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

English version

Autoři: Gautam Vishwakarma ^aff001; Ravi Prakash Sanyal ^aff002; Ajay Saini ^aff002; Parmeshwar Kumar Sahu ^aff004; Ravi Raj Singh Patel ^aff004; Deepak Sharma ^aff004; Ratan Tiwari ^aff005; Bikram Kishore Das ^aff001
Působiště autorů: Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre, Trombay, Mumbai, Maharashtra, India ^aff001; Homi Bhabha National Institute, Anushaktinagar, Trombay, Mumbai, Maharashtra, India ^aff002; Molecular Biology Division, Bhabha Atomic Research Centre, Trombay, Mumbai, Maharashtra, India ^aff003; Department of Genetics and Plant Breeding, Indira Gandhi Krishi Vishwavidyalaya, Raipur, Chhattisgarh, India ^aff004; ICAR - Indian Institute of Wheat and Barley Research, Karnal, Haryana, India ^aff005
Vyšlo v časopise: PLoS ONE 14(11)
Kategorie: Research Article
doi: https://doi.org/10.1371/journal.pone.0224572

Souhrn

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.

Klíčová slova:

Crop genetics – Crops – Gel electrophoresis – India – Plant breeding – Polymerase chain reaction – Rice – Wheat

Zdroje

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

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