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1、毛细管电泳四色荧光检测法分析茶树SSR标记第13卷第3期2009年6月生命科学研究LifeScienceResearchVo1.13No.3June20o9毛细管电泳四色荧光检测法分析茶树SSR标记黄建安,李娟,谭月萍2,刘硕谦,李勤,刘仲华(1.湖南农业大学茶学教育部重点实验室,中国湖南长沙410128;2.湖南省茶业有限公司,中国湖南长沙410005)摘要:将毛细管电泳四色荧光检测技术应用于茶树SSR标记分析.该方法采用三引物PCR扩增SSR位点,三引物即在5端加有M13尾巴序列(5.CACGACGITGTAAAACGAC.3)的特异正向引物,特异反向引物及带有荧光标记的通用型Ml3引物:
2、为了运用四色荧光检测系统使通过一次毛细管电泳能同时检测3个以上的SSR住点.采用蓝,绿,黑3种不同颜色的荧光染料分别对3个M13引一物进行标记.应用该方法对42个茶树品种(系)的16个SSR住点进行遗传分析的结果表明:此法具有简便,可靠,低成本及高通量的优点;且随着所分析SSR位点数的增加,降低成本的效果更加显着.采用建立的方法,还筛选获得了11个多态性丰富的可应用于茶树遗传研究的SSR标记.关键词:微卫星(microsatellite,SSR);通用M13引物;毛细管电泳;四色荧光检测;茶树中图分类号:$571.1:Q523文献标识码:A文章编号:10077847(2009)03.0251.
3、07TeaPlantMicrosatelliteGenotypingbyCapillaryElectr0ph0resis.withFour-ColorFluorescentDetectionTechniqueHUANGJianan,LIJuan,TANYueping,LIUShuoqian,LIQin,LIUZhonghua(1.TheKeyLaboratoryofTeaScienceofMintryofEducation,HunanAgricuhuralUniversity,Changsha410128,Hunan,China;2.HunanTeaCo.,Ltd,Changsha410005
4、,Hunan,China)Abstract:Thecapillaryelectrophoresiswithfour.colorfluorescentdetectiontechniquewasusedtoanalyzethemicrosatellitesinteaplant.ThreeprimerswereusedinonePCRreactionforamplificationofadefinedmicrosatellitelOCUSbythismethod.Thethreeprimerswereasfollows:asequence.specificforwardprimertailedwit
5、hM13一tailatits5end.asequence.specificreverseprimerandauniversalfluorescent.1abeledMl3primer(5.CACGACGTrGTAAAACGAC3).Inordertodetectthreeormoremicrosatellitelocisimultaneouslyineachcapillaryelectrophoresisthroughfour-colorfluorescentdetectionsystem.threeuniversalM13primerswerelabeledwiththreedifferen
6、tfluorescencedyes(blue,greenandblack).Withthismethodthegeneticanalysisof16microsatellitelociamong42teaplantswascaiedout.Theresultsshowedthatthismethodhadmeritsofsimple,reliable,costeffectiveandhighthroughputmicrosatellitegenotyping,andtheeconomybroughtbyitbecamegreaterwithanincreasingnumberofmicrosa
7、tellitemarkers.Also,11polymorphicmicrosatellitemarkersofteaplantwereidentifiedbythismethod.andthesemarkerswouldbeusefulforinvestigatinggeneticquestionsofteaplant(Camelliasinens).Keywords:microsatellite;universalM13primer;capillaryelectrophoresis;fourcolorfluorescent收稿日期:2o080905;修回日期:2009-0507基金项目:国
8、家自然科学基金资助项目(30871572);湖南农业大学人才稳定基金资助项目(07WD22)作者简介:黄建安(1964一),女,湖南沅江人,湖南农业大学教授,主要从事茶叶生物化学与分子生物学研究,E.mail:iian7513sina.c0m;通讯作者:刘仲华(1965一),男,湖南衡阳人,湖南农业大学教授,主要从事茶及植物功能成分化学研究,E-mail:.Receiveddate:20080905;Accepteddate:20090507Foundationitem:ProjectofNationalNaturalScienceFoundationofChina(30871572);Hun
9、anAeulturMUniversityTalentStabilization(O7WD22).Biographies:HUANGJian-an(1964-),female,YuanjiangofHunanprovince,professorofHunanAculturalUniversity,E-mail:jian7513sina.corn;Correspondingauthor:LIUZhonhua(1965-),male,HengyangofHunanprovince,professorofHunanAculturalUniversity,E-mail:larkin-I;1l】63.co
