*Article* **Identification of Major Loci and Candidate Genes for Anthocyanin Biosynthesis in Broccoli Using QTL-Seq**

**Chunqing Liu 1,†, Xueqin Yao 1,†, Guangqing Li 1, Lei Huang 1, Xinyan Wu <sup>2</sup> and Zhujie Xie 1,\***


**Abstract:** Anthcyanins determine the colors of flowers, fruits, and purple vegetables and act as important health-promoting antioxidants. BT 126 represents a broccoli variety with a high content of anthocyanins (5.72 mg/g FW). Through QTL-seq bulk segregant analysis, the present study aimed to determine the quantitative trait loci (QTLs) involved in anthocyanin biosynthesis in the F2 population (n = 302), which was obtained by crossing BT 126 with a non-anthocyanin-containing SN 60. The whole-genome resequencing of purple (n = 30) and green (n = 30) bulk segregates detected ~1,117,709 single nucleotide polymorphisms (SNPs) in the *B. oleracea* genome. Two QTLs, tightly correlated with anthocyanin biosynthesis (*p* < 0.05), were detected on chromosomes 7 (*BoPur7.1*) and 9 (*BoPur9.1*). The subsequent high-resolution mapping of *BoPur9.1* in the F2 population (n = 280) and F3 population (n = 580), with high-throughput genotyping of SNPs technology, narrowed the major anthocyanin biosynthesis QTL region to a physical distance of 73 kb, containing 14 genes. Among these genes, *Bo9g174880*, *Bo9g174890,* and *Bo9g174900* showed high homology with *AT5G07990* (gene encoding flavonoid 3 hydroxylase), which was identified as a candidate gene for *BoPur9.1*. The expression of *BoF3'H* in BT 126 was significantly higher than that in SN60. Multiple biomarkers, related to these QTLs, represented potential targets of marker-assisted selection (MAS) foranthocyanin biosynthesis in broccoli. The present study provided genetic insights into the development of novel crop varieties with augmented health-promoting features and improved appearance.

**Keywords:** *Brassica oleracea*; broccoli; QTL; candidate gene; anthocyanin
