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.
Highlights
Using representative wheat and rice genotypes, amount of genomic DNA, primer concentration and annealing temperature were optimized for Sequence Tagged Microsatellite Site (STMS) Polymerase chain reaction (PCR) amplification
The STMS markers specific to A, B- and D-genomes of wheat yielded clean profiles on PCR amplification from a representative genotype (Fig 1A, 1B and 1C)
The GLADS approach combined the simplicity of PCR, sensitivity of SYBR Green, and informativeness of melt-profiles for a simple, rapid and effective assay for various types of STMS analysis viz. detection/scoring, differentiation, allelic polymorphism, and multiplex analysis
Summary
Conventional breeding utilizes various approaches for improvement of desired traits in crop plants [1,2], which involve laborious and time-consuming manual screening of large. A gel-less approach for detection of STMS markers in wheat and rice. These are not straight forward methods for introgression of complex traits/phenotypes governed by multiple genes [3]. DNA markers ascertains linked traits (at any stage of plant) saves screening time, and are highly desirable for crop breeding applications such as, marker assisted selection (MAS) or marker assisted backcross breeding (MABB) [4]. Polymerase chain reaction (PCR) has revolutionized the development and use of a variety of DNA markers viz. Sequence Tagged Microsatellite Site (STMS), Sequence-Tagged Site (STS), Sequence Characterized Amplified Region (SCAR), Randomly Amplified Polymorphic DNA (RAPD), Arbitrary Primed-PCR (AP-PCR), Inter Simple Sequence Repeat (ISSR), Amplified Fragment Length Polymorphism (AFLP), RetroposonMicrosatellite Amplified Polymorphism (RE-MAP) [5]. Recent advances in genome sequencing has resulted in development of SNP markers for diverse applications including genome wide association studies (GWAS), marker development, genomic selection, germplasm characterization [6,7]
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