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，每年棉花生产的收入估计就达1000亿美元。棉花是研究纤维细胞分化、细胞扩增、纤维素生物合成以及多倍体基因组进化和作物驯化的优良模式系统。高地或美洲棉（Gossypium hirsutum L.）和Pima或埃及棉（Gossypium barbadense L.）已经从异源四倍体物种中独立驯化。提高棉纤维产量和质量将为石油基合成纤维提供可持续的替代品。为驯化的异源四倍体棉花生成参考级基因组序列将使基础和应用研究成为可能，这将为纤维发育和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产生广泛影响。由于大多数作物都是多倍体起源的，因此该项目开发的方法和原理也将推动对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/）和NCBI的SRA（http：//www.ncbi.nlm.nih.gov/geo/info/seq.html）。 植物材料和注释序列的最终版本将通过棉花研究社区网站CottonGen（http：//www.cottongen.org/icgi/home）分发。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。将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密切合作，提高教育和外联的有效性。 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中的基本问题。 物理图谱引导的杂交方法与全基因组鸟枪法（WGS）和测序基因分型（GBS）相结合，是对复杂多倍体物种（包括棉花和其他重要物种）的“金标准”参考级基因组进行测序和组装的最佳策略之一。小麦等重要作物。 该研究小组开发了细菌人工染色体（BAC）资源，包括物理图谱和BAC末端序列（BES），WGS，PacBio长读段和高地棉花（TM-1）的单核苷酸多态性（SNP）数据。 此外，关键的试点数据已经产生，这是必要的，在准备一个成功的战略测序异源四倍体棉花。 具体来说，该项目将从覆盖~85%基因组的最小平铺路径中对单个BAC插入物进行测序和组装。 将通过整合WGS重叠群、配对和PacBio长读段的额外3，000个或更多BAC的第二次迭代来填充和/或减少缺口。 序列重叠群和支架将通过高密度连锁图和BES靶向GBS排序。 最终序列将由十三对同源假分子组成，包括通过靶向GBS和BAC荧光原位杂交进行验证。 参考级序列将由Phytozome使用从约20个棉花组织收集的RNA-seq数据进行注释。 该参考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%的高地棉花。 该序列将用于分析表观基因组及其在异源四倍体棉花纤维发育中的作用。

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.