High resolution melting analysis is a more sensitive and effective alternative to gel-based platforms in analysis of SSR--an example in citrus.

High resolution melting curve analysis (HRM) has been used as an efficient, accurate and cost-effective tool to detect single nucleotide polymorphisms (SNPs) or insertions or deletions (INDELs). However, its efficiency, accuracy and applicability to discriminate microsatellite polymorphism have not...

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Autores principales:	Gaetano Distefano, Marco Caruso, Stefano La Malfa, Alessandra Gentile, Shu-Biao Wu
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2012
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/fb8ec04ca9ad465e880d29325b0b41ef
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spelling	oai:doaj.org-article:fb8ec04ca9ad465e880d29325b0b41ef2021-11-18T07:06:58ZHigh resolution melting analysis is a more sensitive and effective alternative to gel-based platforms in analysis of SSR--an example in citrus.1932-620310.1371/journal.pone.0044202https://doaj.org/article/fb8ec04ca9ad465e880d29325b0b41ef2012-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/22957003/pdf/?tool=EBIhttps://doaj.org/toc/1932-6203High resolution melting curve analysis (HRM) has been used as an efficient, accurate and cost-effective tool to detect single nucleotide polymorphisms (SNPs) or insertions or deletions (INDELs). However, its efficiency, accuracy and applicability to discriminate microsatellite polymorphism have not been extensively assessed. The traditional protocols used for SSR genotyping include PCR amplification of the DNA fragment and the separation of the fragments on electrophoresis-based platform. However, post-PCR handling processes are laborious and costly. Furthermore, SNPs present in the sequences flanking repeat motif cannot be detected by polyacrylamide-gel-electrophoresis based methods. In the present study, we compared the discriminating power of HRM with the traditional electrophoresis-based methods and provided a panel of primers for HRM genotyping in Citrus. The results showed that sixteen SSR markers produced distinct polymorphic melting curves among the Citrus spp investigated through HRM analysis. Among those, 10 showed more genotypes by HRM analysis than capillary electrophoresis owing to the presence of SNPs in the amplicons. For the SSR markers without SNPs present in the flanking region, HRM also gave distinct melting curves which detected same genotypes as were shown in capillary electrophoresis (CE) analysis. Moreover, HRM analysis allowed the discrimination of most of the 15 citrus genotypes and the resulting genetic distance analysis clustered them into three main branches. In conclusion, it has been approved that HRM is not only an efficient and cost-effective alternative of electrophoresis-based method for SSR markers, but also a method to uncover more polymorphisms contributed by SNPs present in SSRs. It was therefore suggested that the panel of SSR markers could be used in a variety of applications in the citrus biodiversity and breeding programs using HRM analysis. Furthermore, we speculate that the HRM analysis can be employed to analyse SSR markers in a wide range of applications in all other species.Gaetano DistefanoMarco CarusoStefano La MalfaAlessandra GentileShu-Biao WuPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 7, Iss 8, p e44202 (2012)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Gaetano Distefano Marco Caruso Stefano La Malfa Alessandra Gentile Shu-Biao Wu High resolution melting analysis is a more sensitive and effective alternative to gel-based platforms in analysis of SSR--an example in citrus.
description	High resolution melting curve analysis (HRM) has been used as an efficient, accurate and cost-effective tool to detect single nucleotide polymorphisms (SNPs) or insertions or deletions (INDELs). However, its efficiency, accuracy and applicability to discriminate microsatellite polymorphism have not been extensively assessed. The traditional protocols used for SSR genotyping include PCR amplification of the DNA fragment and the separation of the fragments on electrophoresis-based platform. However, post-PCR handling processes are laborious and costly. Furthermore, SNPs present in the sequences flanking repeat motif cannot be detected by polyacrylamide-gel-electrophoresis based methods. In the present study, we compared the discriminating power of HRM with the traditional electrophoresis-based methods and provided a panel of primers for HRM genotyping in Citrus. The results showed that sixteen SSR markers produced distinct polymorphic melting curves among the Citrus spp investigated through HRM analysis. Among those, 10 showed more genotypes by HRM analysis than capillary electrophoresis owing to the presence of SNPs in the amplicons. For the SSR markers without SNPs present in the flanking region, HRM also gave distinct melting curves which detected same genotypes as were shown in capillary electrophoresis (CE) analysis. Moreover, HRM analysis allowed the discrimination of most of the 15 citrus genotypes and the resulting genetic distance analysis clustered them into three main branches. In conclusion, it has been approved that HRM is not only an efficient and cost-effective alternative of electrophoresis-based method for SSR markers, but also a method to uncover more polymorphisms contributed by SNPs present in SSRs. It was therefore suggested that the panel of SSR markers could be used in a variety of applications in the citrus biodiversity and breeding programs using HRM analysis. Furthermore, we speculate that the HRM analysis can be employed to analyse SSR markers in a wide range of applications in all other species.
format	article
author	Gaetano Distefano Marco Caruso Stefano La Malfa Alessandra Gentile Shu-Biao Wu
author_facet	Gaetano Distefano Marco Caruso Stefano La Malfa Alessandra Gentile Shu-Biao Wu
author_sort	Gaetano Distefano
title	High resolution melting analysis is a more sensitive and effective alternative to gel-based platforms in analysis of SSR--an example in citrus.
title_short	High resolution melting analysis is a more sensitive and effective alternative to gel-based platforms in analysis of SSR--an example in citrus.
title_full	High resolution melting analysis is a more sensitive and effective alternative to gel-based platforms in analysis of SSR--an example in citrus.
title_fullStr	High resolution melting analysis is a more sensitive and effective alternative to gel-based platforms in analysis of SSR--an example in citrus.
title_full_unstemmed	High resolution melting analysis is a more sensitive and effective alternative to gel-based platforms in analysis of SSR--an example in citrus.
title_sort	high resolution melting analysis is a more sensitive and effective alternative to gel-based platforms in analysis of ssr--an example in citrus.
publisher	Public Library of Science (PLoS)
publishDate	2012
url	https://doaj.org/article/fb8ec04ca9ad465e880d29325b0b41ef
work_keys_str_mv	AT gaetanodistefano highresolutionmeltinganalysisisamoresensitiveandeffectivealternativetogelbasedplatformsinanalysisofssranexampleincitrus AT marcocaruso highresolutionmeltinganalysisisamoresensitiveandeffectivealternativetogelbasedplatformsinanalysisofssranexampleincitrus AT stefanolamalfa highresolutionmeltinganalysisisamoresensitiveandeffectivealternativetogelbasedplatformsinanalysisofssranexampleincitrus AT alessandragentile highresolutionmeltinganalysisisamoresensitiveandeffectivealternativetogelbasedplatformsinanalysisofssranexampleincitrus AT shubiaowu highresolutionmeltinganalysisisamoresensitiveandeffectivealternativetogelbasedplatformsinanalysisofssranexampleincitrus
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High resolution melting analysis is a more sensitive and effective alternative to gel-based platforms in analysis of SSR--an example in citrus.

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