*3.4. Prediction and Analysis of the Candidate Gene*

Based on the *B. oleracea* genome databases (TO1000) (http://plants.ensembl.org/ Brassica\_oleracea) (accessed on 5 May 2021), a total of 3 anthocyanin-related and 14 predicted protein-coding genes were found in the interval of *BoPur7.1* and *BoPur9.1*, respectively (Table 3). According to domain annotations from InterPro and BLASTX (best hit) analyses, two of these fourteen genes have not been annotated (Table 3). The other fifteen candidate genes were as follows: *Bo7g096780* (homologous gene AT5G24520), encoding the transparent testa glabra 1; *Bo7g099150* (homologous gene AT3G49940), encoding the LOB domain-containing protein 38; *Bo7g108300* (homologous gene AT4G22880), encoding the anthocyanin synthase; *Bo9g174870* (homologous gene AT5G08000), encoding the X8-GPI family of proteins; *Bo9g174880*, *Bo9g174890*, and *Bo9g174900* (homologous gene AT5G07990), encoding flavonoid 3 hydroxylase (F3'H), which catalyzed the conversion of dihydrokaempferol to dihydroquercetin in anthocyanin biosynthesis [45]; *Bo9g174920* (homologous gene AT5G07960), encoding the Asterix-like protein; *Bo9g174940* (homologous gene AT5G07890), encoding the myosin heavy chain-like protein; *Bo9g174950* (homologous gene AT5G07830), encoding the glucuronidase 2; *Bo9g174960* (homologous gene AT5G07820), encoding the calmodulin-binding, protein-like protein; *Bo9g174970* (homologous gene AT5G07800), encoding the flavin-containing monooxygenase; and *Bo9g174980*, *Bo9g174990*, and *Bo9g175000* (homologous gene AT5G07740), encoding the actin-binding protein. *BoF3'H* was annotated as a homologue of the anthocyanin biosynthesis gene.



<sup>a</sup> The best hits of the seven *B. oleracea* genes compared to *A. thaliana* (AT). <sup>b</sup> GO annotations for seven Bo to AT best-hit genes obtained from TAIR.

> A primer pair, spanning the full-length CDS of *BoF3'H* was designed, and PCR was performed using the cDNA of BT 126 and SN 60 as a template. DNA sequencing revealed that the full-length of *BoF3'H* in purple-head SN 60 is 1669 bp, whereas it was 1600 bp in green-head SN 60. Compared with *BoF3'H* in BT 126, a deletion of 68 bp and 1bp was found at nucleotide 1080 and 1501, respectively, and nine SNPs were present in SN60 (sequences of BT 126 and SN60 were illustrated in Table S3). The polymorphism of the candidate gene BoF3'H was further confirmed in the segregating population. To delineate the potential candidate genes regulating the anthocyanin biosynthesis in broccoli, differ-ential expression profiling of *BoF3'H* was performed in two parental lines, at three

head developmental stages. The expression of *BoF3'H*, *BoANS2*, and *BoLED38* in BT 126 were up-regulated at three head developmental stages of anthocyanin accumulation, and the expression levels were significantly higher than those in SN60 (Figure 4B–F).

**Figure 4.** Structure identity and expression of broccoli gene *BoF3'H*. (**A**) Comparison of *BoF3'H* promoter and cDNA sequence between parental lines BT 126 and SN 60; (**B**–**E**) relative expression of *BoF3'H*, *BoTTG1*, *BoANS2*, and *BoLED38* at three head developmental stages. Significant differences between BT 126 and SN 60 are indicated by \* based on Students *t* test (\* *p* < 0.05, \*\* *p* < 0.01; \*\*\* *p* < 0.001); (**F**) the phenotype of the head during three developmental stages. The upper part depicts the head of BT 126, while the lower part depicts the head of SN 60. Scale bar = 1 cm.
