rDNA ITS Sequence Analysis and Molecular Phylogeny of Four Fungal Strains Assigned to the Family Rus.docx

rDNA ITS Sequence Analysis and Molecular Phylogeny of Four Fungal Strains Assigned to the Family Rus.docx

《rDNA ITS Sequence Analysis and Molecular Phylogeny of Four Fungal Strains Assigned to the Family Rus.docx》由会员分享，可在线阅读，更多相关《rDNA ITS Sequence Analysis and Molecular Phylogeny of Four Fungal Strains Assigned to the Family Rus.docx（8页珍藏版）》请在冰豆网上搜索。

rDNAITSSequenceAnalysisandMolecularPhylogenyofFourFungalStrainsAssignedtotheFamilyRus

rDNAITSSequenceAnalysisandMolecularPhylogenyofFourFungalStrainsAssignedtotheFamilyRus

　　1BiotechnologyInstitute,ZhejiangAcademyofForestryScience,Hangzhou,Zhejiang?

A310023，China；

　　2LishuiEdibleFungiResearchandDevelopmentCenter，ZhejiangEssenceFungiDevelopment

　　CompanyLtd，Lishui，Zhejiang?

A323000，China

　　Internaltranscribedspacer（ITS）sequencesoffouredibleandmedicinalfungibelongingtotheRussulaceae,collectedfromtheLishuimountainousareaofZhejiangProvince,wereclonedandsequenced.ComparisonswithsequencedatainGenBankwereundertakenusingBLAST,andthefourITSsequences,togetherwith15referencesequencesobtainedfromGenBank,wereusedtoconstructaphylogenetictree.rDNAITSsequencesofthefourstrainsrangedfrom673to766bpinlengthandbetween47.4%and49.48%inGCcontent.PhylogeneticdataindicatedstrainR57tobeRussulacyanoxantha.DifferencesinthesizeandGCcontentofITSsequencesfromstrainsR32andR57（R.cyanoxantha）andstrainL37（Lactariussanguifluus）weresignificant,whereasthecorrespondingdifferencesbetweenstrainsR32andR57wererelativelysmall.Aclosekinship,withonlysmalldetectabledifferencesintheirrespectiveITSsequences,existedbetweenR.mustelina,R.virescensandR.parazurea.AsimilarsituationappliedtostrainsofL.sanguifluusandL.salmonicolor.

　　Russulaceae；?

ArDNA；?

AInternalTranscribedSpacer；?

ASequenceanalysis；?

APhylogeny；?

AMacrofungi

　　ThefamilyRussulaceaeisassignedtotheBasidiomycotina,HymenomycetesandAgaricales,andincludestwogenera,RussulaandLactarius.AccordingtoWANGandSHI[1]andJIetal[2],300speciesofRussulaand400speciesofLactariushavebeenidentifiedworldwide.AbundantmacrofungalresourcesexistintheLishuimountainousarealocatedinsouthwestZhejiangProvince.AccordingtoYANGetal[3],911differentkindsofwildfungicanbefoundintheQingyuanCountyregionofLishui,43ofwhichbelongtothefamilyRussulaceae.MostfungiassignedtotheRussulaceaeareedibleandpossessmedicinalfunctions[4-8].Inordertoproducefruitingbodies,itisessentialforthesefungitoformasymbioticrelationshipwithhostplantrootsand,sincetheyhavenotyetbeencultivatedartificially,thesupplyoffruitbodiesislimited.

　　AlthoughtheRussulaceaehasbeenwidelystudiedwithrespecttoavailableresources,environmentalecology,tissueisolation,nutritionalandfunctionalcomponents,artificialcultivationandsubstratefermentation[9-12],relativelylittleresearchhasbeenundertakenonthemolecularphylogenyandtheextentofgeneticpolymorphismwithinthefamily.Inthisreport,ITSsequencesoffouredible/medicinalfungibelongingtotheRussulaceaeandrecurrentlyfoundinLishuimountainousarea,wereclonedandsequenced.BLASTsearcheswereconductedtoidentifyhomologoussequencesintheGenBankdatabaseinordertoprovideabasisforfutureresearchonphylogeneticrelationshipsandgeneticpolymorphism.

