Identification of a Candidate Gene and Development of a Marker for Anthocyanin-free Florets at Low Temperature in Japanese Broccoli (<i>Brassica oleracea</i> var. <i>italica</i>)

Broccoli produces green florets under normal growth conditions, but produces darkish-colored florets after low-temperature exposure due to the accumulation of anthocyanin, which often reduces its commercial value. Selecting anthocyanin-free genotypes requires exposing the florets to low temperatures, which is laborious and time-consuming. We identified a genomic region on chromosome C09, which was associated with anthocyanin pigments in florets at low temperatures, through quantitative trait locus sequencing (QTL-seq) analysis using the phenotype data obtained from field evaluation of anthocyanin pigments of F2 plants over two years. Among nine differentially expressed genes in the QTL region, one gene encoding flavonoid 3′-hydroxylase (F3′H) was selected as a candidate. Sequence analysis of Brassica oleracea F3′H (BoF3′H) of parental lines revealed the presence of three alleles, with one derived from the anthocyanin-free parents, Bof3′h-2, exhibiting a 43-bp deletion in the second exon, causing a frame-shift mutation. Designing DNA markers capable of distinguishing this mutation, we demonstrated that, among 35 F1 cultivars released in Japan, eight homozygous for the Bof3′h-2 allele showed no anthocyanin pigments and lower anthocyanin contents in florets in the autumn- to over-winter harvesting. The expression level the BoF3′H gene was not necessarily related to the difference in anthocyanin contents. Furthermore, seedlings of the Arabidopsis f3′h mutants exhibited no pigments and a significantly reduced anthocyanin content under low-temperature treatment compared to that in the wild type. These results suggested that the 43-bp deletion in BoF3′H is responsible for the anthocyanin-free trait in broccoli genetic resources, and the developed marker was deemed useful for marker-assisted breeding.