荷花SRAPPCR反应体系的优化与确立孙祖霞

201134653－58JournalofNanjingAgriculturalUniversityhttp//nauxb．njau．edu．cn2011－06－09200903020。*[e-mail]。．SRAP-PCRJ．201134653－58SRAP-PCR11*111121．2100952．210014NelumbonuciferaGaertn‘’L1645SRAP-PCRMg2+、dNTPs、TaqDNA、DNA5SRAP-PCR。SRAP-PCR10μL2．0mmol·L－1Mg2+、300μmol·L－1dNTPs、0．5UTaqDNA、4μmol·L－1、50ngDNA10×PCRBuffer。TaqDNA、Mg2+、、dNTPs、DNA。48SRAP-PCR、。SRAP-PCRS682．32A1000－2030201106－0053－06OptimizationandestablishmentofSRAP-PCRreactionsystemforNelumbonuciferaGaertnSUNZu-xia1LIUZhao-lei1*CHENSu-mei1CHENFa-di1LOUWang-huai1GUOHai-lin121．CollegeofHorticultureNanjingAgriculturalUniversityNanjing210095China2．InstituteofBotanyJiangsuProvinceandChineseAcademyofSciencesNanjing210014ChinaAbstractUsingNelumbonuciferaGaertn‘Xinhong’leavesastheexperimentalmaterialbytheorthogonalexperimentdesignL1645fivefactorsincludingconcentrationforMg2+anddNTPsdosageforTaqDNApolymeraseprimerandtemplateDNAinSRAP-PCRreactionsystemwereoptimizedandtheoptimizationSRAP-PCRreactionsystemsuitableforlotuswerealsoestablished．TheresultshowedthattheoptimalSRAP-PCRreactionsystemwasasfollowstotalvolume10μLincluding2．0mmol·L－1Mg2+300μmol·L－1dNTPs0．5UTaqDNApolymerase4μmol·L－1primer50ngDNAand10×PCRBuffer．TheorderofeachfactorindifferentlevelsaffectingtheresultofPCRwasTaqDNApolymeraseMg2+primerdNTPsDNA．TheoptimalSRAP-PCRreactionsystemwasidentifiedbymeansofgenomicDNAfrom48varietiesofN．nuciferaGaertnandtheamplificationpatternwithclearbandandrichpolymorphismwasobtained．ItisconcludedthattheSRAP-PCRreactionsystemissteadyandreliable．KeywordsNelumbonuciferaGaertnSRAP-PCRorthogonalexperimentdesignoptimizationofreactionsystemNelumbonuciferaGaertnNelumbonaceaeNelumbo、31。78。ISSR、SSRRAPD2－4。sequence-relatedamplifiedpolymorphismSRAPLi52001PCR、、、。6－7、8、9、10QTL11。SRAP12。SRAP、。。13、614。34‘’SRAP-PCRMg2+、dNTPs、TaqDNA、DNA548SRAP-PCRSRAP、。11．1481－70℃。1Table1ListofmaterialsNo．MaterialGermplasmsourcesNo．MaterialGermplasmsources1XinhongA25ZhuoyueA2AijiangnanA261Shanghai1G3SinianA27QinhuairenjiaA4CaiyunB28YixianlianA5JinlingchangxiangA29JingcaiA6YulourenzuiC30BaiheA7XintongshuaiA31YicaiA8YaoniangyujiaoA32MoheH9HongtailianC33GuifeizuijiuA10JuziA34XuanwuhonglianH11XiaojinbianA35GuifeichuyuA12FengbaoA36YunyaA13JinfenghuangA37HuanqingA14DongfanghongA38ShaoxinghonglianH15XiaosajinD39JinhehuanA16YellowAntelopeA40TaoyuanchunseA17PuzheheibaiheD41HongjinqueA18ShenqingA42JinlinghuoduA19KaluolingongzhuA43HongqingtingA20HongfeitianA44HuangmudanA21BaoshihuaE45XiangxuehaiA22XuecuilianF46JinsenianhuaA23LüzhixingA47HuohuaC24JinbihuihuangA48GuanyinlianAANanjingYileenGardenCo．