A Cost‐effective High‐resolution Melting Approach using the EvaGreen Dye for DNA Polymorphism Detection and Genotyping in Plants

Abstract

High-resolution melting (HRM) analysis relies on the use of fluorescent dyes, such as LCGreen, ResoLight, and SYTO9, which bind in a saturated manner to the double-stranded DNAs. These dyes are expensive in use and may not be affordable when dealing with a large quantity of samples. EvaGreen is a much cheaper DNA helix intercalating dye and has been used in quantitative real-time polymerase chain reaction (PCR) and post-PCR DNA melt curve analysis. Here we report on the development of an EvaGreen-based HRM analysis and its performance, in comparison with the popular LCGreen-based HRM analysis, in detection of DNA polymorphism in plants. We found that various polymorphisms ranged from single nucleotide polymorphisms (SNPs) to Indels were equally detected by using EvaGreen-or LCGreenbased HRM. EvaGreen dye was sensitive enough in discovery of SNPs in fivefold pooled samples. Using this economical dye we successfully identified multiple novel mutant alleles of Gln1-3 gene, which produces a cytosolic glutamine synthetase isoenzyme (GS1), in a maize ethyl methanesulfonate (EMS)-mutagenized library, and genotyped rice mapping populations with SNP markers. The current results suggest that EvaGreen is a promising dye for HRM analysis for its ease to use and cost effectiveness.Li YD, Chu ZZ, Liu XG, Jing HC, Liu YG, Hao DY (2010) A cost-effective high-resolution melting approach using the EvaGreen dye for DNA polymorphism detection and genotyping in plants.

Comparative analyses of genetic/epigenetic diversities and structures in a wild barley species (Hordeum brevisubulatum) using MSAP, SSAP and AFLP

“…The bands of MSAP were scored as described by Li et al (2010). Percentage of polymorphic loci, Shannon's information index (I), Nei's (1973) gene diversity index (h), (epi-) genetic distance, relative degree of (epi-)genetic diversity (G ST ) and gene flow (N m ) were calculated using the POPGENE program, version 1.31 (Yeh et al, 1997).…”

“…Furthermore, because DNA methylation patterns are often responsive and prone to alterations under environmental and biological stresses (Kalisz and Purugganan, 2004;Salmon et al, 2005), it is likely that they also play an important role in coping with stress and facilitating ecological adaptation by modulating the expression of critical genes. A suitable scoring criterion for assessing polymorphisms in DNA methylation has been used in measuring epigenetic variation in populations (Keyte et al, 2006;Li et al, 2010). Henceforth, some studies have paid attention to the polymorphism of cytosine methylation patterns in populations (Herrera and Bazaga, 2010;Lira-Medeiros et al, 2010;Yi et al, 2010;Richards, 2011).…”

Targeted mutation breeding of horticultural plants

“…PCR and HRM analysis are conducted on the same samples in multiwell format, which facilitates throughput. HRM has been used to discover induced variability in specific tomato, wheat, and maize genes (Gady et al, 2009, Dong et al, 2009, Li et al, 2010 and natural variability in target genes of almond, barley, lettuce, olive, potato, and peach (Wu et al, 2009;Hofinger et al, 2009;Simko et al, 2009;Muleo et al, 2009;De Koeyer et al, 2009;Chen and Wilde, 2011). Improvement of mismatch detection by HRM and TILLING is an active field of study, largely due to medical diagnostic applications (e.g.…”

Mutation Detection by Analysis of DNA Heteroduplexes in TILLING Populations of Diploid Species

“…Usually up to fivefold pools are used in HRM (Gady et al 2009;Li et al 2010), although up to 12-fold pools were used for SNP detection in peach cultivars (Chen and Wilde 2011). The differences in the pooling sizes for which the correct mutation detection is possible are probably due to the different instruments that are used in different studies or in the genome size of the species that is being studied (Chen and Wilde 2011).…”

“…BRCA1 and BRCA2 genes, Hondow et al 2011). This method has also been used to detect mutations (single-nucleotide polymorphisms, SNPs and small insertions/deletions, InDels) in plant genetic studies, including TILLING in Lycopersicon esculentum (Gady et al 2009), Oryzias latipes (Ishikawa et al 2010), Triticum aestivum (Botticella et al 2011), Brassica rapa (Lochlainn et al 2011) and genetic mapping in Hordeum vulgare (Lehmensiek et al 2008), Zea mays (Li et al 2010) and Oryza sativa (Li et al 2011). HRM has also been used for the identification of transgenic plants (Milner et al 2014) and the detection of DNA methylation (Wojdacz and Dobrovic 2007).…”