A 43 Bp-Deletion in the F3'H Gene Reducing Anthocyanins Is Responsible for Keeping Buds Green at Low Temperatures in Broccoli

Most broccoli cultivars or accessions exhibit green buds under appropriate growth conditions, which turn purple at cold temperatures. However, certain cultivars consistently maintain green buds both during normal growth and at cold temperatures. In this study, we used BSA-seq (bulked segregation analysis-sequencing), along with fine mapping and transcriptome analysis to identify a candidate gene (flavonoid 3'-hydroxylase, F3'H) responsible for reducing anthocyanin accumulation in the mutant GS and HX-16 broccoli (Brassica oleracea L. var. italica), which could retain green buds even at low temperatures. A 43-bp deletion was detected in the coding sequence (CDS) of the F3'H gene in HX-16 and the mutant GS, which significantly decreased F3'H expression and the accumulation of cyanidin and delphinidin in the mutant GS. Furthermore, the expression of F3'H was upregulated at low temperatures in the wild line PS. Our results demonstrated the efficacy of utilizing the 43-bp InDel (Insertion-Deletion) in predicting whether buds in B. oleracea L. will turn purple or remain green at cold temperatures across forty-two germplasm materials. This study provides critical genetic and molecular insights for the molecular breeding of B. oleracea and sheds light on the molecular mechanisms underlying the effect of low temperatures on bud color in broccoli.