Allelic Variation of <i>MYB10</i> Is the Major Force Controlling Natural Variation in Skin and Flesh Color in Strawberry ( <i>Fragaria</i> spp.) Fruit

The fruits of diploid and octoploid strawberry (<i>Fragaria</i> spp) show substantial natural variation in color due to distinct anthocyanin accumulation and distribution patterns. Anthocyanin biosynthesis is controlled by a clade of R2R3 MYB transcription factors, among which MYB10 is the main activator in strawberry fruit. Here, we show that mutations in <i>MYB10</i> cause most of the variation in anthocyanin accumulation and distribution observed in diploid woodland strawberry (<i>F. vesca</i>) and octoploid cultivated strawberry (<i>F</i> ×<i>ananassa</i>). Using a mapping-by-sequencing approach, we identified a <i>gypsy</i>-transposon in <i>MYB10</i> that truncates the protein and knocks out anthocyanin biosynthesis in a white-fruited <i>F. vesca</i> ecotype. Two additional loss-of-function mutations in <i>MYB10</i> were identified among geographically diverse white-fruited <i>F. vesca</i> ecotypes. Genetic and transcriptomic analyses of octoploid <i>Fragaria</i> spp revealed that <i>FaMYB10-2</i>, one of three <i>MYB10</i> homoeologs identified, regulates anthocyanin biosynthesis in developing fruit. Furthermore, independent mutations in <i>MYB10-2</i> are the underlying cause of natural variation in fruit skin and flesh color in octoploid strawberry. We identified a CACTA-like transposon (<i>FaEnSpm-2</i>) insertion in the <i>MYB10-2</i> promoter of red-fleshed accessions that was associated with enhanced expression. Our findings suggest that cis-regulatory elements in <i>FaEnSpm-2</i> are responsible for enhanced <i>MYB10-2</i> expression and anthocyanin biosynthesis in strawberry fruit flesh.