ConservedprimersequencesforPCRamplificationandsequencingfromnuclearribosomal

RNA
Vilgalyslab,DukeUniversity

Overtheyears,ourlabhascompiledausefullistofconservedprimersequencesusefulforamplificationand
sequencingofnuclearrDNAfrommostmajorgroupsoffungi(primarilyEumycota),aswellasothereukaryotes.
Alloftheseprimerswereidentifiedandtestedbyourownlabbasedonconsensusbetweenthepublishedlarge
andsmallsubunitRNAsequencesfromfungi,plantsandothereukaryotessourcesofotherusefulprimer
sequencesfrompublishedliteraturearealsoindicated.Together,theseprimersspanmostofthenuclearrDNA
codingregion(seefigures),permittingamplificationofanydesiredregion.Standardsymbolsareusedforthe
fourprimarynucleotidesvariablepositionsareindicatedasfollows:P=A,G/Q=C,T/R=A,T/V=A,C/
W=G,T.Primersendingwith"R"representthecodingstrand(sameasRNA).Allotherprimersare
complementarytothecodingstrand.Thisinformationisprovidedfreelyandmaybepassedontoanyonewho
wantstouseit.
ThenuclearencodedribosomalRNAgenes(rDNA)offungiexistasamultiplecopygenefamilycomprisedof
highlysimilarDNAsequences(typicallyfrom812kbeach)arrangedinaheadtotoemanner.Eachrepeatunit
hascodingregionsforonemajortranscript(containingtheprimaryrRNAsforasingleribosome),punctuatedby
oneormoreintergenicspacer(IGS)regions.Insomegroups(mostlybasidiomcyetesandsomeascomycetous
yeasts),eachrepeatalsohasaseparatelytranscribedcodingregionfor5SRNAwhosepositionanddirectionof
transcriptionmayvayamonggroups.SeveralrestrictionsitesforEcoRIandBglIIareconservedintherDNAof
fungi.Nearlyallbasidiomyceteswe'vestudiedshareanEcoRIsitewithinthe5.8SRNAgenealongwithaBglII
sitehalfwayintotheLSURNAsequence.Primers5.8SRandLR7includetheserestrictionsites,whichmakes
themconvenientforcloning.
Forthosewhoaren'tfamiliarwithrDNAandfungalsystematics,severalexcellentreviewsareavailableonfungi
(Hibbett,1992)andgenerallyforeukaryotes(HillisandDixon,1991).SeeGerbi(1986)forageneral
introductiontothemolecularbiologyandevolutionofrDNAinothereukaryotes.Anotherusefulsourceof
primerinformationmaybefoundinGargas&Depriest(1996)andattheTomBrunslabwebsite
http://mendel.berkeley.edu/boletus/boletus.html.

SmallsubunitRNA(1718S)primersequences
LargesubunitRNA(2528S)andIGSprimersequences
Internaltranscribedspacer(ITS)regionprimers
Intergenicspacer(IGS)primers(including5SRNAprimersequencesforbasidiomycetefungi)
backtothetopofthispage

SmallsubunitRNA(SR)primers:

Primer
Sequence(5'>3')
PositionwithinS.cereviseae
name
17SRNA
BMB'A'
GRATTACCGCGGCWGCTG
580558
BMB'B'
CCGTCAATTCVTTTPAGTTT
11461127
BMB'C'
ACGGGCGGTGTGTPC
16381624
BMBBR CTTAAAGGAATTGACGGAA
11301148
BMBCR GTACACACCGCCCGTCG
16241640
SR1R
TACCTGGTTGATQCTGCCAGT
121
SR1
ATTACCGCGGCTGCT
578564
SR2
CGGCCATGCACCACC
12771263
SR3
GAAAGTTGATAGGGCT
318302
SR4
AAACCAACAAAATAGAA
838820
SR5
GTGCCCTTCCGTCAATT
11461130
SR6
TGTTACGACTTTTACTT
17601744
SR6R
AAGWAAAAGTCGTAACAAGG
17441763
SR7
GTTCAACTACGAGCTTTTTAA
617637
SR7R
AGTTAAAAAGCTCGTAGTTG
637617
SR8R
GAACCAGGACTTTTACCTT
732749
SR9R
QAGAGGTGAAATTCT
896910
SR10R
TTTGACTCAACACGGG
11811196
NS1
GTAGTCATATGCTTGTCTC

NS2
GGCTGCTGGCACCAGACTTGC

NS3
GCAAGTCTGGTGCCAGCAGCC

NS
CTTCCGTCAATTCCTTTAAG
(similartoBMBB)
NS5
AACTTAAAGGAATTGACGGAAG
(issimilartoBMBBR)
NS6
GCATCACAGACCTGTTATTGCCTC
NS7
GAGGCAATAACAGGTCTGTGATGC
NS8
TCCGCAGGTTCACCTACGGA

BMB="universal"SSUprimersdevelopedbyLaneetal.,1985
SR=primersdevelopedbyVilgalyslab
NS=primersdescribedbyWhiteetal.,1990

comments

backtothetopofthispage

LargesubunitRNA(2528S)primersequences

Note:mostmolecularsystematicsstudiesonlyutilizethefirst600900basesfromtheLSUgene,
whichincludesthreedivergentdomains(D1,D2,D3)thatareamongthemostvariableregions
withintheentiregene(muchoftheLSUisinvariantevenacrosswidelydivergenttaxa).Mostofthe
datainourAgaricalesLSUdatabaseconsistsofthefirst900basesfromtheLSUgene(wetypically
amplifyusingprimers5.8SR+LR7,followedbysequencingusingprimersLR5,LR16,LR0R,and
LR3R).

