物的研究。基因组序列和基因表达数据将通过公共数据库提供，包括GenBank、GEO （http://www.ncbi.nlm.nih.gov/geo/）、miRBase （http://microrna.sanger.ac）。uk/)，以及NCBI的SRA （http://www.ncbi.nlm.nih.gov/geo/info/seq.html）。植物材料和最终版本的注释序列将通过棉花研究社区网站CottonGen （http://www.cottongen.org/icgi/home）发布。将为密西西比三角洲的初中、高中和本科院校开发植物基因组学和生物信息学方面的创新和可扩展的教育和培训模块。通过与奥尔康州立大学和密西西比河谷州立大学这两所历史上的黑人学院和大学的密切合作，将提高教育和推广的有效性。该项目的总体目标是开发高质量的序列资源，以解决具有经济重要性的作物植物的基本问题。物理图谱引导杂交方法与全基因组霰弹枪（WGS）和基因分型测序（GBS）相结合，是对复杂多倍体物种（包括棉花和其他重要作物，如小麦）进行“金标准”参考级基因组测序和组装的最佳策略之一。研究组开发了陆地棉（TM-1）的细菌人工染色体（BAC）物理图谱和BAC末端序列（BES）、WGS、PacBio长reads、单核苷酸多态性（SNP）数据等资源。此外，还获得了关键的试验数据，这对制定成功的异源四倍体棉花测序策略是必要的。具体来说，该项目将从覆盖约85%基因组的最小平铺路径对单个BAC插入进行测序和组装。通过整合WGS序列、配偶对和PacBio长读段，将另外3000个或更多的bac进行第二次迭代，填补或减少这些空白。序列组和支架将通过高密度连锁图和靶向bes的GBS进行排序。最终序列将包括13对同源伪分子，包括靶向GBS和bac荧光原位杂交验证。该参考级序列将由Phytozome用来自约20个棉花组织的RNA-seq数据进行注释。这一参考等级序列将立即发布给研究和育种界，以改善占世界棉花产量95%的陆地棉花。该序列将用于分析异源四倍体棉花的表观基因组及其在纤维发育中的作用。

Cotton is the world's largest source of natural textile fiber and is a main oilseed crop. The revenue resulting from annual cotton production is estimated to be $100 billion in the USA alone. Cotton is an excellent model system for studying fiber cell differentiation, cell expansion, and cellulose biosynthesis, as well as polyploid genome evolution and crop domestication. Upland or American cotton (Gossypium hirsutum L.) and Pima or Egyptian cotton (Gossypium barbadense L.) have been independently domesticated from allotetraploid species. Improving cotton fiber yield and quality will provide a sustainable alternative to petroleum-based synthetic fibers. Generating a reference-grade genome sequence for domesticated allotetraploid cotton will enable both basic and applied research that will provide new insights into fiber development and cellulose and wall biosynthesis with broad impact for biomass crops and biofuel production. Since most crops are of polyploid origin, methods and principles being developed from this project will also advance research in other crops with complex and polyploid genomes. Genomic sequence and gene expression data will be made available through public data depositories, including GenBank, GEO (http://www.ncbi.nlm.nih.gov/geo/), miRBase (http://microrna.sanger.ac.uk/), and the NCBI's SRA (http://www.ncbi.nlm.nih.gov/geo/info/seq.html). Plant materials and the final version of annotated sequence will be distributed through CottonGen (http://www.cottongen.org/icgi/home), the cotton research community website. Innovative and scalable education and training modules in plant genomics and bioinformatics will be developed for middle and high schools and undergraduate institutions in the Mississippi Delta. The effectiveness of education and outreach will be enhanced through close collaborations with Alcorn State University and Mississippi Valley State University, two Historically Black Colleges and Universities. The overall goal of this project is to develop high-quality sequence resources to address fundamental questions in crop plants that are of economic importance. The physical map-guided hybrid approach in combination with whole-genome shotgun (WGS) and genotyping-by-sequencing (GBS) is among the best strategies to sequence and assemble "gold-standard" reference-grade genomes of complex polyploid species including cotton and other important crops such as wheat. The research team has developed bacterial artificial chromosome (BAC) resources including a physical map and BAC-end sequences (BES), WGS, PacBio long reads, and single nucleotide polymorphism (SNP) data of Upland cotton (TM-1). Moreover, the critical pilot data have been generated, which are necessary in preparing a successful strategy for sequencing the allotetraploid cotton. Specifically, the project will sequence and assemble individual BAC inserts from the minimum tiling path that covers ~85% of the genome. The gaps will be filled and/or reduced by the second iteration of additional 3,000 or more BACs with integration of WGS contigs, mate-pair and PacBio long reads. The sequence contigs and scaffolds will be ordered by high-density linkage maps and BES-targeted GBS. The final sequence will consist of thirteen pairs of homoeologous pseudo-molecules, including validation by targeted GBS and BAC-fluorescence in situ hybridization. The reference-grade sequence will be annotated by Phytozome with the RNA-seq data collected from ~20 cotton tissues. This reference-grade sequence will be immediately released to the research and breeding communities to improve Upland cotton that accounts for 95% of the world cotton production. The sequence will be utilized for analyzing epigenomes and their roles in fiber development of allotetraploid cotton.