*3.2. QTL-Seq Analysis*

The high-throughput, whole-genome resequencing of both parents, as well as the LAB and HAB libraries, yielded 158.49 to 261.57 million reads per sample. Based on the *B. oleracea* reference genome (estimated genome size~630 Mb), the read mapping ratio was >93% (Table 1). The mean coverage of the reference genome, across all samples, was 33×. As depicted in Table 1, the 4× coverage of each specimen ranged from 86.85% to 92.43%. Meanwhile, the value of Q20 was above 93% in all specimens (data not shown). The comparative genome sequence analysis of the parental DNA and DNA bulks with the reference genome revealed 1,117,709 polymorphic SNPs.


**Table 1.** Whole-genome mapping statistics for parents, as well as LAB and HAB bulks.

The SNP-index of each SNP, that distinguished LAB from HAB, was determined. The mean SNP-index, covering 1 Mb genomic sequence, was assessed separately in LAB and HAB, by the 1 kb sliding window method, and mapped against all *B. oleracea* reference chromosomes (9 in total). The Δ (SNP-index) was determined by combining the SNPindex data of LAB and HAB and was plotted against the positions (Mb) on the *B. oleracea* genome. The QTL-seq analysis detected two major genomic regions on chromosomes 7 (36,784,249–44,791,849) (*BoPur7.1*) and 9 (46,217,406–52,600,419) (*BoPur9.1*), with significant associations with anthocyanin biosynthesis in broccoli (Figure 2). Moreover, LAB and HAB mapping plants with reduced or elevated anthocyanin biosynthesis had the majority of SNP alleles from SNP 1 to SNP 26 (Table S1). An important genomic region [BoSNP5 (46,217,406 bp) to BoSNP 26 (52,600,419 bp)] harboring the QTL *Pur* on chromosome 9 showed a Δ (SNP-index) that was markedly different from 0 (*p* < 0.05) (Figures 2A and 3B). The QTL-seq data confirmed an important QTL (*BoPur9.1*) located at the 6.38 Mb genomic interval [46,217,406 (BoSNP 5) to 52,600,419 (BoSNP 26) bp] on chromosome 9, which was associated with the regulation of anthocyanin biosynthesis in broccoli.

**Figure 2.** Distribution of two progeny SNP-index on the chromosome length, represented by the horizontal axis (Mb); vertical axis: SNP-index. (**A**) Δ (SNP-index) graphs generated from QTL-seq study; (**B**) SNP-index graphs depicting the HAB (high anthocyanin biosynthesis bulk); (**C**) SNP-index graphs depicting the LAB (low anthocyanin biosynthesis bulk). Gray shaded boxes indicate significant QTLs.

**Figure 3.** Maps of the *Pur* loci on C07 (**A**) and C09 (**B**) constructed using 26 SNP markers.