LtdBChongqingDazuYameijiaAquaticPlantsCompanyCEastLackScenicAreaofWuhanDPuzheheiTourismAreainYunnanProvinceEChineseLotusReaserchCenterFBaiyangdianLotusDaguanyuaninHebeiProvinceGShang-haiBotanicalGardenHHangzhou1．2Mg2+、dNTPsTaqDNA100bpDNAmarkerSRAP2。2DNASRAP-PCRTable2TheprimersequencesusedforSRAP-PCRofgenomicDNAformNelumbonuciferaGaertnPrimer5'→3'5'→3'sequencePrimer5'→3'5'→3'sequenceForwardMe20Me13Me19TGAGTCCAAACCGGTCCTGAGTCCAAACCGGTGTTGAGTCCAAACCGGTAAReverseEm2Em4Em9GACTGCGTACGAATTAACGACTGCGTACGAATTTGAGACTGCGTACGAATTGACPTC－100TMPCRMJResearch、Eppendorf5810REppendorf、JY－SCZ7JS－380。1．31．3．1DNACTAB15DNA。456SRAP-PCRDNA1．2%DNA。DNA50ng·μL－1－20℃。1．3．2SRAP-PCR‘’L164516Mg2+dNTPs、TaqDNA、DNA543。SRAPMe20－Em23。Mg2+1．0、1．5、2．0、2．5mmol·L－1dNTPs100、200、300、400μmol·L－1TaqDNA0．5、1．0、1．5、2．0U2、4、6、8μmol·L－1DNA25、50、75、100ng。10μL10×PCRBuffer。3SRAP-PCRTable3OrthogonaldesignforSRAP-PCRNo．FactorandlevelMgCl2/mmol·L－1dNTPs/μmol·L－1TaqDNA/U/μmol·L－1PrimerDNA/ng11．01000．522521．02001．045031．03001．567541．04002．0810051．51001．0610061．52000．587571．53002．025081．54001．542592．01001．5850102．02002．0625112．03000．54100122．04001．0275132．51002．0475142．52001．52100152．53001．0825162．54000．56501．3．3SRAP-PCR122μL100bpDNAladderDNA8%0．5×TBE200V2～2．5hloadingbuffer。17。1．3．4SRAP-PCR48DNA2SRAPMe13－Em4ME19－Em9SRAP-PCRSRAP-PCR。1．4Excel2003。22．1SRAP-PCR2．1．1DNADNA1DNASRAPDNA。1DNAFig．1ElectrophoresisofgenomicDNAforsometestedsamples1－231。TheNo．oftestedsamplesisthesameasinTable1．2．1．232。1、2、3、4、9、10、13、145534、5、11、15515。、11。、、、16、、1。162、12、4、1、13、11、5、6、10、9、16、8、7、3、15、14。kiR。4SRAP-PCRTaqDNA、Mg2+、、dNTPsDNAMg2+2．0mmol·L－1、dNTP300μmol·L－1、TaqDNA1．0U、4μmol·L－1、DNA50ng。2Fig．2ElectrophoresisoforthogonaldesignM100bp100bpDNAladder1－163。TheNo．ofamplificationsystemsisthesameasinTable3．4Table4StatisticalanalysisoforthogonaldesignNo．FactorandlevelMgCl2/mmol·L－1dNTP/μmol·L－1TaqDNA/U/μmol·L－1PrimerDNA/ngk14．758．0010．754．508．00k28．758．7512．0010．2510．25k310．7510．005．7510．007．50k49．757．255．509．258．25R6．002．756．505．752．75kTheaveragevalueofeachfactorlevelRTherangeofeachfactor2．1．3SRAP-PCR4211TaqDNADNA。TaqDNA1．0Uki、0．5UkiTaqDNA0．5UDNA50ngkiDNA50ng。SRAP-PCR10μLMg2+2．0mmol·L－1、dNTPs300μmol·L－1、TaqDNA0．5U、4μmol·L－1、DNA50ng10×PCRBuffer。2．2SRAP-PCR2Me13－Em4Me19－Em948PCR。3Me13－Em41412PPB85．71%Me19－Em9151493.33%。2、DNASRAP-PCR。3PCR。656SRAP-PC