High resolution melting (HRM)-based STMS marker analysis for rapid identification of radiation induced mutants of sugarcane

Abstract

Molecular marker based selection has become the method of choice to find undiscoverable natural variations and induced mutations in plants. Routine gel based molecular marker screening procedures involve complex steps which require considerable investment of time, money and efforts. Most often, genomic configuration of a plant species complicates the ultimate output of such screening programme. Therefore, a reliable and rapid high-throughput method is required to facilitate ease in identification of variants in a large number of samples. Here we demonstrate the application of a high-resolution melting (HRM) method to detect gamma radiation induced mutants of sugarcane (Saccharum spp.) which is a complex aneu-polyploidy plant. We have screened nine distinct mutants using sequence-tagged microsatellite site (STMS) markers and the analyses based on gel electrophoresis and HRM curve were compared. All the six STMS microsatellite markers coupled with HRM analysis were found highly informative and generated a unique melting curve for each of the marker tested. Of these, two markers NKSCSSR 22 and NKSCSSR 42 exhibited differential melting curve among the mutants and these results were well correlated with the gel based banding pattern. Taken together these results highlighted potential of HRM based marker screening and demonstrated reliability and robustness in detecting induced mutations in a complex polyploidy plant like sugarcane.