香菇分子標(biāo)記

香菇分子標(biāo)記第一頁(yè)，共二十三頁(yè)，編輯于2023年，星期四Abstract：Randomampli?edpolymorphicDNA(RAPD),inter-simplesequencerepeat(ISSR)andsequence-relatedampli?edpolymorphism(SRAP)markerswereusedtoevaluatethegeneticdiversityamong23eliteLentinulaedodesstrainsinChina.Atotalof138,77and144bandsweredetectedby16RAPDprimers,5ISSRprimersand23SRAPprimercombinations,amongwhich58.8%,73.5%and56.3%waspolymorphic,respectively.第二頁(yè)，共二十三頁(yè)，編輯于2023年，星期四ThepurposeofthisworkwasusingRAPD,ISSRandSRAPmarkerstoinvestigategeneticdiversityamong23elitestrains,andtolaythefoundationsfordirectingnewcultivarsbreedinginL.edodes.ByUPGMAclustering,adendrogramwasconstructedbasedoneachanalysis.Thethreedendrogramsshowedthat23L.edodesstrainswereclusteredintothreeorfourgroups.Thegroupingexhibitedsimilarstructureandwasgenerallyconsistentwiththeirpedigrees.第三頁(yè)，共二十三頁(yè)，編輯于2023年，星期四Twenty-threeL.edodesstrainssharedgreatsimilarityindicatedthatthelowlevelofgeneticdiversityofL.edodesstrainsandtheirrelationshipbetweeneachother.Theimportantsourceofbreedingmaterial,suchaswildandexotictypes,mustbeintroducedinordertobroadengeneticbaseanddecreasesgeneticvulnerabilityofL.edodes.第四頁(yè)，共二十三頁(yè)，編輯于2023年，星期四Keywords：LentinulaedodesRandomampli?edpolymorphicDNA(RAPD)Intersimplesequencerepeats(ISSR)Sequence-relatedampli?edpolymorphism(SRAP)EdiblemushroomStrainGeneticdiversityMolecularmarker.Introduction:Lentinulaedodes.Pegler(XiangguorShiitakemushroom)isthesecondhighestcultivatedediblemush-roomintotalproductionyieldallovertheworld,andwidelycultivatedinEasternAsia,suchasChina,JapanandKoreabecauseofits?avor,taste,qualityandmedicinalpurposes.第五頁(yè)，共二十三頁(yè)，編輯于2023年，星期四AlthoughChinaisaleadingproducerandexporterofL.edodes,themajorlatentconstraintinpromotingcommercialproductionofthiscropisexistingcultivatedstrainsusuallyderivedfromalimitednumberofelitelines,whichareoftenusedintheproductionofmanycultivars,resultinginanincreasinglynarrowgeneticbase.Thedevelopmentofnewcultivarsofediblemushroomswithsuperiorpropertiessuchashighfruitingbodyproductivity,diseaseresistance,andhighcontentofeffectiveconstituentthatbene?tshumanhealthtomeetchangingagronomicandeconomicrequirementsinthismodernrapiddevelopmentalsocietywillbeverynecessaryandimportant.第六頁(yè)，共二十三頁(yè)，編輯于2023年，星期四

ThepurposeofthisworkwasusingRAPD,ISSRandSRAPmarkerstoinvestigategeneticdiversityamong23elitestrains,andtolaythefoundationsfordirectingnewcultivarsbreedinginL.edodes.Materialsandmethods:LentinulaedodesstrainsandcultureconditionsTwenty-threeL.edodesstrains,whichplantedinmanyregionsinChinacoveringawiderangeofecologicalconditionsfromnorthtosouthandfrommountainsto?atirrigatedlands,wereusedinthisstudy.