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

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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.