An intronic SNP in the Carotenoid Cleavage Dioxygenase 1 (CsCCD1) controls yellow flesh formation in cucumber fruit (Cucumis sativus L.)

Summary Vitamin A is a crucial yet scarce vitamin essential for maintaining normal metabolism and bodily functions in humans and can only be obtained from food. Carotenoids represent a diverse group of functional pigments that act as precursors for vitamins, hormones, aroma volatiles and antioxidants. As a vital vegetable in the world, elevated carotenoid levels in cucumber fruit produce yellow flesh, enhancing both visual appeal and nutritional value. However, the genetic mechanisms and regulatory networks governing yellow flesh in cucumbers remain inadequately characterized. In this study, we employed map‐based cloning to identify a Carotenoid Cleavage Dioxygenase 1 ( CsCCD1 ) as a key genetic factor influencing yellow flesh in cucumbers. A causal single nucleotide polymorphism (SNP) in the eighth intron of CsCCD1 led to aberrant splicing, resulting in a truncated transcript. The truncated protein has significantly decreased enzyme activity and increased carotenoid accumulation in the fruit. CRISPR/Cas9‐generated CsCCD1 knockout mutants exhibited yellow flesh and significantly higher carotenoid content compared to wild‐type cucumbers. Metabolic profiling indicated a marked accumulation of β‐cryptoxanthin in the flesh of these knockout mutants. The intronic SNP was shown to perfectly segregate with yellow flesh in 159 diverse cucumber germplasms, particularly within the semi‐wild ecotype Xishuangbanna, known for its substantial carotenoid accumulation. Furthermore, transient overexpression of CsCCD1 in yellow‐fleshed Xishuangbanna cucumbers restored a white flesh phenotype, underscoring the critical role of CsCCD1 in determining flesh colour in both cultivated and semi‐wild cucumbers. These findings lay a theoretical foundation for breeding high‐nutrient yellow‐fleshed cucumber varieties.