*2.3. QTL-Seq Analysis*

By applying an established QTL-seq method, based on SNP-index and Δ( SNP-index) estimates, candidate genomic regions harboring the main QTL(s) involved in anthocyanin biosynthesis in broccoli were identified. Only the SNPs homozygous in either parent and polymorphic between the parents were prepared for the further analysis. Then 1,117,709 SNPs, between both parents, were selected. The read depth for the above-mentioned homozygous SNPs, in LAB and HAB bulks, was obtained to evaluate the SNP-index. The SNP-index, calculated according to the reads order-checking depth information, utilized short reads covering the given nucleotide position, calculated the differs reads bar number, and accounted for the ratio of the total number [26]. The genotype of one parent was utilized as a reference to determine the number of reads for the parental genotype, or other genotypes, in the LAB and HAB libraries. Then, the number of the various reads was divided by the total read number, and the ratio constituted the SNP-index of the base sites. The SNP-index points below 0.3, in both libraries, were removed. The sliding window method was utilized to present the SNP-indexes for the entire genome. All SNP-indexes in a given window were averaged to obtain the SNP-index for that particular window. The window size of 1Mb and the step size of 1Kb were routinely utilized. The Δ (SNPindex) was then calculated using the following formula: [SNP index (HAB bulk)—SNP index (LAB bulk)].