第七頁(yè)，共二十三頁(yè)，編輯于2023年，星期四DNAextraction：ExtractionoftotalgenomicDNAwasdonebythesodiumdodecylsulfate-cetyltrimethylammoniumbromide(SDA-CTAB)methodasdescribedbyZeng(2003).TheconcentrationandpurityoftheDNAweredeterminedusingGeneQuantProDNA/RNA(GEHealthcare),andextractedDNAwasstoredat-20℃afterdilutiontotherequiredconcentration.第八頁(yè)，共二十三頁(yè)，編輯于2023年，星期四RAPD,ISSR,SRAPanalysisTheRAPD,ISSRandSRAPproductswereallanalysedbyelectrophoresison1.5%(w/w)agarosegelswhichcontaining0.5lgethidiumbromide/mlin19TAEbufferandthenvisualizedandphotographedunderultravioletlightusingtheJS-380Bautomaticgelimaginganalyzer.TheGeneRulerTM100bpLadderPluswasusedasamolecularsizestandard.AndthethreePCRreactionswereallrepeatedatleasttwicetocon?rmthereproducibilityofeachPCRband.第九頁(yè)，共二十三頁(yè)，編輯于2023年，星期四DataanalysisAmpli?edfragmentsofRAPD,ISSRandSRAPwerescoredaspresent(1),andabsence(0).Dice’ssimilaritycoef?cientsbetweenstrainswerecalculatedbytheNTSYS-pc2.10e.ClusteranalysiswasperformedusingtheUPGMAalgorithm,andadendrogramwasproducedbasedoneachsimplematchingmatrix.Becausefragmentsthatsmallerthan100bpinlengthgavelowreproducibilityandcouldnotbeaccuratelyperformed,DNAfragmentswhichbiggerthan100baseswereusedfordataanalysis.第十頁(yè)，共二十三頁(yè)，編輯于2023年，星期四第十一頁(yè)，共二十三頁(yè)，編輯于2023年，星期四Fig.1TheextentofRAPD(a),ISSR(b)andSRAP(c)polymorphismobservedamong23L.edodesstrains,revealedbyprimerorprimecombinationS1028,ISSR2andMe1-Em16,respectively.Lanes1–23,correspondedtotheL.edodesstrainslistedinTable1;laneM,Molecularruler(GeneRulerTM100bpLadderPlus第十二頁(yè)，共二十三頁(yè)，編輯于2023年，星期四ThedendrogrambasedonRAPDdatawasconstructedbyUPGMAanalysisgroupedthe23strainsintothreemainclusters,withsimilaritycoef?cientrangingfrom0.64to1.00(Fig.2a).ClusterIisthebiggestcluster,comprised17strains,i.e.,Shenxiang-2,Shenxiang-8,Shenxiang-7,Wuxiang-1,Suxiang,Cr04,Shenxiang-12,Shenxiang-10,L26,Shenxiang-4,L66,Cr02,Shenxiang-6,Shenxiang-9,L03,Hunong-1andMinfeng-1.Among17strains,Shen-xiang-2andShenxiang-8,Shenxiang-7andWuxiang,Shenxiang-10andShenxiang-12,Shenxiang-6andShen-xiang-9appearedtobeclosertoeachother.ClusterIIconsistedof241,241-4,Qingke-20,Zhexiang-939-9andZhexiang-939-6.XiangjiuformedaseparateOTUsinclustershowinglesssimilaritycoef?cient(0.64)withotherstrainsstudiedandappearedtobedistinctfromallothers.第十三頁(yè)，共二十三頁(yè)，編輯于2023年，星期四ISSRanalysisThe?veprimerswerescreenedfortheirabilitytogenerateISSRpolymorphicDNAbandsusingeachstrain’sgenomicDNA.Inall,77unambiguousandreproducibleampli?cationproductswerescored.The?veprimersampli?edfragmentsacrossthe23strainsstudied,withthenumberofampli?edfragmentsrangingfromtwelve(ISSR12)toeighteen(ISSR1)andwhichvariedinsizefrom150to2,000bp.Ofthe77ampli?edbands,56werepolymorphic,withanaverageof11.2polymorphicfragmentsperprimer.Percentagepolymorphismrangedfrom55.6%(ISSR1)toamaximumof93.3%(ISSR2),withanaverageof73.5%polymorphism.