真菌檢測鑒定通用引物-Fungal-Primers

ITS1:5’-CCGTAGGTGAACCTGCGG-3’

ITS4:5’-TCCTCCGCTTATTGATATGC-3’Tm55℃

NS17:CATGTCTAAGTTTAAGCAA

NS3:GCAAGTCTGGTGCCAGCAGCC

NS4:CTTCCGTCAATTCCTTTAAG

NS22:AATTAAGCAGACAAATCACT

NS24:AAACCTTgTTACgACTTTTA

LR0R:5’-GTACCCGCTGAACTTAAGC-3’

LR3:5’-CCGTGTTTCAAGACGGG

LR3R:5’-GTCTTGAAACACGGACC(complementarytoRLR3R:GGTCCGTGTTTCAAGAC)

LR5:5’-TTAAAAAGCTCGTAGTTGAAC-3’

LR7:5’-TACTACCACCAAGATCT

LR12:5’-GACTTAGAGGCGTTCAG

Lr0R/LR5:Tm50-52℃

NL1:5’-GCATATCAATAAGCGGAGGAAAAG

NL1:5′-TGCGTTGATTACGTCCCTGC(alsocalledV9:TGCGTTGATTACGTCCCTGC)

NL1:5’-TGCTGGAGCCATGGATC-3

NL2:5’-CTCTCTTTTCAAAGTTCTTTTCATCT

NL2:5’-AACGGCTTCGACAACAGC-3

NL2:5’-CTTGTTCGCTATCGGTCTC(alsoNL2A:5′-CTTGTTCGCTATCGGTCTC)

NL2:5’-TACTTGTTCGCTATCGGTCT-3'

NL3:5’-GAGACCGATAGCGAACAAG(alsoNL3A:5’-GAGACCGATAGCGAACAAG)

NL3:5’-AGACCGATAGCGAACAAGTA

NL3:5’-GCTGTTGTCGAAGCCGTT-3

NL4:5’-GGTCCGTGTTTCAAGACGG（similartoRLR3R：5′-GGTCCGTGTTTCAAGAC)

NL4:5’-TAGATACATGGCGCAGTC-3

ConservedprimersequencesforPCRamplificationandsequencingfromnuclearribosomalRNA(

/fungi/mycolab/primers.htm

)

Vilgalyslab,DukeUniversity

Overtheyears,ourlabhascompiledausefullistofconservedprimersequencesusefulforamplificationandsequencingofnuclearrDNAfrommostmajorgroupsoffungi(primarilyEumycota),aswellasothereukaryotes.AlloftheseprimerswereidentifiedandtestedbyourownlabbasedonconsensusbetweenthepublishedlargeandsmallsubunitRNAsequencesfromfungi,plantsandothereukaryotes;sourcesofotherusefulprimersequencesfrompublishedliteraturearealsoindicated.Together,theseprimersspanmostofthenuclearrDNAcodingregion(seefigures),permittingamplificationofanydesiredregion.Standardsymbolsareusedforthefourprimarynucleotides;variablepositionsareindicatedasfollows:P=A,G/Q=C,T/R=A,T/V=A,C/W=G,T.Primersendingwith"R"representthecodingstrand(sameasRNA).Allotherprimersarecomplementarytothecodingstrand.Thisinformationisprovidedfreelyandmaybepassedontoanyonewhowantstouseit.

Thenuclear-encodedribosomalRNAgenes(rDNA)offungiexistasamultiple-copygenefamilycomprisedofhighlysimilarDNAsequences(typicallyfrom8-12kbeach)arrangedinahead-to-toemanner.Eachrepeatunithascodingregionsforonemajortranscript(containingtheprimaryrRNAsforasingleribosome),punctuatedbyoneormoreintergenicspacer(IGS)regions.Insomegroups(mostlybasidiomcyetesandsomeascomycetousyeasts),eachrepeatalsohasaseparatelytranscribedcodingregionfor5SRNAwhosepositionanddirectionoftranscriptionmayvayamonggroups.SeveralrestrictionsitesforEcoRIandBglIIareconservedintherDNAoffungi.Nearlyallbasidiomyceteswe'vestudiedshareanEcoRIsitewithinthe5.8SRNAgenealongwithaBglIIsitehalfwayintotheLSURNAsequence.Primers5.8SRandLR7includetheserestrictionsites,whichmakesthemconvenientforcloning.

Forthosewhoaren'tfamiliarwithrDNAandfungalsystematics,severalexcellentreviewsareavailableonfungi(Hibbett,1992)andgenerallyforeukaryotes(HillisandDixon,1991).SeeGerbi(1986)forageneralintroductiontothemolecularbiologyandevolutionofrDNAinothereukaryotes.AnotherusefulsourceofprimerinformationmaybefoundinGargas&Depriest(1996)andattheTomBrunslabwebsite

/boletus/boletus.html

.

Internaltranscribedspacer(ITS)regionprimers

TheITSregionisnowperhapsthemostwidelysequencedDNAregioninfungi.Ithastypicallybeenmostusefulformolecularsystematicsatthespecieslevel,andevenwithinspecies(e.g.,toidentifygeographicraces).BecauseofitshigherdegreeofvariationthanothergenicregionsofrDNA(SSUandLSU),variationamongindividualrDNArepeatscansometimesbeobservedwithinboththeITSandIGSregions.InadditiontothestandardITS1+ITS4primersusedbymostlabs,everaltaxon-specificprimershavebeendescribedthatallowselectiveamplificationoffungalsequences(e.g.,seeGardes&Bruns1993paperdescribingamplificationofbasidiomyceteITSsequencesfrommycorrhizasamples).

primername

sequence(5'->3')

comments

reference

ITS1

TCCGTAGGTGAACCTGCGG

Whiteetal,1990

ITS2

GCTGCGTTCTTCATCGATGC

(issimilarto5.8Sbelow)

