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