10、n1.252生命科学研究2009正detection;teaplant(Camelliasinensis)CLCnumber:$571.1;Q523Documentcode:AArticleID:10077847(2009)03025107(L/feScienceResearch,2009,13(3):251257)Microsatelliteanalysis,alsoreferredassimplesequencelengthpolymorphismorshorttandemrepeatpolymorphism,isawidelyusedtechniqueforvarietalidentif
11、ication,fortrackingtraitsinbreeding,andtogenerategeneticmaps_1_.TheprincipleofthemicrosatellitemarkertechniqueistouseprimerpairsthatflankrepetitiveDNAsequencestoamplifysamplesofgenomicDNAandtoexaminethesizeoftheamplifiedallelesthroughseparatingtheproductsonhighresolutionpolyacrylamidegelsbyelectroph
12、oresis2J.anddetectionofamplifiedallelesisachievedbythreedifferentprinciples:1)radioactively,2)silverstainingand3)alaserinducedfluorescencedetectionsystem.Theemploymentoffluorescentlabeledmicrosatelliteprimersandlaserdetection(e.g.automatedsequencer)ingenotypingproced-uressignificantlyimprovesthethro
13、ughputandaut0matization.Theuseofmicrosatellite,however,canbecostlyduetothehighpriceofthefluorescentlabe1.whichmustbecarriedbyoneoftheprimersintheprimerpair.Thecostofmierosatelliteassayincreasessubstantiallyifalargenutuberof1ocihavetobetested.Inordertoovercomethisfinancialdifficulty,Schuelkeintro-duc
14、edanovelprocedureinwhichthreeprimerswereusedfortheamplificationofadefinedmicrosatellitelOCHS:asequence-specificforwardDrimertailedwithM13.tailatits5end,asequencespecificreverseprimerandtheuniversalfluorescent1abeledM13primerE引.ThisM13.tailsequenceisroughly18-21bp,suchastheM13sequence5一CACGACGTFGTAAA
15、ACGAC.3,andisunlikelytooccurinthegenomeofinterest.Todate,thesystemhadbeenusedsuccessfullywitholives引.tigersnake51switchgrass,wheatmicrosatellitemarkersandcouldbeavaluabletoolforgenotypingplants.Tointroduceasimple,lessexpensiveandhighthroughputmethodforamplificationanddetectionofmicrosatelliteinteapl
16、ant,threedifferentfluorescencedyeswereusedtolabelthreeM13primerswhichhavethesamesequenceinthisresearch.Thus,threeormoredifierentmicrosatellitemarkerswithdifferentdyescouldbedetectedsimultaneouslyineachcapillaryelectrophoresisiftheallelesmigratedtononoverlappingsizeranges.Suchamethodwouldallowsignifi
17、cantcostreductionandfacilitatethegenotypingoflargernumberofsamples.1Materialandmethods1.1Teaplantmaterial42varietiesfromtheteacollectionorchardinHunanAgriculturalUniversitywereincludedinthemolecularanalysis.AnalyzedteavarietieswerelistedinTable1.Table1ThevarietiesofanalyzedteaplantsNo.VarietyNoNoVar
18、ietyVarietyMeizhanHuangyeshuixianDonghuzaoDahongpaoXinyangzhongGaoqiaozaoRuchengbainlaochaQiquZaofengchunMaoxieXishuiNo.1FushouMeitanNo.2ZhenghedabaichaXiangfeicuiFuhaoBixiangzaoTieguanyinClone9803GuazijinFudingdabaichaBaiguanyinWuniuzaoHuangyanXishuiNo.5FudingdahaochaBaihaozaoFuyunNo.6LongjingNo.43
19、YingshuangZiyazhongGuangxiantangzhongJinxuanMingfengPingyunNo.1lZhuyeqiBaimaoNo.2ChaoandawuyeYunnandayezhongSoubaiBaxianchaTongtianxiang如鲳加勰123456789mH第3期黄建安等:毛细管电泳四色荧光检测法分析茶树SSR标记2531.2Microsatelliteassay1.2.1DNAisolationGenomicDNAswereextractedfromfreshtealeavesbyamodifiedCTABmethodfollowingthepro
