## 3 Results  
### Analysis of F3'H Homologous Sequences and Development of Specific Markers  
The development of molecular markers associated with a specific agronomic trait is important when breeding new broccoli varieties from different crosses. Previous genetic analysis showed that two QTLs, _Pur7.1_ and _Pur9.1_, located on chromosomes C7 and C9 controlled anthocyanin accumulation. _F3'H_, which is related to anthocyanin biosynthesis, may be a candidate gene on chromosome C9 [47]. The differences in the _F3'H_ gene between the two parents were investigated to confirm the linkage between _F3'H_ and anthocyanin-related trait segregation. The sequence analysis showed that a deletion of the 43-bp fragment existed at the second exon of _F3'H_ in SN60 green broccoli, which formed a premature termination codon (Figure 2). The insertion/deletion site of _F3'H_ was used to design the InDel marker (F3'H-F/R); -187 and 144 bp amplified products were obtained for BT126 and SN60, respectively. PCR-amplified bands indicated that the deleted fragment was heterozygous in the F1 plants, indicating that it could be used as a marker to distinguish _F3'H_/_F3'H_, _F3'H_/_F3'H_, and _f3'h_/_f3'h_ genotypes. It was subsequently validated in 720 F2-generation populations with phenotypic data. The results indicated that all of the f3'h/f3'h genotypes showed a green head consistent with that of SN60, whereas _F3'H_/_F3'H_ and _F3'H_/_f3'h_ genotypes could be obtained in purple individuals (Figure 3a). In addition, the marker was detected in 63 inbred lines. The _f3'h/f3'h_ genotype was detected in green broccoli inbred lines without anthocyanin at low temperatures. In contrast, the _F3'H_/_F3'H_ genotype was obtained in both purple broccoli inbred lines and green broccoli inbred lines with low anthocyanin at low temperatures (Figure 3b). Therefore, the introduction of a premature termination codon led to loss of function in _F3'H_, which is the key gene for determining broccoli coloration. However, both the deep purple and light purple populations had _F3'H_/_F3'H_ and _F3'H_/_f3'h_ genotypes, which indicated that _F3'H_ genotypes were not related to pigmentation grades.  
Examination of the _Pur7.1_ candidates in the two parental lines resulted in the detection of six single-nucleotide polymorphism (SNP) differences. Two KASP primers were designed by selecting SNP sites in the _Pur7.1_ candidate interval (C7:3 6.78 MB-43.70 MB) and used to genotype the 720 F2 individuals. The results showed that one KASP marker (Pur7.1-K1) at the 42,567,442 bp position on chromosome C07 had a C/T mutation in this SNP base (C for BT126 and T for SN60). The purple F2 individuals could be classified into three categories: deep purple, purple, and light purple. The results proved that green individuals had CC, TT, and CT genotype, whereas the deep purple, purple, and light purple individuals had CC, CT, and TT genotypes, which fit a 1:2:1 Mendelian ratio in the F2 generation (Figure 4). In addition, the _Pur7.1_-linked _Pur7.1_-K1 marker was detected in 63 inbred lines. The allele with mutation \"C\" was present in 91.1% of the purple lines and the allele with mutation \"T\" was present in 93.2% of the light purple lines.