Integrated Optimized HPLC-MS/MS Profiling and GWAS Uncover Candidate Genes for Folate Content in Cucumber Fruits

Folate is an essential B vitamin critical for human health. As a frequently consumed raw vegetable, cucumber represents a promising dietary source for folate. Breeding high-folate cucumber varieties has been challenging due to limited knowledge of its folate composition and genetic regulation. In this study, we developed an optimized HPLC-MS/MS method capable of detecting 12 folate derivatives, enabling accurate quantification in crops with high water content. We analyzed the folate content in fruits from a core cucumber germplasm collection over two growing seasons. Our results showed that 5-M-THF is the most abundant folate derivative, with the South of China type exhibiting the highest content. Through GWAS, we identified 35 significant loci associated with folate accumulation, including eight pleiotropic loci. Furthermore, we identified four candidate genes: CsaV3_3G033660 and CsaV3_3G033670 for fa3.3, and CsaV3_1G029540 and CsaV3_1G029550 for fa1.5. This study provides valuable genetic resources and molecular markers for breeding folate-enriched cucumbers.