Identification and functional analysis of three new anthocyanin R2R3-MYB genes in .

We identified three novel members of the R2R3-MYB clade of anthocyanin regulators in the genome of the purple flowering wild accession, and we called them (). Two of these genes, and are inactivated by two different single base mutations in their coding sequence. All three of these genes are absent in the white flowering species and , in the red flowering , and in several lines, including R27 and W115. and other .  lines (M1, V30, and W59) instead harbor functional copies of the genes. Comparative, functional and phylogenic analysis of anthocyanin R2R3-MYB genes strongly suggest that the genes cluster is a duplication of the genomic fragment containing the other three R2R3-MYB genes with roles in pigmentation that were previously defined, the - (--) cluster. An investigation of the genomic fragments containing anthocyanin MYBs in different a accessions reveals that massive rearrangements have taken place, resulting in large differences in the regions surrounding these genes, even in closely related species. Yeast two-hybrid assays showed that the ASR proteins can participate in the WMBW (WRKY, MYB, B-HLH, and WDR) anthocyanin regulatory complex by interacting with the transcription factors AN1 and AN11. All three ASRs can induce anthocyanin synthesis when ectopically expressed in lines, but ASR1 appeared to be the most effective. The expression patterns of and cover several different petunia tissues with higher expression at early stages of bud development. In contrast, is only weakly expressed in the stigma, ovary, and anther filaments. The characterization of these novel MYB genes completes the picture of the MYB members of the petunia anthocyanin regulatory MBW complex and suggests possible mechanisms of the diversification of pigmentation patterns during plant evolution.