High-resolution Melting Approachusing

Journal of Integrative Plant Biology 2010,52(12):1036–1042New TechnologyA Cost-effective High-resolution Melting Approach using the EvaGreen Dye for DNA Polymorphism Detection and Genotyping in PlantsYi-Dan Li 1,Zhi-Zhan Chu 3,Xiang-Guo Liu 1,Hai-Chun Jing 2,Yao-Guang Liu 3∗and Dong-Yun Hao 1∗1BiotechnologyResearch Centre,Jilin Academy of Agricultural Sciences,Changchun 130033,China 2Institute of Botany,the Chinese Academy of Sciences,Beijing 100093,China3College of Life Sciences,South China Agricultural University,Guangzhou 510642,China ∗Corresponding authorsTel:+8643187063195;Fax:+8643187063080;E-mail:dyhao@ Tel:+862085281908;Fax:+862085282180;E-mail:ygliu@Available online on 5October 2010at and /journal/jipb doi:10.1111/j.1744-7909.2010.01001.xAbstractHigh-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 ing this economical dye we successfully identified multiple novel mutant alleles of Gln1-3gene,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.J.Integr.Plant Biol .52(12),1036–1042.IntroductionHigh-resolution melting (HRM)is a new generation of mutation scanning and genotyping technology (Wittwer et al.2003).The advantages of HRM include the high efficiency and labor-saving benefits.Since invented in 2003,this technology has been adopted in clinical chemistry,human pathology and plant sciences (White et al.2007;Croxford et al.2008;Wu et al.2008;Akiyama et al.2009;Dagar et al.2009;Hofinger et al.2009;Sun et al.2010;Ujino-Ihara et al.2010).Several HRM platformsare available from various manufacturers.The key to thistechnology is the use of special fluorescent dyes that can bind to double-stranded DNA in a saturated manner.Popular dyes in use include LCGreen/LCGreen Plus (Idaho Technology Inc.,Salt Lake City,UT,USA),ResoLight Dye (Roche Diagnostics Limited,Shanghai,China)and SYTO9(Invitrogen,Beijing,China),among which LCGreen and LCGreen Plus are by far the most widely used due to its high sensitivity,stability,reliability and compatibility with many popular HRM instruments (Caren et al.2006;Herrmann et al.2006).However,the high cost ofC2010Institute of Botany,Chinese Academy ofSciencesA Cost-effective HRM Approach using EvaGreen 1037these dyes often limits the application of this technology,in particular when dealing with a large number of samples.Eva-Green (Biotium Inc.,Hayward,CA,USA)is a newly developed and low-cost dye,and has been used in quantitative real-time polymerase chain reaction (PCR),post-PCR DNA melt curve analysis and several other applications (Ihrig et al.2006;Sang and Ren 2006;Wang et al.2006;White et al.2007;Akiyama et al.2009;Dagar et al.2009;Sun et al.2010;Ujino-Ihara et al.2010).It is also a so-called saturated dye that intercalates every single nucleotide of the double-stranded DNA.The objective of this paper was to examine whether EvaGreen can be used in HRM analysis as a reliable and economical approach for routine detection of DNA polymorphisms and genotyping in plants.ResultsDNA polymorphism detection in LightScanner RplatformLCGreen Plus (or LCGreen)was the manufacturer-recommended dye for HRM analysis using LightScanner.Weexamined the performance of EvaGreen as a substitute of LCGreen Plus in DNA polymorphism analysis.Three ampli-cons of approximately 300bp in length with known sequence polymorphisms in the maize Gln1-3gene,which producesaFigure parison of the high-resolution melting (HRM)assays using EvaGreen (left)and LCGreen (right).The HRM data are presented as two different parts:the top displays normalized melting curves for the tested amplicons,whereas the bottom shows difference curves displaying the difference in fluorescence ( F )of the amplicons in relation to the wild-type (WT).Amplicons containing single nucleotide polymorphisms (SNPs)or deletions and the wild-type samples used in the assay are color coded and are indicated inside the bottom graphs.The curves are also color coded according to the color of the amplicon.cytosolic glutamine synthetase isoenzyme (GS1)and plays an important role in grain development (Martin et al.2006),were selected.The aliquots of the same amplicons were analyzed using LCGreen Plus dye as the control.As shown in Figure 1,the normalized melting curves and the difference curves using EvaGreen were similar to those using LCGreen Plus.The am-plicons containing one single nucleotide polymorphism (SNP),two SNPs or 4nt deletion were clearly distinguished from the wild-type amplicons at F >0.05,a value that is considered to be significant in HRM analysis.Detection of nucleotide polymorphisms in pooled samplesThe pooling strategy is frequently used in mutation scanning to increase the efficiency whilst reducing costs.To examine the detection efficiency of EvaGreen in pooled samples,the above-mentioned mutant amplicons were mixed in various ratios with the corresponding wild-type amplicons.It can be seen from Figure 2that both EvaGreen and LCGreen HRM analyses showed a comparable performance.As expected,the 1:1(variant:wild-type)amplicon mixtures exhibited the highest difference curve peak.One SNP was also predictable in a 1:2(variant:wild-type)amplicon mixture (Figure 2).However,when the amplicon mixture ratio (variant:wild-type)was 1:3or higher,the F values became less than 0.05,indicating1038Journal of Integrative Plant Biology Vol.52No.122010Figure 2.High-resolution melting (HRM)assays using different mixing ratios of genomic DNAs of the variant and wild-type maize line as the templates.The HRM data are presented as graphs displaying the difference in florescence ( F )of the two different amplicons and wild-type amplicon mixtures in relation to the wild-type (WT).Each variant amplicon was mixed with a WT amplicon at a ratio of 1:1,1:2,1:3,or 1:5,respectively.that the prediction of one SNP was unreliable.For amplicons containing two SNPs,it was reliably detectable in a 1:1,1:2,1:3,or even 1:5(variant:wild-type)mixture.We could also detect the amplicon containing a 4nt deletion (Figure 2).Identification of novel variant alleles of the maize Gln1-3geneTo test the robustness and application of the EvaGreen-based HRM analysis,mutations in the Gln1-3gene in an ethyl methanesulfonate (EMS)-mutagenized maize popula-tions were screened.A total of 528M 2maize lines with defective grain development were selected from 8902maize M 1lines.Primers were designed to amplify four overlapping fragments of the gene for HRM analysis (Figure 3).In total 17mutations were predicted in 12maize lines and were confirmed by Sanger sequencing (Table 1).In addition to identifying various SNPs,a 1nt insertion and a 4nt deletion were found.Nine mutations appeared to be newly induced byEMS,showing the transition of G/C to A/T,as expected for the effects of EMS (Greene et al.2003).The other mutations were likely to be variants introduced by cross-pollination of the surrounding maize cultivars.Together,these results indicated that EvaGreen-based HRM analysis is efficient in detection of SNPs or other mutations when amplicons are around 300bp in length.EvaGreen-based HRM analysis for rice genotypingTo verify the use of EvaGreen-based HRM analysis for geno-typing,we genotyped rice lines with various polymorphic markers including SNPs,Indels and simple sequence repeats (SSR).The results showed that all of these molecular markers could clearly distinguish the genotypes of parental homozy-gotes and heterozygotes of F 2individuals in rice mapping populations.Figure 4shows examples of two markers with one and three SNPs in genotyping of an F 2population by EvaGreen-based HRM analyses.A Cost-effective HRM Approach using EvaGreen1039Figure 3.Schematic representation of the maize Gln1-3and detected nucleotide changes (top)showing the locations and sizes of polymerase chain reaction (PCR)amplicons (bottom).DiscussionThe present study demonstrated that EvaGreen was a satis-factory substitutive dye in LightScanner-based HRM analysis.Cross-platform comparison of instruments and dyes showed that LightScanner is an efficient and high throughput HRM platform,and the LCGreen Plus is a more efficient and sensitive dye for HRM analysis than ResoLight Dye,SYTO9and SYBR-Green (Caren et al.2006;Herrmann et al.2006).We found that in HRM analysis EvaGreen and LCGreen Plus performedTable 1.Summary of the high-resolution melting (HRM)results by using EvaGreen Lines Nucleotide change62538T >C182102G >C,2353G >A,2512A >T 632502insert A792480–24834-nt deletion 861821C >A,2522C >T 892504G >A 902533A >G1052353G >A,2504G >A 1651833C >A 1812513G >T 1832529G >A1842510G >C,2538T >Csimilarly in screening a range of DNA polymorphisms including SNPs,insertion and deletion.We also found that EvaGreen was even more sensitive in detection of the amplicon containing one SNP in pooled samples.The predictions of EvaGreen-based HRM analysis approached 100%accuracy when ampli-cons are around 300bp,which is an ideal size to identify DNA polymorphism in