BANYULS genes from Brassica juncea and Brassica nigra: cloning, evolution and involvement in seed coat colour

Abstract

SUMMARYThe molecular mechanism underlying the yellow seed trait has been a subject of quality breeding in Brassica. Thus, uncovering the biosynthetic pathway of proanthocyanidin (PA) accumulation in the Brassica seed coat is a promising research programme. Arabidopsis thaliana BANYULS (BAN) encodes anthocyanidin reductase, which is involved in seed coat pigmentation. In the current study, 2 and 4 BAN homologues were isolated using one pair of primers from Brassica nigra and Brassica juncea, respectively. Reverse transcription polymerase chain reaction (PCR) analysis showed that BAN was expressed abundantly in the seed coat of black seeds and in the embryos of all lines, but not in the seed coat of yellow seeds. Primers incorporating B genome-specific nucleotide variations were designed according to previously published BAN gene sequences of Brassica species to discern the BAN sequence located in B genome origin of Brassica using allele-specific PCR amplification. Proanthocyanidins were also detected by p-dimethylaminocinnamaldehyde staining and a butanol–hydrochloric acid (BuOH–HCl) colorimetric assay in the seed coat of black seeds, but not in the seed coat of yellow seeds. Anthocyanins were not also detected in the seed coat of Brassica species by the BuOH–HCl assay. Both transcriptional and chemical analyses suggested that BAN genes could be involved in both the biosynthesis of PAs and colour formation in the seed coat of Brassica species, whereas no expression of the BAN gene could block biosynthesis of PAs in the yellow seed coat.