　　1MaterialsandMethods

　　1.1Fungalstrains

　　Strainno.,andthesitewherefruitbodysampleswerecollected,areshowninTable1intheChineseversion.StrainsR29,R32andL37havebeenidentifiedtospecieslevelanddesignatedR.mustelinaFr.,R.cyano-xanthaSchaeff.:

Fr.andL.sanguifluus（Paul.）Fr.,respectively.StrainR57hasstilltobeidentified.Aftercollection,fruitbodieswerecleanedofresidualsediment,transportedtothelaboratoryinliquidnitrogen,andthereafterpreservedat-20℃.

　　FULizhong,?

ALIHaibo,?

AWEIHailong,?

Aetal

　　1.2Methods

　　1.2.1ExtractionofgenomicDNA

　　GenomicDNAwasextractedfrompilei（includinggills）bytheSDS-CTABmethod[13]andpurifiedusingDNApurificationkits（HangzhouBioerTechnologyCo.Ltd.）.Puritywasevaluatedby1.5%（w/v）agarosegelelectrophoresis,andDNAconcentrationsweredeterminedwithaDNA/RNAUVspectrophotometer（GEHealthcare,Amer-sham,England）.

　　1.2.2rDNAITSanalysisITS-PCRamplificationwasperformedinaprogrammableTC-XPthermocycler（HangzhouBioerTechnologyCo.Ltd.）in25μLreactionmixturescontaining:

2.5μL10×PCRBuffer,2μLdNTPs（10mmol/L）,2μLMgCl?

?

2（25mmol/L）,0.3μLTaqDNApolyme-rase（5U/μL）,1μLITS5/ITS4primers[14]（10μmol/L）,3μLtemplateDNA（17ng/μL）and14.2μLddH2Otovolume.Amplificationconditionswereasfollows:

1cycleat94℃for1min；30cyclesat92℃for15s，61℃for15sand72℃for1min；thenafinalextensionat72℃for5min.

　　1.2.3Recovery,cloningandsequencingofITSfragments

　　ITSfragmentsin1.5%agarosegelswererecoveredusingaDNAGelRecoveryKit（HangzhouBioerTechnologyCo.Ltd）,andnucleicacidconcentrationsweredeterminedwithaDNA/RNAultravioletspectro-photometer.TherecoveredfragmentswereligatedintopMD-18TVectorandtransformedintocompetentE.coli（DH5α）cells.Afterblue-whitespotscreeningandidentificationusingPCRamplification,threerandompositiveclonesineachsampleweresequenced（TaKaRaBiotechnologyCo.Ltd.,Dalian）.

　　1.3ComparisonofITSsequencesandanalysisofphylogeny

　　Afterremovalofthecarriersequencesfromthe5’and3’ends,ITSsequencesweretransformedintoFASTAformatandmultiplealignmentsbetweenfourITSsequencesandreferencesequencesobtainedfromaBLASTsearchoftheGenBankdatabasewereconfirmedusingClustalX（Version1.81）[15]software.

　　AphylogenetictreewasconstructedwithMEGA（Version2.1）software[16].GeneticdistanceswerecalculatedusingtheKimura2-parametermodel.Evolutionarydistancesweredeterminedusingtheneighbor-joiningmethod,andthestatisticalsignificanceofeachbranchofthephylogenetictreewasconfirmedusingbootstrapvaluesfrom1000replicates.

　　2Results

　　2.1AnalysisofrDNAITSsequences

　　TheamplifiedrDNAITSregionsofstrainsR29,R32,L37,andR57consistedof701bp,673bp,766bpand673bprespectively,andthecorrespondingGCcontentswere49.22%,47.85%,49.48%and47.4%（seeFig.1intheChineseversion）.Thelargestdifferenceinsequencelength（93bp）wasbetweenstrainL37andstrainsR32andR57,whilethelargestdifferenceinGCcontent（2.08%）wasbetweenstrainsL37andR57.OurdataindicatedthatsignificantdifferencesexistedamongdifferentgeneraincludedinthefamilyRussulaceae.ItisnoteworthythatthesizeoftheITSsequenceinstrainsR32andR57wasidentical（673bp）,andthattheGCcontentdifferedbyonly0.45%.

