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Summary: Brassica napus (oilseed rape) is an important oil crop in temperate regions, which originated from hybridization of Brassica oleracea and Brassica rapa. Owing to its polyploidy, the functional study of single genes is cumbersome. Phytic acid is considered as an antinutritive compound, and we aimed to knock out the underlying synthesis and transporter genes to identify low phytic acid mutants.We implemented a high‐throughput next‐generation sequencing screening protocol for an ethylmethane sulfonate population of 7680 plants in six gene families (BnMIPS, BnMIK, Bn2‐PGK, BnIPK1, BnIPK2, and BnMRP5) with two paralogues for each gene.A total of 1487 mutations were revealed, and the vast majority (96%) were confirmed by Sanger sequencing. Furthermore, the characterization of double mutants of Bn.2‐PGK2 showed a significant reduction of phytic acid contents.We propose to use three‐dimensional pooling combined with amplicon stacking and next‐generation sequencing to identify mutations in polyploid oilseed rape in a fast and cost‐effective manner for complex metabolic pathways. Furthermore, the mutants identified in Bn2‐PGK2 might be a very valuable resource for industrial production of oilseed rape protein for human consumption. [ABSTRACT FROM AUTHOR]

We developed two mutant populations of oilseed rape ( Brassica napus L.) using EMS (ethylmethanesulfonate) as a mutagen. The populations were derived from the spring type line YN01-429 and the winter type cultivar Express 617 encompassing 5,361 and 3,488 M plants, respectively. A high-throughput screening protocol was established based on a two-dimensional 8× pooling strategy. Genes of the sinapine biosynthesis pathway were chosen for determining the mutation frequencies and for creating novel genetic variation for rapeseed breeding. The extraction meal of oilseed rape is a rich protein source containing about 40% protein. Its use as an animal feed or human food, however, is limited by antinutritive compounds like sinapine. The targeting-induced local lesions in genomes (TILLING) strategy was applied to identify mutations of major genes of the sinapine biosynthesis pathway. We constructed locus-specific primers for several TILLING amplicons of two sinapine synthesis genes, BnaX.SGT and BnaX.REF1, covering 80-90% of the coding sequences. Screening of both populations revealed 229 and 341 mutations within the BnaX.SGT sequences (135 missense and 13 nonsense mutations) and the BnaX.REF1 sequences (162 missense, 3 nonsense, 8 splice site mutations), respectively. These mutants provide a new resource for breeding low-sinapine oilseed rape. The frequencies of missense and nonsense mutations corresponded to the frequencies of the target codons. Mutation frequencies ranged from 1/12 to 1/22 kb for the Express 617 population and from 1/27 to 1/60 kb for the YN01-429 population. Our TILLING resource is publicly available. Due to the high mutation frequencies in combination with an 8× pooling strategy, mutants can be routinely identified in a cost-efficient manner. However, primers have to be carefully designed to amplify single sequences from the polyploid rapeseed genome. [ABSTRACT FROM AUTHOR]