title: 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 LCGreen-based 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.
authors.affiliation: Biotechnology Research Centre, Jilin Academy of Agricultural Sciences, Changchun 130033, China.
authors.forename: Yi-Dan
authors.initials: YD
authors.lastname: Li
authors.forename: Zhi-Zhan
authors.initials: ZZ
authors.lastname: Chu
authors.forename: Xiang-Guo
authors.initials: XG
authors.lastname: Liu
authors.forename: Hai-Chun
authors.initials: HC
authors.lastname: Jing
authors.forename: Yao-Guang
authors.initials: YG
authors.lastname: Liu
authors.forename: Dong-Yun
authors.initials: DY
authors.lastname: Hao
mesh_terms: D000483:Alleles; D003362:Cost-Benefit Analysis; D003433:Crosses, Genetic; D018744:DNA, Plant / Q000235:genetics*; D005456:Fluorescent Dyes / Q000378:metabolism*; D017343:Genes, Plant / Q000235:genetics; D005838:Genotype; D009691:Nucleic Acid Denaturation / Q000235:genetics*; D012275:Oryza / Q000235:genetics; D010944:Plants / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000191:economics* / Q000379:methods*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D013698:Templates, Genetic; D003313:Zea mays / Q000235:genetics
chemical_list: D018744:DNA, Plant; D005456:Fluorescent Dyes