Primer
Sequence(5'>3')
PositionwithinS.
comments
name
cereviseaerRNA
5.8S
CGCTGCGTTCTTCATCG
5135(5.8SRNA)
containsEcoRI
site
5.8SR
TCGATGAAGAACGCAGCG
3451(5.8SRNA)
containsEcoRI
site
LR0R
ACCCGCTGAACTTAAGC
2642

LR1
GGTTGGTTTCTTTTCCT
7357

LR2
TTTTCAAAGTTCTTTTC
385370

LR2R
AAGAACTTTGAAAAGAG
374389

LR3
CCGTGTTTCAAGACGGG
651635

LR3R
GTCTTGAAACACGGACC
638654

LR4
ACCAGAGTTTCCTCTGG
854838

LR5
TCCTGAGGGAAACTTCG
964948

LR6
CGCCAGTTCTGCTTACC
11411125

LR7
TACTACCACCAAGATCT
14481432
containsBglII
site
LR7R
GCAGATCTTGGTGGTAG
14301446
containsBglII
site
LR8
CACCTTGGAGACCTGCT
18611845

LR8R
AGCAGGTCTCCAAGGTG
18451861

LR9
AGAGCACTGGGCAGAAA
22042188

LR10
AGTCAAGCTCAACAGGG
24202404

LR10R
GACCCTGTTGAGCTTGA
24022418

LR11
GCCAGTTATCCCTGTGGTAA
28212802

LR12
LR12R
LR13
LR14
LR15

GACTTAGAGGCGTTCAG
CTGAACGCCTCTAAGTCAGAA
CGTAACAACAAGGCTACT
AGCCAAACTCCCCACCTG
TAAATTACAACTCGGAC

31243106
31063126
33573340
26162599
154138

LR16
LR17R
LR20R
LR21
LR22

TTCCACCCAAACACTCG
TAACCTATTCTCAAACTT
GTGAGACAGGTTAGTTTTACCCT
ACTTCAAGCGTTTCCCTTT
CCTCACGGTACTTGTTCGCT

10811065
10331050
29592982
424393
364344

backtothetopofthispage

Internaltranscribedspacer(ITS)regionprimers

TheITSregionisnowperhapsthemostwidelysequencedDNAregioninfungi.Ithastypically
beenmostusefulformolecularsystematicsatthespecieslevel,andevenwithinspecies(e.g.,to
identifygeographicraces).Becauseofitshigherdegreeofvariationthanothergenicregionsof
rDNA(SSUandLSU),variationamongindividualrDNArepeatscansometimesbeobservedwithin
boththeITSandIGSregions.InadditiontothestandardITS1+ITS4primersusedbymostlabs,
everaltaxonspecificprimershavebeendescribedthatallowselectiveamplificationoffungal
sequences(e.g.,seeGardes&Bruns1993paperdescribingamplificationofbasidiomyceteITS
sequencesfrommycorrhizasamples).

primer
sequence(5'>3')
comments
reference
name
ITS1
TCCGTAGGTGAACCTGCGG

Whiteetal,1990
ITS2
GCTGCGTTCTTCATCGATGC
(issimilarto5.8S
Whiteetal,1990
below)
ITS3
GCATCGATGAAGAACGCAGC
(issimilarto5.8SR
Whiteetal,1990
below)
ITS4
TCCTCCGCTTATTGATATGC

Whiteetal,1990
ITS5
GGAAGTAAAAGTCGTAACAAGG (issimilartoSR6R)
Whiteetal,1990
ITS1F
CTTGGTCATTTAGAGGAAGTAA
Gardes&Bruns,
1993
Gardes&Bruns,
ITS4B
CAGGAGACTTGTACACGGTCCAG
1993

5.8S
5.8SR
SR6R

CGCTGCGTTCTTCATCG
TCGATGAAGAACGCAGCG
AAGWAAAAGTCGTAACAAGG

Vilgalyslab
Vilgalyslab
Vilgalyslab

backtothetopofthispage

Intergenicspacer(IGS)primers(including5SRNAprimersequencesforbasidiomycetefungi)