第十四頁(yè)，共二十三頁(yè)，編輯于2023年，星期四3outof5primersshowedmorethan60%polymorphism.Figure1bistherepresentativeoftheextentofpolymorphismobservedamongtheL.edodesstrainsasrevealedbyISSR2.Figure2bshowsthedendrogrambasedontheISSRdata.23L.edodesstrainsfellclearlyinto4mainclusters,withsimilaritycoef?cientrangingfrom0.62to0.99.ThestructureofthetreeisverysimilartotheonefromRAPDanalysis.ThemembersofthelargestgroupareexactlythesameasinRAPD,exceptShenxiang-6andShenxiang-9,whichformedaseparatecluster.第十五頁(yè)，共二十三頁(yè)，編輯于2023年，星期四SRAPanalysisAtotalof153differentcombinationsofprimerswereemployedusing9forwardand17reverseprimers.Abouttwo-thirdsofthemcouldproduceclearbands,butconsideringthepolymorphismandreproducibility,23combinationswerechoseninthefollowingstudy.Among23L.edodesstrains,atotalof144scorablebandswereproduced.Allthechosenprimercombinationsampli?edfragmentsacrossthe23strainsstudied,withthenumberofampli?edfragmentsrangingfromthree(Me7-Em2,Me9-Em14)toten(Me7-Em14)andwhichvariedinsizefrom150to2,000bp.Ofthe144ampli?edbands,81werepolymorphic,withanaverageof3.5polymorphicfragmentsperprimercombination(Table3).第十六頁(yè)，共二十三頁(yè)，編輯于2023年，星期四第十七頁(yè)，共二十三頁(yè)，編輯于2023年，星期四第十八頁(yè)，共二十三頁(yè)，編輯于2023年，星期四Percentagepolymorphismrangedfrom14.3%(Me5-Em16)toamaximumof100.0%(Me1-Em5),withanaverageof56.3%polymorphism.Onlyelevenoutof23primersshowedmorethan60%polymorphism(Table3).Figure1cistherepresentativeoftheextentofpolymorphismobservedamongtheL.edodesstrainsasrevealedbyprimecombinationMe1-Em16.Figure2cshowsthedendrogrambasedontheSRAPdata.23L.edodesstrainsfellintothreemainclusters,withsimilaritycoef?cientrangingfrom0.70to1.00.ThestructureofthetreeisverysimilartotheonefromRAPDanalysis.第十九頁(yè)，共二十三頁(yè)，編輯于2023年，星期四DiscussionAnalysisofgeneticdiversityisveryimportantinfungiinpracticalbreedingprograms,andmolecularmarkersofferanapproachtounveilthegeneticdiversityamongstrainsbasedonnucleicacidpolymorphisms.Inthisstudy,threemakers,RAPD,ISSRandSRAP,weresimultaneouslyusedtoinvestigategeneticdiversityof23eliteL.edodesstrainsinChina.TheseresultsshowedthethreemarkerswereallsuitableforgeneticdiversityresearchonL.edodes.第二十頁(yè)，共二十三頁(yè)，編輯于2023年，星期四Theinformationonthegeneticrelatednessamongstthestrainswillalsobeusefulforplanningeffectivebreedingprogrammeswhichinclude,forexample,theassessmentofgeneticdiversity,thedetectionofduplicatesampleingermplasmcollectionandtheselectionofparentstrainstoavoidinbreedingdepression.Thereweresomedifferencesinthepositioningofsomestrainsamongthethreedendrograms.Itmayre?ectthefarrelationshipbetweenthetwostrainsandtheotherstrains,butitalsosuggeststhesensitivityofISSR.第二十一頁(yè)，共二十三頁(yè)，編輯于2023年，星期四ItisreasonablethattheprincipleandvirtuesofRAPD,ISSRandSRAParedifferent,andtheyareaimtoamplifyadifferentregionofgenome.Morethanonekindofmolecularmarker