Direct Access to Millions of Mutations by Whole Genome Sequencing of an Oilseed Rape Mutant Population.

Induced mutations are an essential source of genetic variation in plant breeding. EMS mutagenesis has been frequently applied, and mutants have been detected by phenotypic or genotypic screening of large populations. In this study, a rapeseed M 2 population was derived from M 1 parent cultivar “Express” treated with EMS. Whole genomes were sequenced from fourfold (4x) pools of 1,988 M 2 plants representing 497 M 2 families. Detected mutations were not evenly distributed and displayed distinct patterns across the 19 chromosomes with lower mutation rates towards the ends. Mutation frequencies ranged from 32/Mb to 48/Mb. On average, 284,442 single nucleotide polymorphisms per M 2 DNA pool were found resulting from EMS mutagenesis. 55% were C→T and G→A transitions, characteristic for EMS induced (‘canonical’) mutations, whereas the remaining SNPs were ‘non-canonical’ transitions (15%) or transversions (30%). Additionally, we detected 88,725 high confidence insertions and deletions (InDels) per pool. On average, each M 2 plant carried 39,120 canonical mutations, corresponding to a frequency of one mutation per 23.6 kb. Roughly 82% of such mutations were located either 5 kb upstream or downstream (~56%) of gene coding regions or within intergenic regions (26%). The remaining 18% were located within regions coding for genes. All mutations detected by whole-genome sequencing could be verified by comparison with known mutations. Furthermore, all sequences are accessible via the online tool “EMS Brassica” ( http://www.emsbrassica.plantbreeding.uni-kiel.de/ ), which enables direct identification of mutations in any target sequence. The sequence resource described here will further add value for functional gene studies in rapeseed breeding.