The application of high resolution melting in the analysis of simple sequence repeat and single nucleotide polymorphism markers in a pea (Pisum sativum L.) population

Abstract

Knopkiewicz M., Gawłowska M., Święcicki W. (2014): The application of high resolution melting in the analysis of simple sequence repeat and single nucleotide polymorphism markers in a pea (Pisum sativum L.) population. Czech J. Genet. Plant Breed., 50: 151-156.The aim of this study was to verify the high resolution melting (HRM) method in the analysis of single nucleotide polymorphism (SNP) and simple sequence repeat (SSR) markers in pea (Pisum sativum L.). A recombinant inbred line population, Carneval × MP1401, was tested for three SNP and 103 SSR markers. HRM analysis was conducted on a LightScanner 96 instrument with LC Green dye. The melting curve shape permitted two polymorphic genotypes to be distinguished. The results were confirmed by gel electrophoresis. Three SSR markers were sequenced and analysed by the melting prediction software. The results confirmed the presence of one polymerase chain reaction (PCR) product with two melting domains. Sequence tagged site (STS) markers produced specific products: Psat_EST_00189_01_1 (300 bp), Pis_GEN_18_2_1 (400 bp), Pis_GEN_7_1-2_1 (600 bp). Amplicons contained one, four and seven single nucleotide polymorphisms, respectively. Melting curve differences enabled the population genotyping except for Psat_EST_00189_01_1 where resolution was too low. Primers for Psat_EST_00189_01_1 were redesigned to obtain a shorter (100 bp) PCR product which increased the resolution. The number of SNPs and amplicon length are crucial for HRM resolution. The HRM method is fast and has a high throughput. The melting analysis of 96 samples takes less than 10 min. Agarose gel analysis confirmed the reliability of HRM, which eliminates laborious post-PCR analysis.Keywords: genotyping; high resolution melting; molecular markers; Pisum sativum L.DNA analysis via high resolution melting (HRM) is a relatively new technique. It is useful for genotyping, mutation scanning and sequence matching. HRM is based on the properties of fluorescent dyes which bind only to double-stranded DNA. These dyes emit strong fluorescence when they bind to DNA. When the sample is heated, DNA slowly denatures and releases the fluorescent dye, which causes the fluorescence to decrease. When the temperature reaches the melting point of the fragment being analysed, a rapid decrease in fluorescence is observed. HRM instruments measure the fluorescence during the entire heating time to generate a melting curve. The melting temperature of a DNA duplex depends on its length and sequence; thus, different sequences generate different melting curves (Reed et al. 2007). et al. 2011). HRM is an alternative to the time consuming and laborious gel techniques; however, in the field of plant genetics it still requires further testing and improvement. There are few reports of simple sequence repeat (SSR) markers analysed by HRM.

Application of the High Resolution Melting analysis for genetic mapping of Sequence Tagged Site markers in narrow-leafed lupin (Lupinus angustifolius L.)

“…The increased number of polymorphic sites of marker Leg435 is probably the explanation of its successful HRM analysis. Knopkiewicz et al (2014) also used the HRM method to successfully analyze 400 bp and 600 bp amplicons covering both three and seven SNPs, respectively.…”

“…The HRM assay has been successfully applied in studies of genetic variability and SNP/ SSR marker genotyping of various plants, e.g. legumes, including alfalfa (Han et al, 2012), pea (Knopkiewicz et al, 2014), soybean (Monteros et al, 2010) and white lupin (Croxford et al, 2008).…”

Discrimination of human papillomavirus genotypes using innovative technique nested-high resolution melting

“…HRM is one of the most powerful methods to study genetic variation such as SNPs, insertions, and/or deletions in the genome. The fundamental aspect of method is that a minor variation in the nucleic acid sequence leads to a detectable alteration in the melting curve, and differences in the amplified PCR sequences 21 , 22 .…”