Fruit color is a key quality trait in strawberry breeding and cultivar development, as it directly influences consumer preference and marketability. Anthocyanins are the pigments responsible for the red coloration in strawberries, and the transcription factor MYB10 gene plays a crucial role in regulating the anthocyanin biosynthetic pathway. Our previous study identified a homoeolog-specific copy, MYB10-1B, located on chromosome 1B, as a key regulator of fruit color. The natural mutation in MYB10-1B, such as in the variety ‘Florida Pearl’ leads to the development of white fruit. Building on this discovery, we applied CRISPR/Cas9-mediated homoeolog-specific editing to target the functional dominant allele, MYB10-1B, in the cultivated octoploid strawberry ‘Florida Brilliance’, successfully altering the fruit color from red to white. Gene expression analysis in the edited lines revealed downregulation of MYB10-1B and key anthocyanin biosynthesis genes (CHS, DFR, and ANS). Furthermore, whole-genome resequencing results showed precise on-target mutations in MYB10-1B with minimal off-target effects. This study highlights the successful application of homoeolog-specific CRISPR/Cas9-mediated gene editing in polyploid species and provides a foundation for functional genomics and advanced breeding strategies in strawberries. Importantly, our findings demonstrate that specific targeting of the dominantly expressed homoeologous copy is essential for inducing phenotypic changes in polyploids. This underscores the importance of precise gene editing in octoploid strawberry to drive trait improvement.

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