Optimization of dot-blot snp analysis for the detection of drought or salinity stress associated marker in foxtail millet (Setaria italica L.)

Foxtail millet (Setaria italica L.) is an underutilized crop grown for its nutritious grains and relative tolerance to drought or salinity stresses. However, variation in the tolerance level exists among foxtail millet genotypes. A simple and reliable technique for SNP genotyping, namely dot-blot SNP analysis, has been applied for practical plant breeding programs and has a potential for accelerating foxtail millet breeding for drought or salinity stress tolerance. The aim of this study was to conduct SNP analysis for the SiDREB2 gene, which is associated with drought or salinity stress tolerance in foxtail millet, using a marker based on polymorphism at the 558^th nucleotide., Two factors that affect the allele-specific detection of the dot-blot SNP analysis i.e. hybridization temperature and competitive probe ratio were optimized in this study. Four hybridization conditions consisting of the combination between two hybridization temperatures (50 and 55 ºC) and two competitive probe ratios (1:5 and 1:10) were optimized. The second hybridization condition (50 ºC hybridization temperature and 1:10 competitive probe ratio) showed the best result for SNP analysis. This optimum condition was then applied for genotyping 26 foxtail millet genotypes with unknown drought or salinity stress tolerance levels. The optimum condition of the dot-blot SNP analysis was effective for genotyping in an allele-specific manner and used for predicting stress tolerance levels of the foxtail millet genotypes. The results of this study are useful for accelerating foxtail millet breeding for drought or salinity stresses in the future.