Differential expression ofCCD4(4B) drives natural variation in fruit carotenoidcontent in strawberry ( Fragaria spp.)

Pigments, mainly anthocyanins and carotenoids, are important contributors to fruits visual appearance and nutritional properties. In strawberry (Fragaria spp.), the genetic and molecular mechanisms regulating fruit carotenoid biosynthesis and its natural variation remain largely unexplored. In this study, we sought to identify genomic loci contributing to variation in yellow flesh pigmentation. A major QTL, qYellow Flesh-4B, accounting for 82% of the total phenotypic variation was identified on F. x ananassa chromosome 4B. Following a candidate gene approach, we determined that CCD4(4B), a carotenoid cleavage dioxygenase (CCD), was the underlying gene. Specific polymorphisms on CCD4(4B) promoter region were associated with the yellow flesh phenotype and with differential regulation of CCD4(4B) expression during fruit ripening. Furthermore, CCD4(4B) expression levels were negatively correlated with violaxanthin, lutein, zeaxanthin, {beta}-carotene and total carotenoid content. The role of CCD4(4B) in carotenoid turnover was confirmed by transient overexpression in F. x ananassa fruits, which led to a decrease in carotenoid accumulation. Notably, a -35 C>T SNP identified in CCD4(4B) promoter was found to be predictive for CCD4(4B) expression, and carotenoid content in fruits of a diverse germplasm collection, which included different octoploid Fragaria species. Taken together, these results provide important genetic insights into the natural variation of carotenoid content in strawberry. The High-Resolution Melting (HRM) DNA test here developed offers a fast and reliable method to predict high fruit carotenoid content, representing a useful tool for breeding projects aiming to enhance the nutritional value of this crop.