3.8. Development and Validation of SNP-Based Molecular Markers

According to the SNP variations in the upstream promoter sequences of the coding region in the cloned HMW-GS genes (Table S4), two pairs of AS-PCR primers were designed (Table S5). Genomic DNA was extracted using the Trans Fast Taq DNA Polymerase system (TransGen Biotech, Beijing, China). PCR cycles consisted of 94 °C for 3 min for activation, followed by 35 cycles of 94 °C for 5 s, 58 °C/55 °C (Table S6) for 15 s, 72 °C for 2/4 s, and finally ended at 72 °C for a 5 min extension step. The materials used for marker development and verification were described in the section of plant materials.