Please use this identifier to cite or link to this item: https://hdl.handle.net/1959.11/11191
Title: High Resolution Melting Analysis Is a More Sensitive and Effective Alternative to Gel-Based Platforms in Analysis of SSR - An Example in Citrus
Contributor(s): Distefano, Gaetano (author); Caruso, Marco (author); La Malfa, Stefano (author); Gentile, Alessandra (author); Wu, Shubiao  (author)orcid 
Publication Date: 2012
Open Access: Yes
DOI: 10.1371/journal.pone.0044202Open Access Link
Handle Link: https://hdl.handle.net/1959.11/11191
Abstract: 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.
Publication Type: Journal Article
Source of Publication: PLoS One, 7(8), p. 1-11
Publisher: Public Library of Science
Place of Publication: United States of America
ISSN: 1932-6203
Fields of Research (FoR) 2008: 060408 Genomics
Fields of Research (FoR) 2020: 310509 Genomics
Socio-Economic Objective (SEO) 2008: 820203 Citrus Fruit
Socio-Economic Objective (SEO) 2020: 260504 Citrus fruit
Peer Reviewed: Yes
HERDC Category Description: C1 Refereed Article in a Scholarly Journal
Appears in Collections:Journal Article

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