Genome‑wide association study identifies GhF3'H affects the spiraeoside biosynthesis in waste cotton (Gossypium hirsutum L.) flowers

Cotton flowers are rich in various flavonoids, exhibiting multiple biological activities, including antioxidant, anti-inflammatory, and anti-tumor effects. The biosynthetic mechanism of flavonoids in cotton flowers remains undefined. In this study, six macro-flavonoids were identified and quantified in the flowers of 373 G. hirsutum accessions based on HPLC technology. These flavonoids included quercetin (0–2.94 mg/g), isoquercetin (9.69–33.61 mg/g), hyperoside (2.54–8.78 mg/g), spiraeoside (0.87–7.10 mg/g), quercimeritrin (5.70–20.29 mg/g), and quercetin-3′-O-β-D-glucoside (4.23–21.87 mg/g). Several candidate genes associated with spiraeoside and quercimeritrin were identified through GWAS analysis. The study also revealed a significant upregulation of GhF3'H expression and an elevated content of spiraeoside in haplotype II compared to haplotype I. The ectopic overexpression of GhF3'H in tobacco led to a significant increase in dihydroquercetin, quercetin, and spiraeoside levels, and a decrease in naringenin content. LUC experiments confirmed the potential positive regulation of GhF3'H by GhTT2. This study provides vital insights into the biosynthetic mechanism of macro-flavonoids in the flowers of upland cotton population. The identified elite haplotype and candidate genes hold great promise as valuable tools for enhancing flavonoid content in cotton, thus facilitating the efficient utilization of cotton by-products in the future.