Conserved primer sequences for PCR amplification and sequencing from nuclear
ribosomal RNA
Vilgalys lab, Duke University
Over the years, our lab has compiled a useful list of conserved primer sequences useful for
amplification and sequencing of nuclear rDNA from most major groups of fungi (primarily
Eumycota), as well as other eukaryotes. All of these primers were identified and tested by our own
lab based on consensus between the published large and small subunit RNA sequences from fungi,
plants and other eukaryotes; sources of other useful primer sequences from published literature are
also indicated. Together, these primers span most of the nuclear rDNA coding region (see figures),
permitting amplification of any desired region. Standard symbols are used for the four primary
nucleotides; variable positions are indicated as follows: P=A,G / Q=C,T / R=A,T / V=A,C /
W=G,T. Primers ending with "R" represent the coding strand (same as RNA). All other primers are
complementary to the coding strand. This information is provided freely and may be passed on to
anyone who wants to use it.
The nuclear-encoded ribosomal RNA genes (rDNA) of fungi exist as a multiple-copy gene family
comprised of highly similar DNA sequences (typically from 8-12 kb each) arranged in a head-to-toe
manner. Each repeat unit has coding regions for one major transcript (containing the primary
rRNAs for a single ribosome), punctuated by one or more intergenic spacer (IGS) regions. In some
groups (mostly basidiomcyetes and some ascomycetous yeasts), each repeat also has a separately
transcribed coding region for 5S RNA whose position and direction of transcription may vay among
groups. Several restriction sites for EcoRI and BglII are conserved in the rDNA of fungi. Nearly all
basidiomycetes we've studied share an EcoRI site within the 5.8S RNA gene along with a BglII site
halfway into the LSU RNA sequence. Primers 5.8SR and LR7 include these restriction sites, which
makes them convenient for cloning.
For those who aren't familiar with rDNA and fungal systematics, several excellent reviews are
available on fungi (Hibbett, 1992) and generally for eukaryotes (Hillis and Dixon, 1991). See Gerbi
(1986) for a general introduction to the molecular biology and evolution of rDNA in other
eukaryotes. Another useful source of primer information may be found in Gargas & Depriest (1996)
and at the Tom Bruns lab web site http://mendel.berkeley.edu/boletus/boletus.html.
Small subunit RNA (17-18S) primer sequences
Large subunit RNA (25-28S) and IGS primer sequences
Internal transcribed spacer (ITS) region primers
Intergenic spacer (IGS) primers (including 5S RNA primer sequences for
basidiomycete fungi)
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Small subunit RNA (SR) primers:
Primer
name
Sequence (5'-->3')
Position within S. cereviseae
17S RNA
BMB-'A'
GRATTACCGCGGCWGCTG
580-558
BMB-'B'
CCGTCAATTCVTTTPAGTTT
1146-1127
BMB-'C'
ACGGGCGGTGTGTPC
1638-1624
BMB-BR
CTTAAAGGAATTGACGGAA
1130-1148
BMB-CR
GTACACACCGCCCGTCG
1624-1640
SR1R
TACCTGGTTGATQCTGCCAGT
1-21
SR1
ATTACCGCGGCTGCT
578-564
SR2
CGGCCATGCACCACC
1277-1263
SR3
GAAAGTTGATAGGGCT
318-302
SR4
AAACCAACAAAATAGAA
838-820
SR5
GTGCCCTTCCGTCAATT
1146-1130
SR6
TGTTACGACTTTTACTT
1760-1744
SR6R
AAGWAAAAGTCGTAACAAGG
1744-1763
SR7
GTTCAACTACGAGCTTTTTAA
617-637
SR7R
AGTTAAAAAGCTCGTAGTTG
637-617
SR8R
GAACCAGGACTTTTACCTT
732-749
SR9R
QAGAGGTGAAATTCT
896-910
SR10R
TTTGACTCAACACGGG
1181-1196
comments
NS1
GTAGTCATATGCTTGTCTC
NS2
GGCTGCTGGCACCAGACTTGC
NS3
GCAAGTCTGGTGCCAGCAGCC
NS
CTTCCGTCAATTCCTTTAAG
(similar to BMB-B)
NS5
AACTTAAAGGAATTGACGGAAG
(is similar to BMB-BR)
NS6
GCATCACAGACCTGTTATTGCCTC
NS7
GAGGCAATAACAGGTCTGTGATGC
NS8
TCCGCAGGTTCACCTACGGA
BMB = "universal" SSU primers developed by Lane et al., 1985
SR = primers developed by Vilgalys lab
NS = primers described by White et al., 1990
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Large subunit RNA (25-28S) primer sequences
Note: most molecular systematics studies only utilizethe first 600-900 bases from the LSU gene,
which includes three divergent domains (D1, D2, D3) that are among the most variable regions
within the entire gene (much of the LSU is invariant even across widely divergent taxa). Most of the
data in our Agaricales LSU database consists of the first 900 bases from the LSU gene (we typically
amplify using primers 5.8SR + LR7, followed by sequencing using primers LR5, LR16, LR0R, and
LR3R).
