Exploring Nodulation through Genome-Wide Association Studies of Medicago truncatula

Genome-wide association studies (GWAS) identify candidate loci and illuminate genomic architecture of complex traits. The Medicago Hapmap Consortium (Univ. Minnesota, NCGR, Boyce Thompson, JCVI, Hamline Univ.) has resequenced 330 diverse accessions of M. truncatula and related taxa to discover >6,000,000 single nucleotide polymorphisms (SNPs). This dataset provides insight into nucleotide diversity (4-6 per kb), recombination (higher in centromeric, high gene density, NBS-LRR regions) and linkage disequilibrium (declines to background <10 kb). To explore the molecular basis of important phenotypes, the Medicago GWAS panel was assayed for nodulation, mycorrhization, growth and development then compared statistically to the SNP dataset. Flowering time and trichome density each exhibited a single major peak, tagging strongly supported candidates (orthologs of Arabidopsis FD for flowering; Petunia UNSHAVEN for trichomes). For nodulation, high scoring SNPs tagged loci genome-wide, including several reported nodulation-related genes (eg, CaML3, NFP, SERK2). Novel candidates include DNA repair, ubiquination, and molecular chaperones, while others are nodule-upregulated with unknown function. Preliminary functional analyses using stable hairpin knockdowns strongly support a role in nodulation for some uncharacterized candidates. Broader collaborations based on the GWAS platform have discovered candidates in Aphanomyces resistance (C. Jacquet; CNRS-Toulouse) and saponin biosynthesis (L. Sumner; Noble Foundation). A subset of 25 lines from the GWAS panel are being deeply sequenced and assembled independently of the published reference. These new Medicago genomes will enable detailed investigations regarding the structure and evolution of dynamic gene families critical to symbiosis, including NBS-LRRs and nodule-specific cysteine-rich peptides (NCRs).