Anthocyanins are the principal color-producing compounds synthesized in developing fruits of strawberry (Fragaria spp.). Substantial natural variation in color have been observed in fruits of diploid and octoploid accessions, resulting from distinct accumulation and distribution of anthocyanins in fruits. Anthocyanin biosynthesis is controlled by a clade of R2R3 MYB transcription factors, among which MYB10 has been shown as the main activator in strawberry fruit. Here, we show that MYB10 mutations cause most of the anthocyanin variation observed in diploid woodland strawberry (F. vesca) and octoploid cultivated strawberry (F. ×ananassa). Using a mapping-by-sequencing approach, we identified a gypsytransposon insertion in MYB10 that truncates the protein and knocks out anthocyanin biosynthesis in a white-fruited F. vesca ecotype. Two additional lossof-function MYB10 mutations were identified among geographically diverse whitefruited F. vesca ecotypes. Genetic and transcriptomic analyses in octoploid Fragaria spp. revealed that FaMYB10-2, one of three MYB10 homoeologs identified, residing in the F. iinumae-derived subgenome, regulates the biosynthesis of anthocyanins in developing fruit. Furthermore, independent mutations in MYB10-2 are the underlying cause of natural variation in fruit skin and flesh color in octoploid strawberry. We identified a CACTA-like transposon (FaEnSpm-2) insertion in the MYB10-2 promoter of red-fleshed accessions that was associated with enhanced expression and anthocyanin accumulation. Our findings suggest that putative cis regulatory elements provided by FaEnSpm-2 are
required for high and ectopic MYB10-2 expression and induction of anthocyanin biosynthesis in fruit flesh. We developed MYB10-2 (sub-genome) specific DNA markers for marker-assisted selection that accurately predicted anthocyanin phenotypes in octoploid segregating populations.