　　2.2HomologousBLASTretrievalofITSsequences

　　FifteenreferencestrainswithITSsequencessimilartothoseoftheteststrainswereidentifiedbyaBLASTsearchoftheGenBankdatabase（seeTable2intheChineseversion）.Fourreferencestrains,twoidentifiedasR.virescensandtheothertwoasR.parazureaandR.mustelina,exhibitedrelativelyhighsimilaritywithstrainR29.Sixreferencestrains,fouridentifiedasR.cyanoxantha,oneasaRussulasp.andthesixthanunculturedfungus,showedrelativelyhighsimilaritytostrainsR32andR57.Fivereferencestrains,fouridentifiedasL.salmonicolorandoneasL.sanguifluus,exhibitedrelativelyhighsimilaritytostrainL37.

　　2.3Phylogeneticanalysis

　　ThefourITSsequences,togetherwith15referencesequencesobtainedfromtheGenBankdatabasewereusedtoconstructaphylogenetictree（seeFig.2intheChineseversion）.ThirteenstrainsofRussulaandsixstrainsofLactariusrespectivelywereclusteredinthetwogenera.StrainL37waslocatedinthesamebranchasAF249289（L.sanguifluus）,andthesetwostrainswerethenclusteredinagroupwithL.salmonicolorstrainsAF140258,AF140259,AF140265andAF249287atthebootstrapvalueof100%.ThissuggestsarelativelysmallsequencedifferenceandacloserkinshipbetweenL.sanguifluusandL.salmonicolor.

　　StrainsR32andR57werelocatedinthesamebranchatabootstrapvalueof100%,andthesetwostrainsformedanotherclusterwithR.cyanoxanthastrainsAF418608,AM087258,AY606960andAY061669,andanunculturedfungusDQ054553,atabootstrapvalueof82%.Therefore,basedonthesedataandthoseshowninFig.1,R57andR32shouldberegardedasgeographicallydifferentstrainsofR.cyanoxantha.StrainR29waslocatedinthesamebranchasstrainAY061693（R.mustelina）atabootstrapvalueof97%,andthesetwostrainswereclusteredinagrouptogetherwithR.virescensstrainsAM087264andAY061727,andstrainAY061704（R.parazurea）atabootstrapvalueof93%.ThisindicatedsmallsequencedifferencesandclosekinshipbetweenR.mustelina,R.virescensandR.parazurea.

　　3Analysis

　　MostfungibelongingtoRussulaandLactariusareectomycorrhizae,andhavenotyetbeengrowninartificialculture.However,puremycelialculturesofthesemacrofungicanbeidentifiedusingITSsequenceanalysis.ITSsequenceanalysishasbeenusedtoidentifymyceliafromR.vinos[17],L.deliciosus[18],Amanitaexitialis[19]andSuillusbovinus[20].OurdataindicatethatITSsequencescanbeusednotonlyforthemolecularidentificationofRussulaandLactarius,fungithatarenoteasytoaccuratelyidentifytospecieslevelusingmorphologicalcharacteristics,butalsoforpreliminaryestimatesofphylogenyandkinshipbetweendifferentspeciesofRussulaceae.

　　StrainR57wasidentifiedinthisstudyasR.cyanoxanthabasedonITSsequencesimilaritiesandthecloseclusteringoftheirrespectiveITSsequencesinthephylogenetictree.TherearenostandarddefinitionsofspeciesbasedonITSsequences[21,22],althoughITSsequencesaregoodmolecularindicatorsforcomparativeinterspeciesandintraspeciesresearch.WeconsiderITSsequenceanalysiscombinedwithfruitbodymorphologicalcharacterizationtobemoresci