*3.3. Validation of QTL-Seq-Derived Anthocyanin Biosynthesis QTL through High-Throughput SNP*

To assess *BoPur7.1* and *BoPur9.1*, detected by QTL-seq, a high-throughput SNP was carried out. The genotyping data of 26 SNP markers were mapped to a highly dense intraspecific genetic region of chromosomes 7 and 9, depicting polymorphisms between parental genotypes and between LAB and HAB, and were combined with phenotypic features of a second F2 mapping population with 280 individuals. Classical QTL analysis, based on interval mapping and composite interval mapping, revealed two highly important genomic regions: [BoSNP37 (32.5 cM) to BoSNP38 (33.22 cM)], harboring a potent (LOD: 13.1) *Pur* QTL (*BoPur9.1*) on broccoli chromosome 9, and [BoSNP1 (0 cM) to BoSNP2 (16.43 cM)], harboring a potent (LOD: 14.6) *Pur* QTL (*BoPur7.1*) on broccoli chromosome 7. The detected QTLs had an interval of 0.72 cM with 72,683 bp [BoSNP22 (51,716,244 bp) to BoSNP23 (51,788,927 bp)] on chromosome 9 and an interval of 16.43 cM with 6,920,903 bp [BoSNP1 (36,784,249 bp) to BoSNP2 (43,705,152 bp)] on chromosome 7 (Figure 3). The proportions of phenotypic variants caused by the *BoPur9.1* and *BoPur7.1* QTLs were 28.19% and 38.12%, respectively (Table 2).

**Table 2.** Quantitative trait loci (*p* < 0.05) associated with anthocyanin biosynthesis in broccoli.


LOD-logarithm of odds; PVE-phenotypic variance explained; Add-additive effect; Dom-dominance effect.

To further delineate the *BoPur9.1* QTL, the genotyping data of 17 selected SNP markers, the most tightly linked to *BoPur9.1* (SNP10–SNP26), were analyzed in 580 F3 mapping individuals. Loci mapping analysis identified a major QTL for anthocyanin biosynthesis, designed by two SNP markers, including SNP22 and SNP23. This result was consistent with the QTL analysis supporting a major anthocyanin biosynthesis QTL *BoPur9.1*, which was located in the genomic interval of 51.71–51.79 Mb on chromosome 9 (Table S2).
