Safflower is featured with time-honored medical and economic values and developing into diverse phenotypic and genetic variations. In this study, to explore the critical genes associated with color phenotypes and flavonoid derivatives biosynthesis of safflower, BSA-seq, conflated with transcriptomic and metabolic methods were performed in two extreme colors (yellow and white) in the population of ‘ZHH0119’ and ‘XHH007.’ After crossing two parent plants reciprocally, the F₄ generation of two accessions were used to construct near-isogenic gene pools for the two extreme traits of yellow and white safflower. BSA-seq results located five QTLs regions on chromosomes 2, 8, 9, 10, and 12 including 6 CtPALs, 3 CtC4Hs, 2 Ct4CLs, 1 CtCHS, 32 CtUGTs, and 70 CtCYPs, which tied to the yellow color phenotype of safflower. Through transcriptome analysis of two accessions and at different flowering stages, 1 CtPAL, 5 CtC4Hs, 4 CtCHSs, 3 CtCHIs, 3 CtFLSs, 48 CtUGTs, 51 CtCYPs, and 75 transcription factors were revealed as significantly upregulated in the yellow accession compared to the white. Integrated analysis identified eight CtUGTs (CtUGT50-57) that exhibited significant positive correlations with chalcone glycosides of yellow safflower. Based on functional characterization, CtUGT52 was found to boost Hydroxysafflor yellow A (HSYA) content in yellow safflower which possessing substrate promiscuity (chalones, flavonols, and flavonoids) and catalytic promiscuity (flavonols and flavonoids), revealing its vital role in the HSYA biosynthesis through transgenic overexpression. Combining catalytic mechanism verification of CtUGT52 towards phloretin, kaempferol, and luteolin, our study to some extent, elucidated the modification function of CtUGTs for flavonoid aglycones in the flavonoid biosynthesis pathway of safflower.

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