**4. Discussion**

Increasing the anthocyanin content in Brassica vegetables represents an important goal for nutriment breeding. This work discovered two important genomic regions harboring anthocyanin biosynthesis QTLs *BoPur7.1* and *Bopur9.1* on chromosomes 7 and 9, based on intra-specific broccoli mapping individuals, through whole-genome NGS-based, high-throughput QTL-seq. The two QTL-seq-derived *Pur* QTLs were subsequently verified by Hi-SNP analysis. In previous studies, a major locus was reported to control anthocyanin pigmentation. In purple cauliflower (*B. oleracea* var *botrytis*), a *Pur* gene, encoding the transcription factor R2R3 MYB, was isolated [43]. Broccoli flower head's purple sepal trait is regulated by a major loci and two minor loci on chromosome C01 [20]. The purple gene of non-heading Chinese cabbage is subject to a single dominant inheritance mode but does not follow the Mendel law [33]. Due to the considerable variations in anthocyanin content, a novel locus was mapped in the linkage group R07 in purple turnip plants [46]. Moreover, a single dominant gene on C09, named *BoPr*, was reported to control anthocyanin pigmentation in leaves. The physical region of the C09 QTL was from 19,018,694 to 24,359,626 (5.3 Mb in internal length), which was different from our major QTLs for head anthocyanin contents. The major locus on C09 from 51,716,244 to 51,788,927 was included in this study. This indicates a population-specific inheritance modality for certain QTLs controlling anthocyanin biosynthesis in the Brassica species. Hence, the integrated approach developed here could be used for rapidly identifying the target QTLs, important

genes, and/or alleles involved in the qualitative and quantitative traits of various crops. Diverse models of purple traits, related to anthocyanins, may come from different genetic backgrounds. The purple gene in ornamental kale (*B. oleracea* L. var. *acephala*) shows a single dominant inheritance pattern on chromosome C09, and *Bo9g058630* encoding dihydroflavonol 4-reductase (DFR) was considered to be a candidate gene [22].

Combining QTL-seq with Hi-SNP analysis, the 0.73 Mb QTL *Bopur9.1* region [BoSNP37 (51,716,244 bp) to BoSNP38 (51,788,927 bp)], encompassing 14 genes, was detected on chromosome 9, which accounted for ∼28.19% of all phenotypic variations in anthocyanin biosynthesis. Among these genes, according to their respective annotations (Table 3), three genes, *Bo9g174880*, *Bo9g174890*, and *Bo9g174900* were homologues of Arabidopsis *F3'H*, encoding flavanone 3 -hydroxylase. The *BoF3'H* gene has been suggested to have played a critical role in modifying plant coloration [45], which contained a coding sequence length of 1536 bp and encoded a protein of 511 amino acids with four exons. All of the 3 candidate genes became aligned to the different regions of *BoF3'H*. An increased expression of *F3'H* was found to be responsible for anthocyanin production in a number of anthocyaninaccumulating mutants. For example, the ectopic expression of apple *MdF3'H* can increase the production of flavonols and cyanidin-based pigments in the Arabidopsis *tt7* mutant, under nitrogen pressure [47]. The *VvF3'H* gene was identified from grapevine, and its ectopic expression results in high accumulation levels of anthocyanin and flavonols in the petunia *ht1* mutant line [48]. *OsF3'H* editing in the Heugseonchal or Sinmyungheugchal variety, using the CRISPR/Cas9 system, might be responsible for ocher seeds with lower anthocyanin contents than wild-type black rice plants [49]. Two copies of the *F3'H* gene were isolated in the barley genome and exhibited a tissue-specific expression pattern of anthocyanin synthesis [50]. Furthermore, nine SNPs and a 68-bp insertion, between the 3rd and 4rd exon of *BoF3'H* CDS sequences, were found, which may cause a gain-of-function mutation (Figure 4A). The 68 bp InDel was in the open reading frame (between E3 and E4), as well as the T InDel in the 3 end. Therefore, a frame shift is expected, resulting in a putative truncated or extended protein. In addition, since most enzymes had conserved regions, anthocyanin discoloration might occur, as a result of *BoF3'H* enzyme inactivity, due to one of the two isoforms. In addition, the 6.92 Mb QTL *Bopur7.1* region [BoSNP1 (36,784,249 bp) to BoSNP2 (43,705,152 bp)], encompassing 3 anthocyanin-related genes, was detected on chromosome 7, which accounted for ∼38.12% of all phenotypic variations in anthocyanin biosynthesis. Among these genes, *BoANS2* and *BoLED38* were activated at three head developmental stages of anthocyanin accumulation [12,13]. However, further experiments involving transformation are needed to verify whether the function of this gene is responsible for the purple head in broccoli.

Most of the agronomic traits are controlled by multiple QTLs. The traditional QTL finemapping method usually requires several generations of backcrossing, screening of a large population for recombinants and exhaustive field phenotyping, which is both time-consuming and labor-intensive [51]. Although the genotyping-by-sequencing (GBS) approach has been shown to be an efficient way to develop high-resolution genetic mapping [52], the genotyping of a large population with genome-wide markers is expensive. It is important to note that the QTL-seq approach has been successfully used to conduct a BSA analysis, as well as deployed for the mapping of a segregated population of homozygous parental lines with opposite phenotypes. Using QTL-seq, only two samples need to be sequenced. The sequence coverage is decided by genome size and complexity, but as an example for broccoli, sufficient data could be generated by sequencing each bulk to 30× coverage. Further, it's an effective method to identify markers most tightly linked to the trait.

In this study, two identified QTLs were distributed on chromosome 7 and 9, respectively. The purple intensity and size of the heading leaves had great variations among F2 individuals. The anthocyanin content of purple head BT 126 was also affected by the temperature condition. Therefore, environmental factors might have an important effect on anthocyanin synthesis. The genetic mechanism of anthocyanin biosynthesis is complex. Candidate genes that control the purple trait may be different in diverse genetic backgrounds and may have different spatial and temporal expression patterns.

In summary, the above findings indicated that combining QTL-seq, Hi-SNP analysis and differential gene expression profiling might help identify candidate genes manipulating anthocyanin biosynthesis at major QTL intervals in broccoli. *Bo9g174880*, *Bo9g174890*, and *Bo9g174900* were first selected as the strong candidate genes at the *BoPur* locus. The functional assessment of the candidate gene homologs, identified within the confidence intervals of both QTLs, would provide novel insights into the detailed mechanisms of anthocyanin biosynthesis in broccoli.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/horticulturae7080246/s1. Table S1: SNPs genetically mapped on chromosome 7 and 9, used for anthocyanin biosynthesis targeted QTL-seq in broccoli; Table S2: quantitative trait loci (*p* < 0.05), associated with anthocyanin biosynthesis in F3 population; Table S3: sequences of BT 126 and SN60.

**Author Contributions:** Conceptualization, Z.X.; methodology, C.L. and X.Y.; investigation, C.L., L.H., and X.W.; resources, X.Y. and G.L.; data curation, C.L.; writing—original draft preparation, C.L.; writing—review and editing, Z.X.; supervision, Z.X.; funding acquisition, Z.X. and X.Y. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Shanghai Agriculture Applied Technology Development Program, grant number G2016060105" and the Youth Talent Development Plan of Shanghai Municipal Agricultural System, grant number 20180116.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** We acknowledge Bin Wang, Shanghai BioWing Applied Biotechnology Company, China for the kind help in data analysis.

**Conflicts of Interest:** The authors declare no conflict of interest.