20、ceduredescribedbyHuang7j.1.2.2Primersynthesis42primerpairsforteamicrosatellitelociweresynthesizedbySIGMACompany.Foreachprimerpair,thesequenceoftheoriginalforwardprimerwasredesignedbyaddinganuniversalM13-tail(5.CACGACGTrGTAAAACGAC.3)totheir5ends【3.TheuniversalM13primerswerelabeledwithdifferentfluores
21、cencedyes,D4(bluedye),D3(greendye)andD2(blackdye)respectively(Table2),allowingmultiplexed4-colorfluorescencedetectionforthroughputgenotypingofteaplantspecies.TheusablemarkerswereshowedinTable3.TheprimerpairsS1一S11weredesignedaccordingtopublicESTsequencesbyPrimer3(http:/biotools.umassmed.edu/bioapps/
22、primer3_www.cgi)andtheS12.S16werefromFreemansarticleEs3.Table2InformationofuniversalM13primerlabeledwithdifferentfluorescentdye1.2-3PCRconditionsIlhePCRwasconductedinatotalvolumeof20L,containing30nggenomicDNA,1suppliedPCRbufferincluding1.5mmol/LMgC12(Promega),0.2mmol/LofeachdNTP(Promega),0.5unitofTa
23、qDNApolymerase(Promega),0.5xmol/Lofsequence-specificreverseprimer,0.45Imol/LoffluorescencelabeleduniversalM13primerand0.05xmol/LofforwardprimerwithM13一tail.AmplificationreactionswereconductedusinganABI一3700PCRthermocyclerunderthefollowingconditions:94.Cfor5min;35cyclesof94.Cfor45s;aprimerspecificopt
24、imalannealingtemperature(seeTable3)for1minand72.Cfor1min;followedbya10minextensionat72.C.GradientPCRwasusedtodetermineoptimalannealingtemperatureforeachprimerpair.1.2.4CapillaryelectrophoresisdetectionThePCRreactionswerecarriedoutseparatelyforeachmicrosatellite,andthemixtureofPCRproductsofthreeormor
25、edifferentmarkerswithdifferentdyeswasmadeforsimultaneousdetectionoftheamplifiedalleles.Samplewasinjectedfromatotalvolumeof25Lofsampleloadingsolution,including0.25#xLoftheCEQ400sizestandardlabeledwithreddyeperwellofa96wellplate.Tothiswasaddedupto1.02.0txLofPCRproductsmixtureperwellandlayeredwithminer
26、aloiltopreventevaporationofthereagentswhilerunningontheBeckmanCoulterCEQTM8000.ThedatawerecollectedautomaticallybydetectingofthedifferentfluorescenceandanalyzedbyGenomeLabTMFragmentAnalysisSystem.TheamountofPCRproductuseddependedontheefficiencyofthePCRreactionsandtherelativesignalstrengthofamplifica
27、tionproducts,thelattervarieddependingonthesortofdyelabeled.TheroughlyequalsignalstrengthcouldbeobtainedwhentherelativevolumeofbluedyePCRproduct,greendyePCRproduct,blackdyePCRproductwasinaratioof1:2:4inthePCRmixture.1.2.5StatisticalanalysisPOPGENEsoftware【http:/www.ualberta.ca/-fyeh/fyeh】wasusedtocal
28、culatetheeffectivenumberofalleles(Ne),Neis(1973)genediversity(H),Shannonsinformationindex(I).Thepolymorphieinformationcontent(PIC)wascalculatedusingtheonlinePICcalculatorhttp:/www.agri.huji.ae.il/welledHayindparent/PIC.2Resultsanddiscussion2.1Four.colorfluorescentdetectionmethodToreducethecostofauto
