KASP Genotyping Assay for SNP Markers

Detailed KASP assay was used as reported by Agarwal et al.^{15 (link)}, and briefly described as follow. Sequences of SNP markers flanking QTL for TSWV on chromosome A01 were converted to KASP markers. The KASP genotyping assay is fluorescence (FRET) based assay that enables identification of biallelic SNPs^{15 (link)}. Two allele-specific forward primers along with tail sequences and one common reverse primer were synthesized by LGC Genomics (http://www.lgcgroup.com) (Supplementary Table S3). The reaction mixture was prepared following the manufacturer’s instructions with minor modifications in number of cycles (KBioscience; http://www.lgcgroup.com/products/kasp-genotypingchemistry/#.VsZK7PkrKM8). Briefly, KASP assays^{15 (link)} were run with 10 µL final reaction volume containing 5 µL KASP master mix, 0.14 µL primer mix, 2 µL of 10–20 ng/µL genomic DNA, and 2.86 µL of water. The following thermal cycling conditions were used: 15 min at 95 °C followed by 10 touchdown cycles of 20 s at 94 °C and 1 min at 61–55 °C (dropping 0.6 °C per cycle), and then 26 cycles of 20 s at 94 °C and 1 min at 55 °C. For each assay 26 cycles were used. The fluorescent endpoint genotyping method was carried out using Roche Light Cycler 480-II instrument (Roche Applied Sciences, Indianapolis, IN, USA).