美國長木植物園黃楊優(yōu)良品種的分子鑒定

美國長木植物園黃楊優(yōu)良品種的分子鑒定

0u3000sin-roinciparcie非普通規(guī)劃ueling.非普通規(guī)劃ueling.speciec.非織造sib.案例見表3b.云l。這是一個由一個由砂巖組成的家族（b）。它以收取費用、數(shù)量、路線和犧牲的形式存在。年輕的側(cè)柏也有自己的名字。1753年，評價值（歐洲）、surtic（亞洲和美國）和特洛伊木馬（美國、卡羅西州、卡羅西州和東道爾）的名字。這些計劃是自然的。Taxonomically,thephylogeneticrelationshipswithinBuxusandthenumberofitsspeciesarecontradictoryandconfusing.Someresearchersindicatedthattherewereapproximately30species,whileothersincludedmorethan70species.BuxussempervirensL.(CommonBox)andB.microphyllaSieb.EtZucc.(LittleleafBoxorBoxwood)arethemostimportanttaxainornamentals.Theyarewidelycultivatedforhedges,screens,borders,andtopiaryandforspecimenplantingsofpendulousorprostrateform.However,bothspeciesareextremelyvariablewhichhasgivenrisetomanycultivarswithoverlappedmorphologicalcharacteristics.“Cultivarsarealmostindistinguishableandoncetheyareputtotheshearsnoonecanreliablyseparatethem”.Obviously,thecultivaridentification,thespeciesdelineation,andthegenuscircumscription,hadcreatedtheproblems.Thedifferencesamongthetaxonomistsandhorticulturistscouldbenotresolvedwithlimitedmorphologicalcharacteristicsanditisimportanttoclarifythiscontradictoryinformationwithmodernmoleculartechnologies.Deoxyribonucleicacid(DNA)fingerprintingtechniques(DNAmarkers)arethepreferredmethodsforidentifyingcultivarsorgenotypesandinvestigatingthegeneticvariabilitywithinspeciesbecausetheDNAmarkersarenotinfluencedbyenvironmentalorculturalfactors,suchasgeographicallocation,microclimate,andnutrition.TherapidaccurateinformationderivedfromDNAcanbeusedtodistinguishcloselyrelatedplants,especiallyamorphologicallyhomogenousgroupofplants.Currently,severalDNAfingerprintingtechniquesareavailable.Thewidelyusedtechniquesarerestrictionfragmentlengthpolymorphism(RFLP)andthepolymerasechainreaction(PCR)-basedrandomamplifiedpolymorphicDNA(RAPD).RFLPshavebeenusedtoinvestigategeneticdiversityincultivatedplantsandtheirwildrelatives.Hubbardetal.successfullyappliedRFLPtechniquestotheidentificationofrosecultivars.TheRAPDassayislessexpensiveandovercomessometechnicallimitationsofRFLPs.Itcanbeusedforcultivardiscrimination,suchasclonalidentificationofredmaple(Krahletal.,1993)andAmericanelm.Michelmoreetal.reportedthatRAPDswereusefulintheconstructionofgeneticmapsandcouldbeusedaslinkagemarkerstodownymildewresistanceinlettuce.Amplifiedfragmentlengthpolymorphism(AFLP)isaningeniouscombinationofRFLPanalysisandPCRthatresultsinhighlyinformativefingerprints.ComparedwiththeRFLPsorRAPDs,AFLPisthemostuseful,reliable,andpromisingmolecularmarkertechniqueforgenotypiccomparisonswithinspecies.Zhangreported99.9%reproducibilityamongthreereplicationsof27bermudagrassgenotypes.HealsoconcludedthattheAFLPtechniquenotonlyhadhighreproducibilityandrevealedahighfrequencyofpolymorphism,butalsocouldbeusedforcultivarandhybrididentificationandprotection.Obviously,theAFLPassayoffersmorereproducibleinformationandincreasedopportunitiestoresearchmolecularstudiesonornamentaltaxa.SincenoDNAworkhasbeendoneforBuxus.ThisstudywillconductthepreliminaryexplorationofgeneticdiversityandclonediscriminationusingBuxuscollectionfromLongwoodGardens.Furtherstudiescanleadtoscreeningofallavailableclonesandidentifyingthebestclonesforourgardens.1杏仁杏仁1.1lectedofficientFreshleavesof20BuxusaccessionswerecollectedfromthetrialbedsofLongwoodGardensinKennettSquare,Pennsylvania,USA.ThesourceinformationislistedinTable1.1.2u2004范圍TotalgenomicDNAwasisolatedfromfreshleavesfollowingtheproceduressuggestedbyDNeasyPlantMiniKit(QIAGENInc.,Valencia,California,USA).Theextratwostepswerecentrifugingat5000r·min-1foroneminuteafterincubationat65℃andat12000r·min-1forfiveminutesaftericeincubation.IsolatedDNAwasvisualizedon1%Agarosegelwith312nmVariableIntensityTransilluminator(FBTIV-816,FisherScientific,Pittsburgh,Pennsylvania,USA)andthehighyieldedsampleswereusedforthefollowingAFLPexperiment.1.3lymorphismrectingelogen-algorithmsrecte-sratchs軟件sragPerkinElmer(FosterCity,California,USA)LargePlantGenomeKitwaspurchasedtoconductthisexperiment.AmplifiedfragmentlengthpolymorphismreactionswereconductedastherecommendedbytheAFLPPlantMapping.Sampleswereelectrophoresed(2500V)for4hoursat48℃in1(TBEbufferonanautomaticDNAsequencer(modelABI377,Perkin-ElmerAppliedBiosystems)equippedwithGeneScanAnalysissoftware(version3.1,Perkin-ElmerAppliedBiosystems).FragmentsizingwerecalculatedautomaticallyusingthelocalSouthernsizingalgorithms.1.4通過paun反應reporeninvib.stunge-branch,ats國際普通規(guī)制,即價5.3.4.3.4.3.4.3.4.3.4.3.4.3.3.3.4.3.4.3.3.3.3.3.3.3.3.3.3.3.5.5.3.5.5.5.5.5.5.5.5.5.3.3.3.5.3.5.3.5.3.3.3.3.3.3.3.3.3.3.3.3.3.5.3.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.4.5.4.5.3.4.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.DatafilescontainingsizingdataforallsampleswerecreatedusingFragmentBinner[NancyGarnhart,personalcomm.]foreachprimercombination.Thethresholdvalueforfragmentdetectionwas50.Alldatafileswerereviewedbyeyes,thenexportedintoPAUP.Pairwisedistancecomparisonsweregeneratedandunweightedpairgroupmethodwitharithmeticaverage(UPGMA)treewasderivedusingPAUP.Toshowthestrengthofeachbranch,weconducted1000replicatesofbootstrapanalysisandlistedthenumberofcharactersthatchangedunambiguouslyoneachbranch.2cc服務(wù)平臺相關(guān)內(nèi)容Fromthethree-primer-paircombinationsof20BuxusaccessionscollectedfromLongwoodGardenstrialbeds,atotalof212usefulbands(markers)between75and500bpsfragmentsizesweregenerated.Primercombinationslabeledwithblue,green,andyellowdyecolorsproduced90,69,and53bands,respectively.Theaveragenumberofmarkersforeachaccessionwas74andthetotalmarkersforeachaccessionvariedfrom50to108(Table3).Theuniquebands,whichcanbethepotentialdiagnosismarkers,wereamongzeroto15.Obviously,thegeneticdiversityamongthese20accessionswereverylittle,excepttheaccession#1(8uniquebands),#2(14),#3(7),#13(15),and#20(8)[Table1,3].Itisimportanttoindicatethattheaccession#8,#10,#11,#15,#16,#17,and#19didnothaveanyuniqueband[Table1,3],whichimpliedthatthegeneticinformationoftheseaccessionswasverysimilartooneormoretaxa(includedtheabove7accessions)inthisstudy.Itisnotnecessarytokeepsomeofthesetaxaforfurtherevaluation.BasedonallusefulAFLPmarkers,geneticdistances(pairwisedistancecomparisons)andtotalcharacter(band)differencesbetweenBuxusaccessionsweregeneratedbyPAUP(Table4).Geneticdistancesrangedfrom0.028to0.439.Accession#16,#17,#18showedminimalgeneticdifferences,whileaccession#2and#20hadthehighestone.Generally,highgeneticdistancesamongthedifferentspeciesandlowgeneticdistancesamongthecloneswereexpected.Thevaluesofgeneticdistancesamongthevarietiesandcultivarswereintermediatetovaluesamongthespeciesandclones.Thetotalcharacter(band)differencesshowedthesametrendasthegeneticdistance(Table4).OnlyoneUPGMA(unweightedpairgroupmethodwitharithmeticaverage)treewasgeneratedusingPAUP(Fig.1).Basedon1000replicatesofbootstrapanalysis,thenumberofcharactersthatchangedunambiguouslyoneachbranch,andthegeneticdistances(Table4),the20accessionsmightderivefromfivedifferentspeciesorgrex.Accession#1,#2and#13shouldbeoriginatedfromthreedifferentspecies(maybegrex),respectively.Morphologically,thesethreeaccessionshaveroundedorlanceolateleaves,whichcanbedistinguishedfromtherestofplants.Accession#4and#20derivedfromthefourthdistinguishedspecies,Buxusmicrophylla.Therestofthemwerefromthefifthspecies,Buxussempervirens.Morphologicaldatasupportthesetwospecies.Thetaxasubordinatetospecies,especiallycultivarsandclones,areveryimportanttothehorticulturalindustries,plantbreeding,andnaturalconservation.Normally,agroupofindividualswithoneormoreuniquetraitscanbepublishedasanewcultivar(orclone)ifthetrait(s)canbereproducedasexuallyorsexually.FromFig.1,CladAhadthelowsupportofbootstrapvalues,excepttheaccessions#10and#11.Theaccession#10and#11wereverysimilarwithonly0.038geneticvariations.Otherthreeaccessionsinthiscladhadrelativelyhighergeneticdistances(0.189,0.193,and0.203).TheAFLPdatasupportedthattherewerefourdistinguishedcultivarsinthisclad,theaccession#6wasclosertoaccessions#10and#11thanothertwoaccessions.Morphologically,theirhabitswerevariable,butleavesweresimilar,especiallyAccession#10andAccession#11,whichbothshouldbecalledB.sempervirens‘Myrtifolia’.CladBincludedCladCandAccession#5withhighbootstrapvalueat88%.ThegeneticdistancesbetweenAccession#5andeachaccessionofCladCwererelativelyhightorecognizetheAccession#5asadistinguishedcultivar.WithinCladC,Accessions#9and#15hadverylowgeneticdistance(at0.08).Otheraccessionsinthiscladwererelativelyclosetothesetwo.TheAFLPdatasupportedthesesixaccessionsinCladCshouldbederivedfromacultivar.Morphologically,leafshapesamongtheseaccessionssignificantlyoverlappedandcouldnotbedistinguishedifmixingthem.BothmolecularandmorphologicaldatasupportthatthesesixaccessionsoriginatedfromacultivarandcouldbecalledBuxussempervirens‘Suffruticosa’.CladDincludedAccessions#16,#17,and#18.Thesethreeaccessionsformedaverystrongcladwith100%bootstrapsupport.Thegeneticdistancesandthenumberofcharactersthatchangedunambiguouslyoneachbranchwereverylow.Obviously,thesethreeaccessionswerefromacultivarandtheaccessions#16and#17werealmostidenticalfromAFLPdata.Allthreeaccessionshadelliptictorounded,darkgreenfoliageandweshouldcallthemBuxussempervirens‘VardarValley’.CladEonlyhadAccessions#4and#20.Thesetwoaccessionsweregroupedwithhighgeneticdistance(0.189).However,the96%bootstrapvalueindicatedthattheyweremuchclosethananyotheraccession.TheAFLPdataconcludedthatthesetwoaccessionsweretwodistinguishedcultivarseveniftheymightderivefromthesimilargenepoollongtimeago.CladFconsistedofAccessions#2and#13with84%bootstrapsupport.Genetically,theywerenotclosetoeachotherwith0.274geneticdistance.Accession#2had24unambiguouscharacterchangeswhileAccession#13had22.Thesedataindicatedtheyweretwodifferentcultivars,whichcouldbederivedfromdifferentspeciesorgrexorhybridizations.However,theywererelativelyclosetoeachothercomparedwithallotheraccessions.Furtherstudiesshouldbeconductedtoclarifytheirrelati