Functional Characterization of Anthocyanin Biosynthesis-Related () Genes in Blueberries ().

() genes contribute greatly to anthocyanin biosynthesis in plants. Up to now, however, research on the gene family and the key anthocyanin-related members in blueberries () has been limited. In this study, we performed a genome-wide identification of the blueberry gene family, identifying 36 genes categorized into five subfamilies. Gene expression analysis showed that three Subfamily III members () and four Subfamily V members () are highly expressed in blueberry fruits, particularly at late ripening stages. Transient overexpression analysis in apple fruits verified the contributions of and to anthocyanin biosynthesis, with showing better promoting effects. Blueberry fruit-based transient overexpression further confirmed the promoting effects of on anthocyanin accumulation and the expression of anthocyanin-related structural genes (especially its downstream () and () genes). The promoter contains binding sites for both bHLH and MYB transcription factors (TFs). Consistently, yeast one-hybrid and dual-luciferase assays confirmed that anthocyanin-related VcMYB-1 and VcbHLHs can bind to and activate the promoter. Furthermore, co-overexpressing / with led to much higher anthocyanin accumulation than overexpressing alone, indicating that these TFs positively regulate anthocyanin biosynthesis by upregulating . In summary, our study characterized the blueberry gene family and demonstrated the role of in anthocyanin biosynthesis.