Tomato SNP data were retrieved from the SOL Genomics portal, specifically from the “150 Tomato Genome Resequencing Project” (Aflitos et al., 2014) and the “AGIS Tomato 360 Resequencing Project” (Lin et al., 2014). SNPs were selected based on: (i) alternative cohort-wise allele count >8 (from homozygous or heterozygous loci), (ii) intron and UTR SNPs spaced at least 15 kbp from each other or SNPs in CDS, (iii) CDS SNPs spaced at least 5 kbp apart, and (iv) SNPs residing on anchored chromosomes. Eligible polymorphic sites were randomly selected for probe design by NuGen, resulting in filtered panels tested for sequencing performance and reproducibility, with about 5,000 probes finalized for genotyping via SPET (commercialized as Allegro®).
DNA was extracted using Qiagen, LGC Sbeadex, or a modified CTAB method, and libraries prepared with Ovation Rapid Library Systems. Sequencing was performed using the Illumina HiSeq 2500 platform with 75SE and 150SE chemistry for probe testing and genotyping. Base calling and demultiplexing were conducted with the Illumina pipeline, quality checks and adapter trimming with ERNE and Cutadapt, and alignment to reference genomes with BWA-MEM (default parameters). SNP calling was performed using GATK 4.0, with uniquely aligned reads (mapping quality >10)