scholarly journals High Resolution Melting (HRM) for High-Throughput Genotyping—Limitations and Caveats in Practical Case Studies

2017 ◽  
Vol 18 (11) ◽  
pp. 2316 ◽  
Author(s):  
Marcin Słomka ◽  
Marta Sobalska-Kwapis ◽  
Monika Wachulec ◽  
Grzegorz Bartosz ◽  
Dominik Strapagiel
Keyword(s):  
High Resolution ◽  
Case Studies ◽  
High Throughput ◽  
High Resolution Melting ◽  
Practical Case

scholarly journals Development and Validation of High-Resolution Melting Markers Derived from Rysto STS Markers for High-Throughput Marker-Assisted Selection of Potato Carrying Rysto

2016 ◽  
Vol 106 (11) ◽  
pp. 1366-1375 ◽  
Author(s):  
Xianzhou Nie ◽  
Darcy Sutherland ◽  
Virginia Dickison ◽  
Mathuresh Singh ◽  
Agnes M. Murphy ◽  
...  
Keyword(s):  
High Resolution ◽  
High Throughput ◽  
Marker Assisted Selection ◽  
High Resolution Melting ◽  
Chromosome Region ◽  
Sts Markers ◽  
Reverse Primer ◽  
Selection For ◽  
Development And Validation ◽  
Selection Of

Sequence analysis of the chromosome region harboring the sequence-tagged site (STS) markers YES3-3A and YES3-3B for Rysto, a gene responsible for extreme resistance to Potato virus Y (PVY) in potato, was performed in tetraploid potato ‘Barbara’ (Rrrr) and ‘AC Chaleur’ (rrrr) as well as their progeny selections. Three and two sequence variants were identified in Barbara resistant (R) selections and AC Chaleur susceptible (S) selections, respectively. Further analysis indicates that the variant with a 21-nucleotide (nt) deletion is likely the chromosome copy harboring the STS markers. Two primer pairs, one targeting the region containing a 20-nt deletion and the other targeting the region anchoring the YES3-3A reverse primer, were designed. As anticipated, pair one produced two visible fragments in Barbara-R bulk and one visible fragment in AC Chaleur-S bulk; pair two produced one visible fragment in all samples. When subjected to high-resolution melting (HRM) analysis, two distinct melting profiles for R and S samples were observed. Analysis of 147 progeny of Barbara × AC Chaleur revealed 72 and 75 progeny with R and S melting profiles, respectively, which was consistent with YES3-3A and YES3-3B assays and phenotyping analysis, thus demonstrating the potential of HRM profiles as novel molecular markers for Rysto. The efficacy of the newly developed HRM markers for high-throughput marker-assisted selection for Rysto-conferred resistance to PVY was validated further with three populations involving Barbara as the R parent.

Development of a multiplex high-throughput diagnostic assay for the detection of strawberry crown rot diseases using high-resolution melting analysis

2021 ◽  
Author(s):  
Nan-Yi Wang ◽  
Andre Bueno Gama ◽  
Marcus Vinicius Marin ◽  
Natalia Peres
Keyword(s):  
High Resolution ◽  
High Throughput ◽  
High Throughput Screening ◽  
High Resolution Melting ◽  
High Specificity ◽  
Macrophomina Phaseolina ◽  
Disease Diagnosis ◽  
Management Program ◽  
Crown Rot ◽  
Individual Species

Rapid and accurate disease diagnosis is a prerequisite for an effective disease management program in strawberry production. In Florida, Colletotrichum spp., Phytophthora spp, and Macrophomina phaseolina are the primary microorganisms causing strawberry crown rot. Even though the diseases can be caused by different pathogens, symptoms are indistinguishable and equally devastating. To timely inform strawberry growers of diagnostic results for effective deployment of chemical control practices, we developed a multiplex high-resolution melting (HRM) assay to rapidly and accurately detect the above-mentioned pathogens. The multiplex HRM assays using three pre-designed primer pairs showed high specificity for individual species by generating specific melting peaks without cross-reaction between primers or with other common strawberry pathogens. The amplification limit of the assay was 1 pg of Colletotrichum and Phytophthora and 100 pg of M. phaseolina DNA per 10 μl reaction. However, the presence of different melting peaks was observed in mixed DNA samples and was concentration- and target DNA-dependent. A crude DNA extraction protocol was developed to allow high-throughput screening by minimizing the inhibitory effects. Moreover, we applied the HRM assay to 522 plant samples and found high correlations between conventional pathogen isolation and HRM and between singleplex and multiplex assays. Altogether, this multiplex HRM assay is specific, cost-effective, and reliable for the timely detection of strawberry crown rot pathogens.

scholarly journals Application of High-Resolution Melting to Large-Scale, High-Throughput SNP Genotyping

2010 ◽  
Vol 15 (6) ◽  
pp. 623-629 ◽  
Author(s):  
Alessandro Martino ◽  
Tommaso Mancuso ◽  
Anna Maria Rossi
Keyword(s):  
High Resolution ◽  
High Throughput ◽  
Large Scale ◽  
High Resolution Melting ◽  
Fluorescent Dyes ◽  
Gc Content ◽  
Snp Genotyping ◽  
Nucleotide Polymorphisms ◽  
Experimental Conditions ◽  
Discrimination Power

Because of the wide use of single-nucleotide polymorphisms (SNPs) as markers of genetic variation, several high-throughput genotyping methods have been developed and applied during the past decades. High-resolution melting (HRM) is a very attractive, advanced, fast, and cost-effective SNP genotyping technology based on the analysis of the melting profile of PCR products, using intercalating fluorescent dyes to monitor the transition from unmelted to melted DNA. The authors used HRM for genotyping 215 human DNA samples for SNPs in the ABCB1, NQO1, and SLC19A1 genes and 96 samples for SNPs in the IL1A and IL12B genes with the aim of assessing HRM sensitivity and accuracy in comparisons with the TaqMan® assay in view of large-scale, high-throughput SNP-typing applications. The potential effect of PCR product size, TM, GC content, and SNP position on HRM performances was explored with amplicons that were heterogeneous for these factors. Discrimination power ranged from 91.4% to 98.4%, being significantly lower only when the number of rare homozygotes dropped to 1 or few units. The availability of specific and validated assays, in addition to a better standardization of HRM experimental conditions, can considerably reduce time and costs of large-scale genotyping studies with a negligible risk of failure or misclassification.

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