Rapid and convenient gel‐free screening of SCAR markers in wheat using SYBR green‐based melt‐profiling

Sequence characterized amplified region (SCAR) markers that are highly desirable in crop breeding for marker‐assisted selection (MAS) are routinely analysed by gel‐based methods that are low‐throughput, time‐consuming and laborious. In this study, we showed a rapid and convenient method for analysis of SCAR markers in a gel‐free manner. Seven SCAR markers, linked to rust resistance genes (Sr24, Sr26 and Sr31) and seed quality traits (Pina, Pinb and Glu‐D1) in wheat (Triticum aestivum), were amplified on a real‐time PCR machine using custom reaction mixture. Subsequently, melting curve analysis was performed, to assess the specificity of amplicons. Using the amplicon‐specific melt‐profiles, the presence/absence of SCAR markers was analysed in fifteen genotypes and five F₂ populations. Unlike the fluorescence‐based in‐tube detection methods, the present method used the amplicon‐specific melt‐profiles to evaluate the status of the SCAR markers, thus eliminating the need for gel‐based analysis. Results also showed feasibility of multiplex analysis of two markers with well‐separated melting profiles. Overall, the approach is a rapid, convenient and cost‐effective method for high‐throughput screening of SCAR markers.