Differences in the CaMYBA genome between anthocyanin-pigmented cultivars and non-pigmented cultivars in pepper (Capsicum annuum)

Anthocyanin in pepper is beneficial as a food antioxidant compound and as a pigment for ornamentals, while unexpected anthocyanin accumulation in fruit, known as black spots, reduces the commercial quality of some cultivars. Previous studies demonstrated that the Anthocyanin (A) locus determines the anthocyanin accumulation in pepper fruits, and an MYB transcription factor, CaMYBA, was found to be located near the A locus. However, the causal gene sequence of the A locus has not yet been identified. With progress regarding genome information in pepper, two other homologous MYB genes were found to be located near CaMYBA, and they are also considered to be candidate genes for the A locus. In this study, we attempted to identify the causal gene sequence of the A locus by performing linkage analysis, genomic sequence analysis, and gene expression analysis of the three candidate MYB genes. A crossing experiment between pigmented 'Peruvian Purple' and non-pigmented cultivars confirmed that anthocyanin accumulation in the pigmented cultivar was controlled by a single locus. Gene expression analysis demonstrated that a basic helix-loop-helix transcription factor, CaMYC, and CaMYBA were expressed abundantly in pigmented cultivars, but the other two MYB genes were not. Genotyping of the Fsub(2) population derived from the cross demonstrated that the anthocyanin accumulation phenotype was highly linked to CaMYBA, but not to CaMYC. The DNA sequence of CaMYBA in pigmented cultivars had an insertion of a 4.3 kb retrotransposable element LINE-1 in the first intron, but that of non-pigmented cultivars did not. No pigmented cultivar-specific sequence was found in the promoter region of CaMYBA. Therefore, it was suggested that CaMYBA, but not the other two homologous MYB genes, is the A locus gene, and insertion of LINE-1 in CaMYBA appeared to be important for the regulation of anthocyanin accumulation, although the mechanism by which the LINE-1 insertion induces CaMYBA expression is unknown.