29、matedlaserdetectionoffluorescentlabeledPCRfragments,anovel生命科学研究2009芷approach引wastouseaM13一tailedprimer,whereeveryforwardprimerwas5一tailedwiththeM13sequence.Bothmicrosatelliteprimerswereunlabelled,butathirdprimer,calledM13primer,wassynthesizedwhichhadthesamesequenceastheM13-tailsequence.butwasdye1ab
30、eledatits5end.ByusingthreeprimersinthePCRreaction,thetailedforwardandnormalreverseprimersspecificallyamplifythemicrosatelliteintheearlystagesofthereaction.However,a(1015):1molarratiooflabeledM13primerversusaM13-?tailedforwardprimerwereinthePCRreactionsystemandoncetheM13一tailedforwardprimerwasexhaust
31、ed,thefluorescentlabeledM13primertookoverthefunctionoftheforwardprimer,incorporatingfluorescentlabelintothePCRproductforallowingthelaserinducedfluorescencedetection.ThefluorescencelabeleduniversalM13primercouldbeusedforanyM13一tailedforwardprimertogeneratealabeledamplifiedallele.AndtheM13一tailedforwa
32、rdprimerscouldbeobtainedinexpensivelybycustomsynthesis,butthisprocedureonlyusedasingledyetoallowonemicrosatellitelocuswasanalyzedineachcapillaryelectrophoresis.Toreducethecostfurther,threedifferentfluorescentdyeswereusedtolabelthreeM13primersrespectively:dyeD4(blue),dyeD3(green),anddyeD2(black)(Tabl
33、e2,Fig.1).AndthedyeD1(red)wasforinternalstandard.ItthereforeallowedthemultiplexedanalysisofthreeormoredifierentmicrosateUitemarkersineachcapillaryelectrophoresisiftheallelesmigratedtononoverlappingsizeranges.TheFig.2-3showedtheresultsoffluorescencedetectionthatPCRproductsfrom5differentmierosatellite
34、primers,incorporatingM13with2differentcolors,weremixedtogetherandinjectedintoCEQasonesample.ThevarietyinFig.2wasMingfengandinFig.3wasBaihaozhao.Fig.4wastheresultsoffluorescencedetectionthat5differentmicr0satelliteprimersPCRproductsincorporatedM13with3differentcolorsweremixedintoonecapillaryinjection
35、.ThevarietyinFig.4wasXiangfeicui.Besidesthehighthroughputmicrosatellitegenotypingby4一colorfluorescentdetectionwhencomparedtostandardfluorescentmethod,thecostdecreasingwiththismethodbecomegreaterwithincreasingnumbersofmicrosatellitemarkers.Forexample,withthestandardfluorescentmethod,atacostofRMB$1.30
36、/baseandRMB$3o0.00/fluorochromeendlabelingofeachforwardprimer,thecostofprimersforamicrosatelliteamplifiedwithtwo20.baseprimerswouldbeRMB352.00.Thecostofprimersforstandardfluorescence.baseddetectionof10microsatellitemarkerswouldbeRMB3,520.O0.Ontheotherhand.withtheM13.tailedprimermethodthecostoftheunl
37、abeledprimerspermicrosatellitemarkerwouldbeRMB$76.70(ie39basesfortheM13-tailedforwardprimerand20basesforthereverse,atotalof59basesatRMB1.30/base).For4一colorfluorescencedetection,onlythreeuniversallabeledMl3primerswereneeded,eachwith19bases.Theuniversalprimerswouldbeusedforeverymicrosatellitemarker,a
