Genotyping-by-Sequencing of Pumpkin Cultivars

GBS libraries of 48 F₁ cultivars were prepared according to the protocol described by Elshire et al.^{26 (link)}. The 200 ng of genomic DNA for each cultivar were digested using a methylation-sensitive restriction enzyme, ApeKI (NEB, Ipswich, MA, USA). After digestion, the DNA fragments were ligated to different barcode adapters that were assigned to each cultivar. These DNA samples were pooled and amplified by PCR to generate GBS libraries. The libraries were sequenced with the pair-end method in the HiSeq 2500 platform (Illumina Inc., San Diego, CA, USA). For SNP calling, the filtered, high-quality sequencing reads were mapped to the C. maxima (Rimu) genome^{24 (link)} using the Burrows-Wheeler Alignment (BWA) method^{35 (link)} in the TASSEL-GBS pipeline^{36 (link)}. The resulting bi-allelic SNPs with 5x of minimum depth were filtered based on >5% of minor allele frequency and <10% of missing data for further analysis.