Genome-wide Annotation of Mutations in a Phenotyped Mutant Library Provides an Efficient Platform for Discovery of Causal Gene Mutations

Ethyl methanesulfonate (EMS) efficiently generates high-density mutations in genomes. Conventionally, these mutations are identified by techniques that can detect single-nucleotide mismatches in heteroduplexes of individual PCR amplicons. We applied whole-genome sequencing to 256-phenotyped mutant lines of sorghum (Sorghum bicolor L. Moench) to 16x coverage. Comparisons with the reference sequence revealed >1.8 million canonical EMS-induced G/C to A/T mutations, 22% of which were in genic regions, affecting >95% of genes in the sorghum genome. The vast majority (97.5%) of the induced mutations were distinct from natural variations. By applying the mutation database to phenotype analysis, we identified four causal gene mutations affecting drought tolerance, two mutations affecting heat tolerance, and two mutations affecting seed size that corresponded to previously reported seed size QTLs. Our results demonstrate that this collection of sequenced mutant lines can be used to efficiently discover new traits and their underlying causal mutations, thereby accelerating sorghum breeding.