38、ndthuswouldhavetobesynthesizedonalargerscale,thenthecostperbasewentupfromRMB$1.30t01.50/base.ThecostofsynthesisofthethreeuniversalM13primerswouldbeRMB85.5O.plusRMB$90O.O0f0rthethreefluorochromeendlabeling,yieldingatotalofRMB$985.50.Thecostofprimersfor4.colorfluorescencebaseddetectionof10microsatel1i
39、temarkerswouldbeatotalofRMB$1.752.50.aneconomyof50%whencomparedtothestandardfluorescentmethod.Fortheanalysisofalargersetofmicrosatellites,thecostofthestandardfluorescentmethodwasincreased1inearly.forexamplefora100一markerproject.itwouldbe100x352=RMB35,200.00.Whereas.bytheM13.tailedforwardprimercombin
40、ationwith4.colorfluorescencedetectionthecostincreasewasnotlinear.forexampleforasetof100markersthecostwouldbe10076.70=RMB$7670.00fortheunlabeledprimersandthesameRMB$985.50forthethreelabeleduniversalM13primers,totaling第3期黄建安等:毛细管电泳四色荧光检测法分析茶树SSR标记255CodeRepeatmotifPrimersequences(5to3)Optimalannealing
41、temp./oCSizerange/bpNo.ofNeHallelesPICvalueS1(CT)15S2(GA)14S3(CT)12S4(CT)10S5(GA)14S6(CT)22S7(GA)l2S8manyS9(GA)11S10(CT)10S11(GA)1OS12(CT)15(CA)12S13(GT)16$14(CA)12S15(GT)12(GA)18S16(TG)13F:M13*-CTTCrrAACTGGAAAGGGGnGGTR:CAAAGTrI,CCTGGAG1rIlAAACGAF:Ml3.ACCTCGAAGCTGCATITGTR:ACAATCATIlGCCACCACATCFMI3-C
42、ACAAATCTCATcCCCAAACATACR:TCTcTCTCTGGAAATrGAAGAACAF:M13-CCACAAAAACAAAGAATCGGCnTrR:CCTrGAAC1TITCCTCTTCCCATCCF:Ml3.ACAAGCTCGGGCAACCACCATAR:GGATCCAAAGACTTCTrAAGAGCAGF:Ml3.AAAAGAGCAGGAAAAGATGATGIGR:TIrrAITGAAGCAGAGGTGAAGAF:M13.GGGATTGTTGAAG兀rCTCTCTAR:TCCTCCACACCAAATCCTCTCF:M13.AAAGGAAAGGAAAGGAAAGGAAGR:TC
43、ACAAATCAAACAGATTCCAGATF:M13GGATCCACAGTAGTACACTCTAATCR:GGATGCCTCATGTAAGGGACTrrGF:M13-ACGACGGAACTTGCAAAGTAATAR:TCTrGTrGCGTAGGATGATATrJ,CF:M13.1GCnATCTC1BTCCATCCTR:GCCACTCTGCAAAGGAACTCnM13-CTlcATlGGAGCCAAGGAAGCR:AAAGCAGrCTGGAACCTrGCF:M13-TrACATCTCTr兀CAGCTGTGGR:CTTCGGGAACTICTGCTTCATCF:M13.GCATCATTCCACCA
44、CTCACCR:GTCATCAAACCACTGGCTCAF:M13.CATrATCGTCACTTGCAAAGAGGTR:CGAGAAGAAGAGCrCTATrCCTTF:M13-CACATIlCTTGGCGTGTrATrAATITR:ACA.rIlGGCTATCTC1A1A1G555855555560464760454655556O5555l71-206141.16-4-0.190.120.120.22-4-0.160.8523l-2982O1.11-4-0.110.090.080.180.120.90274-306141.150.160.120.110.224-0.】60.8615418ll
45、21.174-0-260.120.130.224-0.170.78214240131.194-0.170.150.110.264-0.170.87143179151.124-0.120.090.080.190.120.87129-14261.284-0.340.184-0.170.300.220.6518O一18121.594-0.350.364-0.140.544-0.160_33148170101.200.280.140.150.250.200.76228256121.160.250.1l4-0.120.204-0.170.773l821.8l4-0.220.444-0.070.644-0
46、.070.44l76216l71.17士O.200.134-0.1】0.244-0.14O.881602o0161.154-0.130.124-0.090.234-0.130.9010517391.084-0.110.074-0.080.134-0140.80I16一l7ll61.22士O.240.184-0.150_350.220.901582O72l1.224-0.220.164-0.120.280.170.91Notes:M13,CACGACGTYGTAAAACGAC;thesizerangeofallelesforthesegenotypeswasbasedonautomatedGenotyperdata;No.ofalleles,thenumberofdifferentallelesdetectedamong42teaplantaccessions.35o003000o250002Oo0Ol5【x】O10o0o5000l50155160165l70175l80l851901952o0205210Size/nt225o020O00175(x】I5000l25001OOoo