Restriction Site Associated DNA Sequencing (RAD-Seq) and Genotyping

The RAD protocol was employed as described by Baird et al. (2008). The enzymes and restriction fragment sizes were evaluated based on the reference genome sequence (ftp://cucurbitgenomics.org/pub/cucurbit/genome/watermelon/97103/v1/) (Guo et al., 2013). MseI was selected for RAD library construction. The library for Illumina sequencing was constructed from 200 ng of each DNA sample. All libraries were sequenced using Illumina HiSeq X Ten at Shanghai Major Biological Medicine Technology Co., Ltd.

For single nucleotide polymorphism (SNP) calling, the Burrows-Wheeler Aligner (Li and Durbin, 2009) was applied for sequence alignment between the individual reads and the reference genome sequence, Genome AnalysisToolKit (McKenna et al., 2010) was used to detect SNP loci, and SAMtools (Li et al., 2009) was used to filter out SNP loci. The filtering of SNP loci was based on three criteria: (i) an average sequence depth of < fivefold in the parents and < threefold in the progeny; (ii) no polymorphism between the parents; and (iii) heterozygosity in the parents.