Sequencing-Powered QTL Analysis, Association Mapping and Reverse Genetics in the Legume Lotus japonicus

Characterization of monogenic symbiotic mutant phenotypes found by forward genetic screens has been instrumental for establishing the framework of genes coordinating nodule formation in the model legumes Lotus japonicus and Medicago truncatula. To complement this approach it is now important to investigate more complex traits governing symbiosis. We are in the process of transforming the genomic resources of the model legume L. japonicus to allow QTL and genome-wide association studies of multigene inherited traits to be combined with efficient validation by reverse genetics. Our results and experiences with genome assembly and annotation, SNP marker generation, QTL and association analysis, as well the development of a sequenced insertion mutant population will be discussed. The reference genome sequence is being updated by integrating Illumina reads with existing shotgun and BAC Sanger data. By sequencing 30 additional Lotus accessions, a dense set of markers has been generated, allowing us to perform combined SHOREmap QTL and association analysis of nodulation traits in L. japonicus. To improve reverse genetic resources, we are generating an insertion mutant population based on the activity of the lotus retrotransposon 1 (LORE1). A pilot experiment with 3,500 plants yielding 8,000 insertion sites through sequencing of a single Illumina library consisting of pooled and barcoded samples has been completed. We are expanding the sequenced mutant population size at a rapid pace, aiming to quickly produce a comprehensive reverse genetic resource with ~60,000 lines. An update on the progress will be presented.