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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{"title"=>"High Resolution Melting Analysis Is a More Sensitive and Effective Alternative to Gel-Based Platforms in Analysis of SSR - An Example in Citrus", "type"=>"journal", "authors"=>[{"first_name"=>"Gaetano", "last_name"=>"Distefano", "scopus_author_id"=>"56247874600"}, {"first_name"=>"Marco", "last_name"=>"Caruso", "scopus_author_id"=>"23099345100"}, {"first_name"=>"Stefano", "last_name"=>"La Malfa", "scopus_author_id"=>"56253214000"}, {"first_name"=>"Alessandra", "last_name"=>"Gentile", "scopus_author_id"=>"8106706500"}, {"first_name"=>"Shu Biao", "last_name"=>"Wu", "scopus_author_id"=>"55901700400"}], "year"=>2012, "source"=>"PLoS ONE", "identifiers"=>{"sgr"=>"84865612672", "doi"=>"10.1371/journal.pone.0044202", "pui"=>"365542935", "issn"=>"19326203", "pmid"=>"22957003", "scopus"=>"2-s2.0-84865612672"}, "id"=>"62ebd817-ddc6-393f-b147-b30ee0721794", "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.", "link"=>"http://www.mendeley.com/research/high-resolution-melting-analysis-more-sensitive-effective-alternative-gelbased-platforms-analysis-ss", "reader_count"=>78, "reader_count_by_academic_status"=>{"Unspecified"=>1, "Professor > Associate Professor"=>7, "Researcher"=>23, "Student > Doctoral Student"=>3, "Student > Ph. D. Student"=>11, "Student > Postgraduate"=>6, "Student > Master"=>14, "Other"=>1, "Student > Bachelor"=>4, "Professor"=>8}, "reader_count_by_user_role"=>{"Unspecified"=>1, "Professor > Associate Professor"=>7, "Researcher"=>23, "Student > Doctoral Student"=>3, "Student > Ph. D. Student"=>11, "Student > Postgraduate"=>6, "Student > Master"=>14, "Other"=>1, "Student > Bachelor"=>4, "Professor"=>8}, "reader_count_by_subject_area"=>{"Unspecified"=>3, "Environmental Science"=>4, "Biochemistry, Genetics and Molecular Biology"=>6, "Agricultural and Biological Sciences"=>56, "Medicine and Dentistry"=>5, "Chemistry"=>1, "Social Sciences"=>1, "Computer Science"=>2}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>5}, "Chemistry"=>{"Chemistry"=>1}, "Social Sciences"=>{"Social Sciences"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>56}, "Computer Science"=>{"Computer Science"=>2}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>6}, "Unspecified"=>{"Unspecified"=>3}, "Environmental Science"=>{"Environmental Science"=>4}}, "reader_count_by_country"=>{"Greece"=>1, "United States"=>2, "Uruguay"=>1, "Japan"=>1, "Brazil"=>2, "Italy"=>1, "Australia"=>1, "France"=>1}, "group_count"=>2}

Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/584861"], "description"=>"<p>A panel of markers selected for CE and HRM analyses of 15 citrus genotypes.</p>", "links"=>[], "tags"=>["markers", "ce", "hrm", "analyses", "15", "citrus"], "article_id"=>255344, "categories"=>["Biotechnology", "Genetics", "Plant Biology"], "users"=>["Gaetano Distefano", "Marco Caruso", "Stefano La Malfa", "Alessandra Gentile", "Shu-Biao Wu"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0044202.t003", "stats"=>{"downloads"=>10, "page_views"=>11, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_A_panel_of_markers_selected_for_CE_and_HRM_analyses_of_15_citrus_genotypes_/255344", "title"=>"A panel of markers selected for CE and HRM analyses of 15 citrus genotypes.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2012-08-30 01:29:04"}
  • {"files"=>["https://ndownloader.figshare.com/files/584817"], "description"=>"<p>Citrus genotypes used for EST- SSRs analysis by capillary electrophoresis and high resolution melting.</p>", "links"=>[], "tags"=>["genotypes", "est-", "ssrs", "capillary", "electrophoresis"], "article_id"=>255301, "categories"=>["Biotechnology", "Genetics", "Plant Biology"], "users"=>["Gaetano Distefano", "Marco Caruso", "Stefano La Malfa", "Alessandra Gentile", "Shu-Biao Wu"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0044202.t002", "stats"=>{"downloads"=>4, "page_views"=>41, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Citrus_genotypes_used_for_EST_SSRs_analysis_by_capillary_electrophoresis_and_high_resolution_melting_/255301", "title"=>"Citrus genotypes used for EST- SSRs analysis by capillary electrophoresis and high resolution melting.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2012-08-30 01:28:21"}