Lighscanner platform (Hofinger et al.2009).Hence,EvaGreen is an efficient,sensitive,and low-cost dye in detection of DNA polymorphism,genotyping and perhaps other applications.There are a few reports recently assessing the efficiency of different dye-based HRM analysis (White et al.2007;Ujino-Ihara et al.2010),in which EvaGreen was not used.However,these reports hardly described the running cost of this approach.In our laboratory,for example,the cost was estimated at approximately US $30per 96reactions (one plate)for HRM-PCR using LCGreen Plus and Master Mixture supplied by the vender in China.One way to reduce the cost would be to prepare the PCR reactions manually instead of using the Master Mixture kit.Still,the cost for LCGreen Plus alone would exceed US $15per 96reactions (one plate).Other dyes usable for HRM analysis are even more expensive than LCGreen Plus.Such a high cost limits the application of HRM technology for dealing with a large-number of samples in crop molecular breeding programs.In case of using EvaGreen;however,the running cost is only about 1/7or less than those of other dyes usable for HRM analysis,at about US $2per 96reactions.1040Journal of Integrative Plant Biology Vol.52No.122010Figure 4.Genotyping of rice F 2plants by EvaGreen high-resolution melting (HRM)with two single nucleotide polymorphism (SNP)markers (G/C >T/A variations).Top parts display normalized melting curves of the amplicons of 96F 2plants,of which the genotypes P1(ZS97A),P2(MH63),and heterozygote are identified.Bottom parts show derivative melting peaks of the same amplicons.In summary,EvaGreen is a competitive alternative for LCGreen Plus in HRM analysis.Shortly after this manuscript was first submitted,another report was published by Ujino-Ihara et al.(2010)that also describes the application of Eva-Green for HRM analysis of SNPs.At present,the EvaGreen HRM approach is routinely used in our laboratories.The EvaGreen-based HRM protocol described in this work may be adopted by other molecular biology and molecular breeding laboratories.Materials and MethodsPlant materialsMaize (Zea Mays cv.8902)pollen was mutagenized with EMS and applied to the silks of cv.8902ears (Okagaki et al.1991).Ears were harvested at 5–6weeks post pollination.M 1seeds were planted and a whole young leaf harvested from each plant for DNA sampling.An F 2population of rice was derived from a cross between two indica lines (ZS97A and MH63).Preparation of ampliconsFragments of the maize Gln1-3gene (GenBank:EU338535.1)was amplified by use of forward primer (position 2393)5 -AGTTCCAAGTCGGGCCTTCC-3 and reverse primer (po-sition2681)5 -TCCTCATCGACTCCGTGCTG-3 .Four ampli-cons derived respectively from three maize variants and maize cultivar Mo17were used in this study,including a single nucleotide polymorphism (SNP)mutant (A/T to G/C),a double-SNP mutant (A/T to G/C,G/C to T/A)and a 4nt deletion,together with a wild-type amplicon as the reference sample.Primer pairs of two rice SNP markers presented in Figure 4are (marker-1)5 -AGTAGCTTGGAATGGGCAGTAG-3 and 5 -CAGTCTCTTTCCATCCTGACAG-3 (for an 86-bp amplicon with 1SNP)and (marker-2)5 -GGTCTATTGATTA CAATGACATG-3 and 5 -CTCACATGGAGATTTGAATTCTT-3 (for an 86-bp amplicon with 3SNPs).PCR and HRM proceduresPCR amplifications using LCGreen Plus (Idaho Technology Inc.)were carried out in 10µL volumes containing 5µL HotShotA Cost-effective HRM Approach using EvaGreen1041Master Mix(Cadama Medical Ltd.,UK),1µL10×LCGreen Plus,1µL each5µM primer,and approximately50ng template DNAs of the maize parent or pooled mixture as the templates.In order to compare the detection effects,we mixed each mutant amplicon with the wild-type amplicon in different ratios,i.e.1:1, 1:2,1:3,and1:5.PCR amplifications using EvaGreen were carried out in10µL volumes containing5µL rTaq Premix(Takara-Bio,Dalian, China),0.5µL20×EvaGreen(Biotium Inc.),1µL each5µM primer,and approximately50ng(for maize)or approximately 10ng(for rice)template DNAs,including the pooled ones of maize.PCR reactions were performed using96well plates(Abgene, ),and the reactions were covered with15µL mineral oil.PCR profile for maize DNA samples was one cycle of 95◦C for5min,30cycles of95◦C for30s,64.2◦C for 30s,and72◦C for30s,one cycle of72◦C for10min, one cycle95◦C for30s,and then down to40◦C holding at the0.1◦C/s.PCR profile for rice genotyping was the same as above except that the re-nature temperature was62◦C. 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