Identification of phytic acid mutants in oilseed rape (Brassica napus) by large scale screening of mutant populations through amplicon sequencing.

Abstract

Brassica napus (oilseed rape) is an important oil crop in temperate regions, which originated from hybridization of B. oleracea and B. rapa. Due to its polyploidy, the functional study of single genes is cumbersome. Phytic acid is considered as an anti-nutritive compound and we aimed to knockout the underlying synthesis and transporter genes to identify low phytic acid mutants. We implemented a high throughput NGS screening protocol for an EMS population of 7680 plants in 6 gene families (BnMIPS, BnMIK, Bn2-PGK, BnIPK1, BnIPK2 and BnMRP5) with two paralogs 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 rapeseed in a fast and cost-effective manner for complex metabolic pathways. Furthermore, the identified mutants in Bn2-PGK2 might be a very valuable resource for industrial production of rapeseed protein for human consumption.