南方、试验站和花生根结线虫随机扩增多态性dna标记的研究

南方、试验站和花生根结线虫随机扩增多态性dna标记的研究

rootknotnemat组（meloidol.）是一个缓慢的马蹄莲类组，属于马蹄莲类。开普敦运营和运营的权限与ses%以上的权限之差有关。而且，80%的原始移动生态行为中存在壳和碎片超。而且，80%的移动生态行为中存在壳和碎片中的竞争行为。米。Effectivemanagementofroot-knotnematodesoftenrequiresrapidandaccuratespeciesidentificationoftargetnematodepopulations.Meloidogynespeciesaretraditionallydifferentia-tedfromeachotherbymorphologicalcharactersandisozymephenotypes.Morphologicalidentificationdemandsconsiderableskillandcouldbeunreliableduetosignificantintraspecificmorpholo-gicalvariationsinMeloidogynespp.IsozymephenotypinghasbeenshowntobeavaluabletoolforpreciseidentificationofmajorMeloidogynespecies.Alimitationofthistechniqueisthatjuveniles,malesandeggscannotbereliablydiagnosed,whichhindersitsuseinroutineexaminationofsoilsamplesthatoftencontainonlyse-cond-stagejuveniles.RecentprogressindevelopmentofPCR-basedmoleculardiagnosticshasofferedapotentialreplacementforthetraditionalprocedures.PowersandHarrisdiscriminatedsinglejuvenilesoffivemajorspeciesofMeloidogynebyamplificationandrestrictionoftheintergenicregionbetweentheCOIIandlrRNAgenesinthemitochondrialgenome(mtDNA-PCR-RFLP).Adi-sadvantageofthemtDNAprocedureisthatrestrictionenzymedigestionofthePCRproductsisneeded.Morerecently,Zijlstraetal.achievedrapididentificationofM.incognita,M.javanicaandM.arenariabydevelopingspecies-specificsequencecharacterizedamplifiedregion(SCAR)primers.However,SCAR-basedPCRproceduresfordirectdiagnosisofsinglejuvenilesofthethreeMeloidogynespecieshavenotbeenreported.ThepresentstudyconstitutesacontinuedefforttowardthedevelopmentofSCARprimersforidentificationofM.incognita,M.javanicaandM.arenaria.Wesequencedseveralspecies-specificrandomamplifiedpolymorphicDNA(RAPD)markersandscreenedforSCARprimerswithhighamplificationspecificityandefficiency.OurobjectivewastodevelopsensitiveSCAR-basedPCRassaysforidentificationofsinglenematodesofthethreemajorroot-knotnematodes.1杏仁杏仁1.1非模糊主义医学上的part3.3.3NematodepopulationsofMeloidogynespp.usedinthisstudyarelistedinTable1.Allthepopulationswerederivedfromsingleeggmassesfromfieldpopulations.Speciesidentificationwasbasedonfemalemalatedehydrogenase(Mdh)andesterase(Est)phenotypes(Table1).Nematodeswereroutinelymaintainedontomatocv.Sukang8at20-28℃inagreenhouse.1.2风-pcr公车比选Nematodeegg-masseswerehand-pickedfrominfectedtomatorootsandjuvenileswerehatchedinwaterat25℃.TotalgenomicDNAwasthenextractedfromjuvenilesusingamodifiedprotocoloftheminiprepmethodofCenis.ForPCRamplificationfromsinglenematodes,individualsecond-stagejuveniles,malesorfemaleswerehand-pickedandrupturedwithasteelneedleina6μLdropoflysisbuffer(1×ExTaqPCRbuffer,100μg/mLproteinaseK;TaKaRaBiotech,Dalian,China)onaglassslide.NematodelysateswerethentransferredtoPCRtubesandfrozenfor30minat-80℃.Afterfreezing,sampleswereincubatedfor1hat60℃and15minat95℃beforebeingprocessedforPCRamplification.1.3rapd-pcr和pcr合作,计算公式与程序总药Sixty-sixprimers(OP26,OPFandOPG,OperonTechnologies,Alameda,USA)werescreenedintherandomamplifiedpolymorphicDNA(RAPD)analysis.RAPDanalysiswasinitiallyperformedonfourbulkDNAsampleseachmadefromfourrepresentativepopulationsofM.incognita,M.javanica,M.arenariaandM.hapla,respectively.Primersamplifyingputativespecies-specificfragmentswerefurthertestedonadditionalpopulationsofMeloidogynespp.toverifythecorrelationofthefragmentstoagivenspecies.RAPD-PCRwasconductedin25μLreactionvolumescontaining10ngoftemplateDNA,0.8μmol/Lprimer,0.2mmol/LdNTPs,1×PCRbuffer(with2mmol/LMgCl2)and1unitofTaKaRaTaqDNApolymerase(TaKaRaBiotech).Reactionmixturesweresubjectedtoapreheatingat94℃for4min,followedby45cyclesof30sat94℃,1minat36℃and2minat72℃,andafinalincubationat72℃for10minusingaMastercyclerPersonalthermalcycler(Eppendorf,Hamburg,Germany).1.4日生非织造材料的表征CandidateRAPDfragmentswereexcisedfromagarosegelsandpurifiedusinganUNIQ-10DNApurificationkit(SangonBiotechnology,Shanghai,China).Thegel-purifiedfragmentswerethenclonedintothepMD18-Tvector(TaKaRaBiotech).SequencingwasdoneonanABIPRISM377-96sequencerusingBigDyeterminatorchemistry(PerkinElmerAppliedBiosystems,FosterCity,USA).SequenceswerecompiledusingBioEditVersion5(T.A.Hall,NorthCarolinaStateUniversity).1.5scarptirindex,mi-f/r,op.4.BasedonthesequencesoftheRAPDmar-kers,anumberofsequencecharacterizedamplifiedregion(SCAR)primersof18-25nucleotidesweredesignedwiththeaidofthesoftwareFastPCR(R.Kalendar,UniversityofHelsinki,Finland).TodetermineprimerpairsthatenablereliableidentificationofsinglenematodesofM.incognita,M.javanicaandM.arenaria,theSCARprimerswerescreenedforamplificationspecificityandefficiencyagainstallthepopulationsofMeloidogynespp.listedinTable1.SCARamplificationswithprimersetsMI-F/R,MJ-F/RandMI-F/MT-Rwereconductedin12.5μLvolumesthatincluded5ngoftemplateDNA,0.4μmol/Lofeachprimer,0.2mmol/LdNTPs,1×ExTaqPCRbuffer(with2mmol/LMgCl2)and0.5unitofExTaqDNApolymerase(TaKaRaBiotech).PCRwasprogrammedas:4minat94℃,followedby35cyclesof30sat94℃,30sat62℃and30sat72℃,andafinalincubationfor10minat72℃.ForSCARamplificationfromsinglenematodes,2μLofnematodelysatewasusedastemplateDNA,andthePCRprogramwasadjustedto40cycleswithanextensiontimeof45s.AfterPCR,5μLofamplificationproductswereelectrophoresedthrougha1.5%agarosegel,stainedwithethidiumbromideandvisulizedunderUVillumination.2opg/op领域,清责于meSixty-sixRAPDprimerswerescreenedfortheirabilitytodisplayDNApolymorphismsamongthefiveMeloidogynespeciesofM.incognita,M.javanica,M.arenaria,M.haplaandM.enterolobii.InitialscreeningandfurtherconfirmationtestsusingallthepopulationslistedinTable1revealedfourteenprimersthatamplifiedelevenandsixdistinctfragmentsspecificforM.incognitaandM.javanica,respectively(datanotshown).Fig.1showsanexampleofRAPDpatternsgeneratedwithprimerOP26-01.OftheRAPDmarkers,OPF-01600,OPF-01800,OPG-11740andOP26-011200,specificforM.incognita,andOPF-12600,OPG-18850andOP26-12690,speci-ficforM.javanica,weresubsequentlyclonedandsequencedforfurtheranalysis(Fig.1;datanotshown).1:MHSD1;2:MHHO1;3:MAJS1;4:MATW1;5:MAHO1;6:MAUS1;7:MJFJ1;