Czech J. Genet. Plant Breed., 2014, 50(2):151-156 | DOI: 10.17221/113/2013-CJGPB
The application of high resolution melting in the analysis of simple sequence repeat and single nucleotide polymorphism markers in a pea (Pisum sativum L.) populationOriginal Paper
- Institute of Plant Genetics, Polish Academy of Sciences, Poznan, Poland
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.
Published: June 30, 2014 Show citation
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References
- Croxford A.E., Rogers T., Caligari P.D., Wilkinson M.J. (2008): High-resolution melt analysis to identify and map sequence-tagged site anchor points onto linkage maps: a white lupin (Lupinus albus) map as an exemplar. New Phytologist, 180: 594-607.
Go to original source... 
Go to PubMed... - Distefano G., Caruso M., LA Malfa S., Gentile A., Wu S-B. (2012): High resolution melting analysis is a more sensitive and effective alternative to gel-based platforms in analysis of SSR - an example in citrus. PLoS ONE, 7: e44202. Go to original source... Go to PubMed...
- Dwight Z., Palais R., Wittwer C.T. (2011): uMELT: prediction of high-resolution melting curves and dynamic melting profiles of PCR products in a rich web application. Bioinformatics, 27: 1019-1020. Go to original source... Go to PubMed...
- Han Y., Khu D.M., Monteros M. J. (2012): High-resolution melting analysis for SNP genotyping and mapping in tetraploid alfalfa (Medicago sativa L.). Molecular Breeding, 29: 489-501. Go to original source... Go to PubMed...
- Hofinger B.J., Jing H.C., Hammond-Kosack K.E., Kanyuka K. (2009): High-resolution melting analysis of cDNA-derived PCR amplicons for rapid and cost-effective identification of novel alleles in barley. Theoretical and Applied Genetics, 119: 851-65. Go to original source... Go to PubMed...
- Li J., Wang X., Dong R., Yang Y., Zhou J., Yu C., Cheng Y., Yan C., Chen J. (2011): Evaluation of high-resolution melting for gene mapping in rice. Plant Molecular Biology Reporter, 29: 979-985. Go to original source...
- Lochlainn S.Ó., Amoah S., Graham N.S., Alamer K., Rios J.J., Kurup S., Stoute A., Hammond J.P., Østergaard L., King G.J., White P.J., Broadley M.R. (2011): High Resolution Melt (HRM) analysis is an efficient tool to genotype EMS mutants in complex crop genomes. Plant Methods, 7: 43. Go to original source... Go to PubMed...
- Loridon K., MCphee K., Morin J., Dubreuil P., Pilet-Nayel M. L., Aubert G., Rameau C., Baranger A., Coyne C., Lejeune-Henaut I., Burstin J. (2005): Microsatellite marker polymorphism and mapping in pea (Pisum sativum L.). Theoretical and Applied Genetics, 111: 1022-1031. Go to original source... Go to PubMed...
- Neff M.M., Turk E., Kalishman M. (2002): Web-based primer design for single nucleotide polymorphism analysis. Trends in Genetics, 18: 613-615. Go to original source... Go to PubMed...
- Reed G.H., Kent J.O., Wittwer C.T. (2007): High-resolution DNA melting analysis for simple and efficient molecular diagnostics. Pharmacogenomics, 8: 597-608. Go to original source... Go to PubMed...
- Semagn K., Babu R., Hearne S., Olsen M. (2014) Single nucleotide polymorphism genotyping using Kompetitive Allele Specific PCR (KASP): overview of the technology and its application in crop improvement. Molecular Breeding, 33: 1-14. Go to original source...
- Tar'an B., Warkentin T., Somers D.J., Miranda D., Vandenberg A., Blade S., Woods S., Bing D., Xue A., DeKoeyer D., Penner G. (2003): Quantitative trait loci for lodging resistance, plant height and partial resistance to mycosphaerella blight in field pea (Pisum sativum L.). Theoretical and Applied Genetics, 107: 1482-1491. Go to original source... Go to PubMed...
- Van K., Kang Y.J., Han K.S. Lee Y.H. Gwag J.G., Moon J.K., Lee S.H. (2013): Genome wide SNP discovery in mungbean by Illumina HiSeq. Theoretical and Applied Genetics, 126: 2017-2027. Go to original source... Go to PubMed...
- Yu R.H., Shan X.H., Wang S., Li X.H., Jiang Y., Tan H., Li Y.D. (2011): A screening method for detecting simple sequence repeat (SSR) polymorphism of Zea mays using high resolution melting-curve analysis. African Journal of Biotechnology, 10: 16443-16447. Go to original source...
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