ThegreatestamountsequencevariationinrDNAexistswithintheIGSregion(sometimesalsoknownasthe
nontranscribedspacerorNTSregion).ThesizeoftheIGSregionmayvaryfrom2kbupwards.Itisnotunusual
tofindhypervariabilityforthisregion(necessitatingcloningofindividualrepeathaplotypes).Severalpatternsof
organizationcanbefoundindifferentgroupsoffungi:
1. MostfilamentousascomyceteshaveasingleuninterruptedIGSregion(betweentheendoftheLSUand
startofthenextSSUsequence),whichmayvaryinlengthfrom25kbormore.Amplificationoftheentire
IGSregionrequiresusingprimersanchoredinthe3'endoftheLSUgene(e.g.,LR12R)and5'endofthe
SSURNAgene(e.g.,invSR1R).
2. Inmanyascomycetousyeastsandnearlyallbasidiomycetes,theIGSalsocontainsasinglecodingregion
forthe5SRNAgene,whichdividestheIGSintotwosmallerregionsthatmaybemoreeasilyamplified
using.Dependingontheorientationandpositionofthe5SRNAgene,thePCRmaybeusedto
sequentiallyamplifyeitheraportionoftheintergenicspacerregion(IGS)beyondthelargesubunitRNA
codingregion.

primer
name

sequence(5'>3')

LR12R

GAACGCCTCTAAGTCAGAATCC

invSR1R
5SRNA
5SRNAR

comments

locatedwithintheLSURNA(see
above)
locatedwithintheSSURNA
ACTGGCAGAATCAACCAGGTA
(positions211)
ATCAGACGGGATGCGGT
(complementaryto5SRNA
positions4626)
ACQGCATCCCGTCTGAT
(5SRNApositions2646)

reference
Vilgalys
lab
Vilgalys
lab
Vilgalys
lab
Vilgalys
lab

backtothetopofthispage

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:analysesofnuclearsmall
subunitrRNAsequences.Molec.Phylog.Evol.1:231241.
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.NumerousgroupIintronswithvariabledistributionsintheribosomal
DNAofalichenfungus.J.Mol.Biol.228:315321.
Elwood,H.J.,G.J.Olsen,andM.L.Sogin.1985.ThesmallsubunitribosomalRNAgenesequencesfromthe
hypotrichousciliatesOxytrichanovaandStylonychiapustula.Mol.Biol.Evol.2:399410.
Gardes,M.,andT.D.Bruns.1993.ITSprimerswithenhancedspecificityforbasidiomycetesapplicationtothe
identificationofmycorrhizaeandrusts.Mol.Ecol.2:113118.
Gargas,A.,andP.T.DePriest.1996.AnomenclatureforfungalPCRprimerswithexamplesfromintron
containingSSUrDNA.
Mycologia88:745748
Gargas,A.,andJ.W.Taylor.1992.Polymerasechainreaction(PCR)primersforamplifyingandsequencing18S
rDNAfrom
lichenizedfungi.Mycologia84:589592.
Gerbi,S.A.1986.Chapter7EvolutionofribosomalDNA.Pp.419517In:Molecularevolution,ed.McIntyre,
R.
Hibbett,D.S.1991.PhylogeneticrelationshipsoftheBasidiomycetegenusLentinus:evidencefromribosomal
RNAandmorphology.Ph.D.Thesis,DukeUniversity,1991.
Hibbett,D.S.1992.RibosomalRNAandfungalsystematics.Trans.Mycol.Soc.Jpn.33:533556.
Hibbett,D.S.,andR.Vilgalys.1991.EvolutionaryrelationshipsofLentinustothePolyporaceae:evidencefrom
restrictionanalysisofenzymaticallyamplifiedribosomalDNA.Mycologia83:425439.
Hibbett,D.S.,andR.Vilgalys.1993.PhylogeneticrelationshipsoftheBasidiomycetegenusLentinusinferred
frommolecularandmorphologicalcharacters.Syst.Bot.18:409433.
Hillis,D.M.,andM.T.Dixon.1991.RibosomalDNA:molecularevoluitonandphylogeneticinference.Quart.
Rev.Biol.66:411453.
Hopple,J.S.,Jr.,andR.Vilgalys.1994.Phylogeneticrelationshipamongcoprinoidtaxaandalliesbasedondata
fromrestrictionsitemappingofnuclearrDNA.Mycologia86:96107.
Lane,D.J.,B.Pace,G.J.Olsen,D.A.Stahl,M.L.Sogin,andN.R.Pace.1985.Rapiddeterminationof16S
ribosomalRNAsequencesforphylogeneticanalyses.Proc.Natl.Acad.Sci.,U.S.A.82:69556959.
Vilgalys,R.,andD.Gonzalez.1990.OrganizationofribosomalDNAinthebasidiomyceteThanatephorus
praticola.Curr.Genet.18:277280.
Vilgalys,R.,J.S.Hopple,Jr.,andD.S.Hibbett.1994.Phylogeneticimplicationsofgenericconceptsinfungal
taxonomy:Theimpactofmolecularsystematicstudies.MycologicaHelvetica6:7391.
White,T.J.,T.Bruns,S.Lee,andJ.W.Taylor.1990.Amplificationanddirectsequencingoffungalribosomal
RNAgenesforphylogenetics.Pp.315322In:PCRProtocols:AGuidetoMethodsandApplications,eds.Innis,
M.A.,D.H.Gelfand,J.J.Sninsky,andT.J.White.AcademicPress,Inc.,NewYork.
W
backtothetopofthispage
gotoVilgalyslabhomepage
gotoagaricphylogenyhomepage