8:MJYN1;9:MJBE1;10:MJIR1;11:MIFJ1;12:MISD1;13:MIAU1;14:MIIT1;M:DNAmarkerDL2000(TaKaRaBiotech).ThearrowindicatestheOP26-011200markerspecificforM.incognita.BasedonthesequencesofthesevenRAPDmarkers,28SCARprimersof18-25nucleotidesweredesignedandtestedfortheiramplificationspecificityandefficiencyagainstallthepopulationsofthefiveMeloidogynespecies(datanotshown).Thiscomprehensiveanalysisrevealedthreeprimerpairs,i.e.MI-F/R,MI-F/MT-RandMJ-F/RderivedfromthesequencesofOP26-011200andOPF-12600,respectively(Table2),thatwhenusedincombinationenabledrapidandsensitiveidentificationofM.incognita,M.javanicaandM.arenaria.UsingtheprimersetMI-F/R,afragmentof955bpwasamplifiedspecificallyfrompurifiedDNAoftheM.incognitapopulations(Fig.2A;Table1);usingtheprimersetMJ-F/R,afragmentof517bpwasamplifiedspecificallyfrompurifiedDNAoftheM.javanicapopulations(Fig.2B;Table1);andusingtheprimersetMI-F/MT-R,afragmentof779bpwasproducedfrompurifiedDNAofthepopulationsofM.incognita,M.javanicaandM.arenaria,noamplificationwasobservedfromthepopulationsofM.haplaandM.enterolobii(Fig.2C;Table1).ThesensitivityoftheSCAR1:MIFJ1;2:MISD1;3:MIAU1;4:MIIT1;5:MJFJ1;6:MJYN1;7:MJBE1;8:MJIR1;9:MAJS1;10:MATW1;11:MAHO1;12:MAUS1;13:MHSD1;14:MHSD2;15:MHHO1;16:MEHN1;M:DL2000DNAmarker.assayswasexaminedbyamplificationtestsusingcrudenematodelysates.ThethreeSCARmar-kerswerereadilyamplifiedfromonethirdofasinglesecond-stagejuvenile(Fig.3),maleorfemale(datanotshown).1-3:Meloidogyneincognita;4-6:M.javanica;7-9:M.arenaria;M:DL2000DNAmarker3mtnda-pcr-rflpe基本信息TheobjectiveofthisstudywastodeveloparapidandsensitivemethodforidentificationofMeloidogyneincognita,M.javanicaandM.arenaria.Toachievethis,weobtainedthesequencesofseveralspecies-specificRAPDmarkersanddesignedvariousSCARprimers.Arigorousscree-ningoftheSCARprimersledtotheidentificationofthreeprimerpairs,MI-F/R,MJ-F/RandMI-F/MT-R,thatwhenusedincombinationinPCRassaysenabledsensitiveidentificationofsinglenematodesofthethreemajorroot-knotnematodes(Fig.2andFig.3).ThereliabilityoftheSCAR-basedPCRassayshasbeenvalidatedbyanalysisof42populationsoffiveMeloidogynespeciesfromdifferentgeographiclocations(Table1).ComparedtothemtDNA-PCR-RFLPmethod,thetechniquedevelopedinthepresentstudyisfasterandlessexpensiveasrestrictionoftheamplificationproductsisnotneeded;andcomparedtothepreviouslyreportedSCAR-basedmethod,thepresenttechniqueissimplerandmoresensitiveassinglenematodescanbedirectlydiagnosed.Root-knotnematodesarecurrentlycontrolledbymeansofchemicalnematicides,cropresistanceandplantquarantine.Rapidandreliablespeciesidentificationisessentialfortheeffectiveimplementationofthelattertwocontrolmeasures.ThePCRassaysdescribedherearesimpleandreliable.TheycanbeexploitedasaconvenienttoolforroutineidentificationofM.incognita,M.javanicaandM.arenariainsoilandrootsamplestoassistcontrolofcroproot-knotdiseases.Inpracticalapplication,Meloidogynejuveniles,malesorfemalesinsoilandrootsamplesmightfirstberecoveredbytechniquessuchassucroseflotatio