Genome-wide SNP Discovery via GBS in Cucumis

To obtain genome-wide SNPs, GBS analyses [24 (link)] were conducted for 91 di- and 102 tetraploid individuals, with one of the latter included twice as a replicate. For the library preparation, 200 ng of genomic DNA was used and cut with the two restriction enzymes PstI-HF (NEB) and MspI (NEB). Library preparation, individual barcoding, and single-end sequencing on the Illumina NovaSeq were performed following Wendler et al. [28 (link)].
Barcoded reads from the 194 samples were de-multiplexed using the Casava pipeline 1.8 (Illumina). Adapter trimming of GBS sequence reads was performed with Cutadapt [29 (link)] within ipyrad v.0.9.58 [30 (link)] and reads shorter than 60 bp after adapter removal were discarded. GBS reads were clustered using the ipyrad 0.7.5 [30 (link)] pipeline with a clustering threshold of 0.85. We tested diverse ipyrad settings but at the end the default settings of parameter files generated with ipyrad were optimal for the other parameters. We generated one output that included the outgroup C. malyi, which was used for phylogenetic analyses, and a second output without C. malyi, which was used for principal component analysis (PCA) and population assignment analyses.