Whiteetal,1990

ITS3

GCATCGATGAAGAACGCAGC

(issimilarto5.8SRbelow)

Whiteetal,1990

ITS4

TCCTCCGCTTATTGATATGC

Whiteetal,1990

ITS5

GGAAGTAAAAGTCGTAACAAGG

(issimilartoSR6R)

Whiteetal,1990

ITS1-F

CTTGGTCATTTAGAGGAAGTAA

Gardes&Bruns,1993

ITS4-B

CAGGAGACTTGTACACGGTCCAG

Gardes&Bruns,1993

5.8S

CGCTGCGTTCTTCATCG

Vilgalyslab

5.8SR

TCGATGAAGAACGCAGCG

Vilgalyslab

SR6R

AAGWAAAAGTCGTAACAAGG

Vilgalyslab

Intergenicspacer(IGS)primers(including5SRNAprimersequencesforbasidiomycetefungi)

ThegreatestamountsequencevariationinrDNAexistswithintheIGSregion(sometimesalsoknownasthenon-transcribedspacerorNTSregion).ThesizeoftheIGSregionmayvaryfrom2kbupwards.Itisnotunusualtofindhypervariabilityforthisregion(necessitatingcloningofindividualrepeathaplotypes).Severalpatternsoforganizationcanbefoundindifferentgroupsoffungi:

MostfilamentousascomyceteshaveasingleuninterruptedIGSregion(betweentheendoftheLSUandstartofthenextSSUsequence),whichmayvaryinlengthfrom2-5kbormore.AmplificationoftheentireIGSregionrequiresusingprimersanchoredinthe3'endoftheLSUgene(e.g.,LR12R)and5'endoftheSSURNAgene(e.g.,invSR1R).

Inmanyascomycetousyeastsandnearlyallbasidiomycetes,theIGSalsocontainsasinglecodingregionforthe5SRNAgene,whichdividestheIGSintotwosmallerregionsthatmaybemoreeasilyamplifiedusing.Dependingontheorientationandpositionofthe5SRNAgene,thePCRmaybeusedtosequentiallyamplifyeitheraportionoftheintergenicspacerregion(IGS)beyondthelargesubunitRNAcodingregion.

primer

sequence(5'->3')

comments

reference

LR12R

GAACGCCTCTAAGTCAGAATCC

locatedwithintheLSURNA(seeabove)

Vilgalyslab

invSR1R

ACTGGCAGAATCAACCAGGTA

locatedwithintheSSURNA(positions21-1)

Vilgalyslab

5SRNA

ATCAGACGGGATGCGGT

(complementaryto5SRNApositions46-26)

Vilgalyslab

5SRNAR

ACQGCATCCCGTCTGAT

(5SRNApositions26-46)

Vilgalyslab

REFERENCES

Bruns,T.D.,R.Vilgalys,S.M.Barns,D.Gonzalez,D.S.Hibbett,D.J.Lane,L.Simon,S.Stickel,T.M.Szaro,W.G.Weisburg,andM.L.Sogin.1992.Evolutionaryrelationshipswithinthefungi:analysesofnuclearsmallsubunitrRNAsequences.Molec.Phylog.Evol.1:231-241.

Bruns,T.D.,T.J.White,andJ.W.Taylor.1991.Fungalmolecularsystematics.Ann.Rev.Ecol.Syst.22:525-564.

DePriest,P.T.,andM.D.Been.1992.NumerousgroupIintronswithvariabledistributionsintheribosomalDNAofalichenfungus.J.Mol.Biol.228:315-321.

Elwood,H.J.,G.J.Olsen,andM.L.Sogin.1985.ThesmallsubunitribosomalRNAgenesequencesfromthehypotrichousciliatesOxytrichanovaandStylonychiapustula.Mol.Biol.Evol.2:399-410.

Gardes,M.,andT.D.Bruns.1993.ITSprimerswithenhancedspecificityforbasidiomycetes-applicationtotheidentificationofmycorrhizaeandrusts.Mol.Ecol.2:113-118.

Gargas,A.,andP.T.DePriest.1996.AnomenclatureforfungalPCRprimerswithexamplesfromintron-containingSSUrDNA.

Mycologia88:745-748

Gargas,A.,andJ.W.Taylor.1992.Polymerasechainreaction(PCR)primersforamplifyingandsequencing18SrDNAfrom

lichenizedfungi.Mycologia84:589-592.

Gerbi,S.A.1986.Chapter7-EvolutionofribosomalDNA.Pp.419-517In:Molecularevolution,ed.McIntyre,R.

Hibbett,D.S.1991.PhylogeneticrelationshipsoftheBasidiomycetegenusLentinus:evidencefromribosomalRNAandmorphology.Ph.D.Thesis,DukeUniversity,1991.

Hibbett,D.S.1992.RibosomalRNAandfungalsystematics.Trans.Mycol.Soc.Jpn.33:533-556.

Hibbett,D.S.,andR.Vilgalys.1991.EvolutionaryrelationshipsofLentinustothePolyporaceae:evidencefromrestrictionanalysisofenzymaticallyamplifiedribosomalDNA.Mycologia83:425-439.

Hibbett,D.S.,andR.Vilgalys.1993.PhylogeneticrelationshipsoftheBasidiomycetegenusLentinusinferredfrommolecularandmorphologicalcharacters.Syst.Bot.18:409-433.

Hillis,D.M.,andM.T.Dixon.1991.RibosomalDNA:molecularevoluitonandphylogeneticinference.Quart.Rev.Biol.66:411-453.

Hopple,J.S.,Jr.,andR.Vilgalys.1994.Phylogeneticrelationshipamongcoprinoidtaxa