GLADS: A gel-less approach for detection of STMS markers in wheat and rice

PLoS One. 2019 Nov 5;14(11):e0224572. doi: 10.1371/journal.pone.0224572. eCollection 2019.

Abstract

Sequence tagged microsatellite site (STMS) are useful PCR based DNA markers. Wide genome coverage, high polymorphic index and co-dominant nature make STMS a preferred choice for marker assisted selection (MAS), genetic diversity analysis, linkage mapping, seed genetic purity analysis etc. Routine STMS analysis involving low-throughput, laborious and time-consuming polyacrylamide/agarose gels often limit their full utility in crop breeding experiments that involve large populations. Therefore, convenient, gel-less marker detection methods are highly desirable for STMS markers. The present study demonstrated the utility of SYBR Green dye based melt-profiling as a simple and convenient gel-less approach for detection of STMS markers (referred to as GLADS) in bread wheat and rice. The method involves use of SYBR Green dye during PCR amplification (or post-PCR) of STMS markers followed by generation of a melt-profile using controlled temperature ramp rate. The STMS amplicons yielded characteristic melt-profiles with differences in melting temperature (Tm) and profile shape. These characteristic features enabled melt-profile based detection and differentiation of STMS markers/alleles in a gel-less manner. The melt-profile approach allowed assessment of the specificity of the PCR assay unlike the end-point signal detection assays. The method also allowed multiplexing of two STMS markers with non-overlapping melt-profiles. In principle, the approach can be effectively used in any crop for STMS marker analysis. This SYBR Green melt-profiling based GLADS approach offers a convenient, low-cost (20-51%) and time-saving alternative for STMS marker detection that can reduce dependence on gel-based detection, and exposure to toxic chemicals.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alleles
  • Genetic Markers / genetics
  • Microsatellite Repeats / genetics*
  • Oryza / genetics*
  • Plant Breeding / methods*
  • Polymerase Chain Reaction / methods
  • Sequence Tagged Sites*
  • Triticum / genetics*

Substances

  • Genetic Markers

Grants and funding

This study was supported by the institutional funding of Bhabha Atomic Research Centre, Mumbai, India. No separate funding was obtained from any other National/International funding body for this study.