A key SNP in the flavonoid biosynthesis pathway gene CsaV3_6G009030 is highly correlated with cucumber fruit bitterness

Cucumber fruit bitterness is a prominent issue affecting fruit quality and seriously compromises the commercial value of cucumbers. In this study, near-isogenic lines (NILs) were developed through six consecutive backcross generations using the bitter cucumber inbred line ‘5’ (P2) as the recurrent parent and the non-bitter cucumber inbred line ‘WK15’ (P1) as the donor parent, generating a BC6F2 population. Bulk segregant RNA-seq (BSR-seq) combined with metabolomics analysis was performed to screen for candidate regions and metabolic pathways significantly associated with fruit bitterness. A 5.8 Mb (2.7–8.5 Mb) candidate region for bitterness was mapped to chromosome 6. Functional analysis of the target region revealed significant enrichment of the flavonoid biosynthesis pathway. Metabolic network analysis indicated that six flavonoid differential metabolites, including 4-methoxycinnamic acid and 2′-hydroxychalcone, were significantly enriched in bitter cucumbers, and their expression levels were highly consistent with the bitter phenotype. Through gene functional annotation and SNP validation, a key gene, CsaV3_6G009030 , involved in the flavonoid biosynthesis pathway was identified, and a functional dCAPS marker was developed based on this gene. This study reveals a potential association between flavonoid metabolism and bitterness formation in cucumber, providing a theoretical basis and technical support for the further application of marker-assisted selection in breeding high-quality, non-bitter cucumber varieties.