Systematic identification of R2R3-MYB S6 subfamily genes in Brassicaceae and its role in anthocyanin biosynthesis in Brassica crops

Abstract The Brassicaceae family includes Arabidopsis thaliana, various vegetables and oil crops. The R2R3-MYB genes of the S6 subfamily are crucial for regulating anthocyanin biosynthesis, however, their systematic identification in Brassicaceae plants is still incomplete. Here, we systematically identified homologous genes of R2R3-MYB transcription factors from the S6 subfamily across 31 Brassicaceae species. A total of 92 homologous genes were identified, with species representation ranging from 0 to 10 genes per species. Phylogenetic analysis classified these homologous genes into six distinct groups. Notably, approximately 70% of the homologous genes were found within the G6 group, indicating a high degree of evolutionary conservation. Furthermore, a phylogenetic analysis was conducted on 35 homologous genes obtained from six species within the U's triangle Brassica plants. The findings provided evidence of significant conservation among orthologous genes across species and demonstrated strong collinearity on subgenomic chromosomes, with notable tandem duplications observed on chromosomes A7 and C6. Subsequently, we predicted the cis-acting elements of these 35 homologous genes, and analyzed their structures, conserved motifs, and characteristic conserved domains, confirming the significant similarities between orthologous genes. Additionally, we employed white and purple flower rapeseed specimens to conduct qRT-PCR validation of the key genes and transcriptional regulators associated with the anthocyanin synthesis pathway. The results revealed significant differential expression of BnaPAP2.A7.b in purple flowers, alongside the differential expression of BnaPAP2.C6.d. Ultimately, based on previous research and the findings of this study, we propose a transcriptional regulatory framework to govern anthocyanin accumulation in distinct tissues or organs of B. napus. Our findings offer a novel perspective on the functional diversification of R2R3-MYB transcription factors within the S6 subfamily homologous genes, while also shedding light on the regulatory network governing anthocyanin biosynthesis in Brassicaceae species.