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. [ABSTRACT FROM AUTHOR]