*2.2. DNA Purification, Library Generation, and Whole-Genome ReSequencing*

Total DNA was extracted from fresh leaves using the CTAB method [35]. A total of 1.5 μg DNA, per specimen, was utilized for DNA sample preparations. The DNA quality was assessed by 1% agarose gel electrophoresis. Among the F2 segregating population, composed of 302 plants, 30 with purple heads (high anthocyanin biosynthesis, HAB) and 30 with green heads (low anthocyanin biosynthesis, LAB) were selected to extract equal amount of DNA, pooled to construct the DNA bulks. The DNA bulks, together with the two parental DNA, were used for whole-genome resequencing.

By ultrasonication, 350-bp fragments were obtained from the tested DNA sample. A sequencing library was generated using the Truseq Nano DNA HT Sample Preparation Kit (Illumina, San Diego, CA, USA), according to the manufacturer's instructions. The constructed library was sequenced on the Illumina HiSeq4000 platform (Illumina, CA, USA), and 150-bp paired-end reads were produced with approximately 350-bp inserts. Stringent quality control (QC) steps were applied to ensure the reliability and accuracy of the reads. Then, the filtered, high-quality sequences from the DNA bulks and parental genotypes were aligned and mapped to the public *B. oleracea* genome database (TO1000) (http://plants.ensembl.org/Brassica\_oleracea) (accessed on 5 May 2021) with the Burrows-Wheeler alignment (BWA) tool [36,37]. Alignment files were converted into BAM files with SAMtools (Wellcome Trust Genome Campus, Cambridge, UK) [38]. SNP detection was carried out for each specimen, utilizing the UnifiedGenotyper function in the GATK3.8 software (The Broad Institute of Harvard and MIT, Cambridge, MA, USA) [39]. ANNOVAR (Children's Hospital of Philadelphia, Philadelphia, PA, USA) was employed for SNP annotation, based on the GFF3 file of the reference genome [40].