  • {"files"=>["https://ndownloader.figshare.com/files/584238"], "description"=>"<p>The HRM analysis produced consistent result with CE when only length polymorphisms were present in the amplicon. <b>A</b>. CE profiles of 4 different genotypes, two homozygous (128/128 bp, and 138/138 bp) and two heterozygous (128/129 bp, and 129/138 bp). Fragment size includes the M13 primer tail (19 bp). <b>B</b>. HRM melting profiles showing 4 distinct genotypes in normalized melting plot which are consistent with the CE results. The exact genotypes are indicated in the rectangular boxes.</p>", "links"=>[], "tags"=>["hrm", "profiles", "genotypes"], "article_id"=>254731, "categories"=>["Biotechnology", "Genetics", "Plant Biology"], "users"=>["Gaetano Distefano", "Marco Caruso", "Stefano La Malfa", "Alessandra Gentile", "Shu-Biao Wu"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0044202.g003", "stats"=>{"downloads"=>3, "page_views"=>4, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_CE_and_HRM_profiles_of_the_genotypes_analyzed_with_the_marker_1527_/254731", "title"=>"CE and HRM profiles of the genotypes analyzed with the marker 1527.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-08-30 01:18:51"}
  • {"files"=>["https://ndownloader.figshare.com/files/584506"], "description"=>"<p>. CE profiles of 10 different genotypes. Seven different alleles (220 bp, 222 bp, 228 bp 232 bp, 238 bp, 240 bp and 246 bp) form 2 homozygous and 8 heterozygous genotypes. Fragment size includes the M13 primer tail (19 bp). <b>B</b>. Normalized HRM plot showing two similar melting curves produced from different genotypes (arrows). <b>C</b> difference plot - two ambiguous genotypes were resolved (arrows) thus 10 genotypes were distinguished which was in agreement with the CE results. The exact genotypes are indicated in the rectangular boxes.</p>", "links"=>[], "tags"=>["hrm", "profiles", "genotypes", "482", "10", "shown", "ce"], "article_id"=>254988, "categories"=>["Biotechnology", "Genetics", "Plant Biology"], "users"=>["Gaetano Distefano", "Marco Caruso", "Stefano La Malfa", "Alessandra Gentile", "Shu-Biao Wu"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0044202.g005", "stats"=>{"downloads"=>1, "page_views"=>14, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_CE_and_HRM_profiles_of_the_genotypes_analyzed_with_the_marker_482_showing_10_genotypes_shown_by_both_CE_and_HRM_analysis_A_/254988", "title"=>"CE and HRM profiles of the genotypes analyzed with the marker 482 showing 10 genotypes shown by both CE and HRM analysis. A", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-08-30 01:23:08"}
  • {"files"=>["https://ndownloader.figshare.com/files/307093"], "description"=>"<div><p>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 <em>Citrus spp</em> 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.</p> </div>", "links"=>[], "tags"=>["melting", "gel-based", "platforms", "ssr", "citrus"], "article_id"=>120590, "categories"=>["Biotechnology", "Genetics", "Cell Biology"], "users"=>["Gaetano Distefano", "Marco Caruso", "Stefano La Malfa", "Alessandra Gentile", "Shu-Biao Wu"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0044202", "stats"=>{"downloads"=>5, "page_views"=>20, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/High_Resolution_Melting_Analysis_Is_a_More_Sensitive_and_Effective_Alternative_to_Gel_Based_Platforms_in_Analysis_of_SSR_An_Example_in_Citrus/120590", "title"=>"High Resolution Melting Analysis Is a More Sensitive and Effective Alternative to Gel-Based Platforms in Analysis of SSR – An Example in Citrus", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2012-08-30 00:09:50"}
  • {"files"=>["https://ndownloader.figshare.com/files/584344"], "description"=>"<p>While normalized HRM plot shows ambiguous genotype separation, further analysis with difference curve resolves the genotypes clearly. <b>A</b>. CE profile of 5 different genotypes. Four different alleles (155 bp, 157 bp, 159 bp and 169 bp) form 2 homozygous and 3 heterozygous genotypes. Fragment size includes the M13 primer tail (19 bp). <b>B</b>. Normalized HRM melting curves showing 4 genotypes - two ambiguous genotypes not resolved. <b>C</b>. Difference plot showing 5 distinct genotypes which resolved two ambiguous genotypes shown in B, which was consistent with CE results. The exact genotypes are indicated in the rectangular boxes.</p>", "links"=>[], "tags"=>["hrm", "profiles", "genotypes"], "article_id"=>254832, "categories"=>["Biotechnology", "Genetics", "Plant Biology"], "users"=>["Gaetano Distefano", "Marco Caruso", "Stefano La Malfa", "Alessandra Gentile", "Shu-Biao Wu"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0044202.g004", "stats"=>{"downloads"=>1, "page_views"=>5, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_CE_and_HRM_profiles_of_the_genotypes_analyzed_with_the_marker_115_/254832", "title"=>"CE and HRM profiles of the genotypes analyzed with the marker 115.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-08-30 01:20:32"}
