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

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 ST...

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Autores principales: Vishwakarma, Gautam, Sanyal, Ravi Prakash, Saini, Ajay, Sahu, Parmeshwar Kumar, Singh Patel, Ravi Raj, Sharma, Deepak, Tiwari, Ratan, Das, Bikram Kishore
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6830750/
https://www.ncbi.nlm.nih.gov/pubmed/31689318
http://dx.doi.org/10.1371/journal.pone.0224572
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author Vishwakarma, Gautam
Sanyal, Ravi Prakash
Saini, Ajay
Sahu, Parmeshwar Kumar
Singh Patel, Ravi Raj
Sharma, Deepak
Tiwari, Ratan
Das, Bikram Kishore
author_facet Vishwakarma, Gautam
Sanyal, Ravi Prakash
Saini, Ajay
Sahu, Parmeshwar Kumar
Singh Patel, Ravi Raj
Sharma, Deepak
Tiwari, Ratan
Das, Bikram Kishore
author_sort Vishwakarma, Gautam
collection PubMed
description 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.
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spelling pubmed-68307502019-11-12 GLADS: A gel-less approach for detection of STMS markers in wheat and rice Vishwakarma, Gautam Sanyal, Ravi Prakash Saini, Ajay Sahu, Parmeshwar Kumar Singh Patel, Ravi Raj Sharma, Deepak Tiwari, Ratan Das, Bikram Kishore PLoS One Research Article 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. Public Library of Science 2019-11-05 /pmc/articles/PMC6830750/ /pubmed/31689318 http://dx.doi.org/10.1371/journal.pone.0224572 Text en © 2019 Vishwakarma et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Vishwakarma, Gautam
Sanyal, Ravi Prakash
Saini, Ajay
Sahu, Parmeshwar Kumar
Singh Patel, Ravi Raj
Sharma, Deepak
Tiwari, Ratan
Das, Bikram Kishore
GLADS: A gel-less approach for detection of STMS markers in wheat and rice
title GLADS: A gel-less approach for detection of STMS markers in wheat and rice
title_full GLADS: A gel-less approach for detection of STMS markers in wheat and rice
title_fullStr GLADS: A gel-less approach for detection of STMS markers in wheat and rice
title_full_unstemmed GLADS: A gel-less approach for detection of STMS markers in wheat and rice
title_short GLADS: A gel-less approach for detection of STMS markers in wheat and rice
title_sort glads: a gel-less approach for detection of stms markers in wheat and rice
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6830750/
https://www.ncbi.nlm.nih.gov/pubmed/31689318
http://dx.doi.org/10.1371/journal.pone.0224572
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