A functional genomics resource for Brassica napus: Development of an EMS mutagenized population and discovery of FAE1 point mutations by TILLING

Abstract

• Two ethylmethanesulfonate (EMS) mutant populations of the semi-winter rapeseed cv. Ningyou7 were constructed with high mutant load, to provide a TILLING platform for functional genomics in Brassica napus, and for introduction of novel allelic variation in rapeseed breeding. • Forward genetic screening of mutants from the M2 populations resulted in identification of a large number of novel phenotypes. Reverse genetic screening focused on the potentially multi-paralogous gene FAE1 (fatty acid elongase1), which controls seed erucic acid synthesis in rapeseed. A B. napus BAC library was screened, and loci in a reference mapping population (TNDH) were mapped to conclude that there are two paralogous copies of FAE1, one on each of the B. napus A and C genomes. • A new procedure is demonstrated to identify novel mutations in situations where two or more very similar paralogous gene copies exist in a genome. The procedure involves TILLING of single plants, using existing SNPs as a positive control, and is able to distinguish novel mutations based on primer pairs designed to amplify both FAE1 paralogues simultaneously. • The procedure was applied to 1344 M2 plants, with 19 mutations identified, of which three were functionally compromised with reduced seed erucic acid content. © National Key Laboratory of Crop Genetic Improvement (2008).