Expanding the Medicago truncatula Hapmap as a Platform for Exploring the Genetics of Legume Symbioses

项目负责人：Nevin Young（明尼苏达大学）合作负责人：Michael Sadowsky， Robert Stupar和Peter Tiffin（明尼苏达大学），Maria Harrison （Boyce Thompson植物研究所），Betsy Martinez-Vaz （Hamline大学），Jason Rafe Miller （J. Craig Venter研究所），Joann Mudge（国家基因组资源中心）本项目使用功能分析，重新组装基因组和生物信息学数据挖掘来表征模式豆科植物Medicago truncatula的共生基因。豆科植物值得注意的是它们与根瘤菌和丛枝菌根（AM）真菌形成复杂的共生关系。然而，现有的关于共生的知识主要来自于敲除突变体，这种方法往往会忽略一些微妙但有重要影响的基因，尤其是在当代进化中可能很重要的基因。在早期的研究中，通过全基因组关联分析（GWAS）发现了几个强有力支持的候选共生位点，对候选基因的支持通常包括独立的证据，如表达谱、与多个性状的相关性或与已知共生表型的共定位。目前的项目将通过包括Tnt1插入和RNAi“敲低”植物系的反向遗传实验来测试大约100个这些候选基因座。有希望的基因将通过涉及先前定义的Sinorhizobium和AM菌株的相互作用分析来检查，并使用一组48个测序的Sinorhizobium菌株来测试“基因对基因”的关系。尽管已知结构变异（SVs）和拷贝数变异（cnv）对基因组变异有重大影响，但早期的GWAS仅针对SNP变异进行定位。这与共生的基因组学特别相关，因为大的基因家族，如NB-ARC结构域基因和结节富含半胱氨酸肽（ncr）在共生中起着关键作用。本项目将对30个节点M. truncatula片段进行深度测序和重新组装，以发现SVs和cnv。svv和cnv将在全基因组范围内植入，从而导致新一轮的GWAS来发现在早期定位阶段缺失的共生位点。该项目的主要成果将是鉴定与当代共生进化相关的基因，以及M. truncatula基因组多样性的结构。这项研究工作将通过涉及哈姆林大学（一所位于明尼苏达大学附近的四年制大学）和波多黎各大学（UPR）的本科生来扩展。哈姆林的学生将在整个学年中工作，然后在夏季与UPR学生一起作为明尼苏达州生命科学暑期本科生研究计划（LSSURP）计划的一部分。他们的工作将针对重要但大部分未表征的sinorhizobbial酶，ACC脱氨酶，学生也将参与反向基因突变的筛选。这些经历将为学生提供发展他们自己的假设驱动项目的机会。通过项目pi的联合指导，学生培训还将帮助哈姆林大学的本科生研究项目在未来的外部研究计划中更具竞争力。基因组序列资源将通过medicagohapmap.org以及Genbank的Short Read Archive、dbSNP和FTP站点向公众开放。重要的是，Medicago Hapmap GWAS平台（可在medicagohapmap.org上获得）将为更广泛的研究界提供长期资源，以发现控制农业和生物学利益的数量变异的豆类基因，特别是美国第四大种植作物苜蓿（Medicago sativa）的性状变异

PI: Nevin Young (University of Minnesota)Co-PIs: Michael Sadowsky, Robert Stupar, and Peter Tiffin (University of Minnesota), Maria Harrison (Boyce Thompson Institute for Plant Research), Betsy Martinez-Vaz (Hamline University), Jason Rafe Miller (J. Craig Venter Institute), Joann Mudge (National Center for Genome Resources) This project uses functional assays, de novo genome assembly and bioinformatic data-mining to characterize symbiosis genes in the model legume Medicago truncatula. Legumes are noteworthy for the sophisticated symbioses they form with rhizobial bacteria and arbuscular mycorrhizal (AM) fungi. However, existing knowledge about symbioses comes primarily from knockout mutants, an approach that often misses genes of subtle yet significant effect, especially genes likely to be important in contemporary evolution. In earlier work, several strongly supported candidate symbiosis loci were discovered through genome-wide association analysis (GWAS) and support for candidate genes often included independent evidence like expression profile, correlation with multiple traits or co-localization with known symbiotic phenotypes. The current project will test ~100 of these candidate loci through reverse genetic experiments involving Tnt1 insertion and RNAi "knockdown" plant lines. Promising genes will be examined through interaction assays involving previously defined Sinorhizobium and AM strains and tested for "gene-for-gene" relationships using a panel of 48 sequenced Sinorhizobium strains. Earlier GWAS mapping only targeted SNP variation, even though structural variants (SVs) and copy number variants (CNVs) are known to have major impacts on genome variation. This is especially relevant to the genomics of symbiosis because the large gene families such as the NB-ARC domain-containing genes and nodule cysteine rich peptides (NCRs) play critical roles in symbiosis. This project will deeply sequence and de novo assemble 30 nodal M. truncatula accessions, in order to discover SVs and CNVs. SVs and CNVs will be imputed genome-wide, leading to a new round of GWAS to discover symbiotic loci missed in the earlier phase of mapping. The primary outcomes of this project will be the identification of genes associated with the contemporary evolution of symbiosis as well as the architecture of M. truncatula genomic diversity. This research effort will be extended by involving undergraduates from Hamline University, a four year institution located near the University of Minnesota, and from the University of Puerto Rico (UPR). Students at Hamline will work throughout the academic year and then together with UPR students during the summer as part of Minnesota's Life Sciences Summer Undergraduate Research Program (LSSURP) program. Their work will target the important but largely uncharacterized Sinorhizobial enzyme, ACC deaminase, and students will also participate in the screening of reverse genetic mutants. These experiences will provide the students the opportunity to develop their own hypothesis-driven projects. Through joint mentoring by project PIs, student training will also assist the undergraduate research program at Hamline to become more competitive for its own future external research initiatives. Genomic sequence resources will be available for public through medicagohapmap.org, and the Short Read Archive, dbSNP and FTP sites at Genbank. Importantly, the underlying Medicago Hapmap GWAS platform, available at medicagohapmap.org, will provide a long-term resource for the broader research community to discover legume genes controlling quantitative variation of agricultural and biological interest, especially trait variation in alfalfa (Medicago sativa), the fourth most widely cultivated crop in the U.S.