An alternative splicing caused by a natural variation in <i>BnaC02.VTE4</i> gene affects vitamin E and glucosinolate content in rapeseed (<i>Brassica napus</i> L.)

Vitamin E (VE) is essential for plants and animals. Rapeseed oil is rich in α-tocopherol (α-T), which is the most bioactive form of VE in human body. This study demonstrated that VE in rapeseed seeds was mainly controlled by embryo genotype through incomplete diallel hybridization. By genome-wide association study, the QTL-qVE.C02 associated with VE and α-T contents was detected in a Brassica napus association population, and the phenotypic contribution rate was up to 18.71%. BnaC02.VTE4, encoding gama-tocopherol methyltransferase, was proved as the target gene of qVE.C02 by genetic complementation. Two BnaC02.VTE4 haplotypes were identified in the population. Compared with BnaC02.VTE4^HapH, a point mutation from A to G at the 3' splicing site of the second intron were found in BnaC02.VTE4^HapL, resulting in alternative splicing and early termination of translation. HapL^1052(G-A), the site-directed mutagenesis fragment of BnaC02.VTE4^HapL, was introduced into Arabidopsis vte4 mutant and 8S088 (a BnaC02.VTE4^HapL accession), and the contents of VE and α-T in atvte4-4 and 8S088 seeds were increased by 90.10% to 307.29%. These demonstrated the point mutation as the causal for the difference in VE biosynthesis in rapeseed. Further, this variation also led to the significant difference in glucosinolate content between BnaC02.VTE4^HapH and BnaC02.VTE4^HapL accessions. Multi-omics analysis suggested that the expression of some genes and the accumulation of several metabolites related to the glucosinolate biosynthesis pathway were significantly increased in BnaC02.VTE4^HapL group. Moreover, by functional marker identification, the BnaC02.VTE4^HapH was found to be selected during domestication. Our findings offered promising opportunities for enhancing rapeseed quality traits.