  • {"files"=>["https://ndownloader.figshare.com/files/584022"], "description"=>"<p>A monomorphic marker in the population under study is shown polymorphic by HRM analysis. <b>A</b>. Monomorphic CE profile obtained from the analysis of 15 citrus genotypes. <b>B</b>. Polymorphic HRM melting curves showing 4 genotypes in normalized melting plot (upper) and difference melting curve (lower). The curves with different color represent different genotypes which are also shown in <b>A</b>. The exact genotypes are indicated in the rectangular boxes. <b>C</b>. Sequence alignment of the amplicons from 4 distinct genotypes. Two SNPs are shown and highlighted in the yellow boxes.</p>", "links"=>[], "tags"=>["hrm", "alignment"], "article_id"=>254519, "categories"=>["Biotechnology", "Genetics", "Plant Biology"], "users"=>["Gaetano Distefano", "Marco Caruso", "Stefano La Malfa", "Alessandra Gentile", "Shu-Biao Wu"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0044202.g002", "stats"=>{"downloads"=>2, "page_views"=>8, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_CE_and_HRM_profiles_and_sequence_alignment_of_genotypes_alleles_analyzed_with_the_marker_93_/254519", "title"=>"CE and HRM profiles, and sequence alignment of genotypes/alleles analyzed with the marker 93.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-08-30 01:15:19"}
  • {"files"=>["https://ndownloader.figshare.com/files/583902"], "description"=>"<p>The HRM analysis revealed a novel genotype, 155C/155T, which was not recognized by CE. <b>A</b>. CE profiles of 3 distinct genotypes with 2 alleles (155 bp and 158 bp). Fragment size includes the M13 primer tail (19 bp). <b>B</b>. HRM curves showing 4 genotypes in normalized melting plot, two homozygous with single and two heterozygous with double melting phases. A new allele was detected in the analysis. Curves with different color represent different genotypes which are also shown in <b>A</b>. The exact genotypes are indicated in the rectangular boxes. <b>C</b>. Alignment of the 3 identified alleles by the bidirectional sequencing which identified a new allele which includes a SNP (red).</p>", "links"=>[], "tags"=>["hrm", "profiles", "15", "citrus", "genotypes"], "article_id"=>254394, "categories"=>["Biotechnology", "Genetics", "Plant Biology"], "users"=>["Gaetano Distefano", "Marco Caruso", "Stefano La Malfa", "Alessandra Gentile", "Shu-Biao Wu"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0044202.g001", "stats"=>{"downloads"=>0, "page_views"=>1, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_CE_and_HRM_profiles_of_15_citrus_genotypes_analyzed_with_the_marker_1388_/254394", "title"=>"CE and HRM profiles of 15 citrus genotypes analyzed with the marker 1388.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-08-30 01:13:14"}
  • {"files"=>["https://ndownloader.figshare.com/files/584772"], "description"=>"<p>Comparison of the allele and genotype information of the markers between CE and HRM analyses.</p>", "links"=>[], "tags"=>["allele", "genotype", "markers", "ce", "hrm"], "article_id"=>255259, "categories"=>["Biotechnology", "Genetics", "Plant Biology"], "users"=>["Gaetano Distefano", "Marco Caruso", "Stefano La Malfa", "Alessandra Gentile", "Shu-Biao Wu"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0044202.t001", "stats"=>{"downloads"=>9, "page_views"=>4, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Comparison_of_the_allele_and_genotype_information_of_the_markers_between_CE_and_HRM_analyses_/255259", "title"=>"Comparison of the allele and genotype information of the markers between CE and HRM analyses.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2012-08-30 01:27:39"}
  • {"files"=>["https://ndownloader.figshare.com/files/584627"], "description"=>"<p>Genetic distance analysis allowed discrimination of most of the 15 citrus genotypes except those belonging to the same species (3 sweet orange and 3 clementine varieties). Citrus genotypes sharing same parental origin clustered in the same group. This fact is especially evident in HRM dendrogram in which all mandarin and clementine genotypes (except Satsuma) are grouped together. The genetic distance was calculated by Powermarker (shared alleles), and clustering was produced using Neighbor-joining method.</p>", "links"=>[], "tags"=>["relationships", "15", "citrus", "genotypes", "ce", "hrm"], "article_id"=>255109, "categories"=>["Biotechnology", "Genetics", "Plant Biology"], "users"=>["Gaetano Distefano", "Marco Caruso", "Stefano La Malfa", "Alessandra Gentile", "Shu-Biao Wu"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0044202.g006", "stats"=>{"downloads"=>4, "page_views"=>34, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Dendrogram_representing_the_structure_of_genetic_diversity_and_relationships_among_15_citrus_genotypes_by_CE_A_and_HRM_B_/255109", "title"=>"Dendrogram representing the structure of genetic diversity and relationships among 15 citrus genotypes by CE (A) and HRM (B).", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-08-30 01:25:09"}

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Relative Metric

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