Primer
name
5.8S
Sequence (5'-->3')
comments
CGCTGCGTTCTTCATCG
Position within S.
cereviseae rRNA
51-35 (5.8S RNA)
5.8SR
TCGATGAAGAACGCAGCG
34-51 (5.8S RNA)
contains EcoRI
site
LR0R
ACCCGCTGAACTTAAGC
26-42
LR1
GGTTGGTTTCTTTTCCT
73-57
contains EcoRI
site
LR2
TTTTCAAAGTTCTTTTC
385-370
LR2R
AAGAACTTTGAAAAGAG
374-389
LR3
CCGTGTTTCAAGACGGG
651-635
LR3R
GTCTTGAAACACGGACC
638-654
LR4
ACCAGAGTTTCCTCTGG
854-838
LR5
TCCTGAGGGAAACTTCG
964-948
LR6
CGCCAGTTCTGCTTACC
1141-1125
LR7
TACTACCACCAAGATCT
1448-1432
contains BglII
site
LR7R
GCAGATCTTGGTGGTAG
1430-1446
contains BglII
site
LR8
CACCTTGGAGACCTGCT
1861-1845
LR8R
AGCAGGTCTCCAAGGTG
1845-1861
LR9
AGAGCACTGGGCAGAAA
2204-2188
LR10
AGTCAAGCTCAACAGGG
2420-2404
LR10R
GACCCTGTTGAGCTTGA
2402-2418
LR11
GCCAGTTATCCCTGTGGTAA
2821-2802
LR12
GACTTAGAGGCGTTCAG
3124-3106
LR12R
CTGAACGCCTCTAAGTCAGAA
3106-3126
LR13
CGTAACAACAAGGCTACT
3357-3340
LR14
AGCCAAACTCCCCACCTG
2616-2599
LR15
TAAATTACAACTCGGAC
154-138
LR16
TTCCACCCAAACACTCG
1081-1065
LR17R
TAACCTATTCTCAAACTT
1033-1050
LR20R
GTGAGACAGGTTAGTTTTACCCT 2959-2982
LR21
ACTTCAAGCGTTTCCCTTT
LR22
CCTCACGGTACTTGTTCGCT
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424-393
364-344
Internal transcribed spacer (ITS) region primers
The ITS region is now perhaps the most widely sequenced DNA region in fungi. It has typically
been most useful for molecular systematics at the species level, and even within species (e.g., to
identify geographic races). Because of its higher degree of variation than other genic regions of
rDNA (SSU and LSU), variation among individual rDNA repeats can sometimes be observed
within both the ITS and IGS regions. In addition to the standard ITS1+ITS4 primers used by most
labs, everal taxon-specific primers have been described that allow selective amplification of fungal
sequences (e.g., see Gardes & Bruns 1993 paper describing amplification of basidiomycete ITS
sequences from mycorrhiza samples).
primer
name
ITS1
sequence (5'->3')
comments
TCCGTAGGTGAACCTGCGG
reference
White et al, 1990
ITS2
GCTGCGTTCTTCATCGATGC
(is similar to 5.8S
below)
White et al, 1990
ITS3
GCATCGATGAAGAACGCAGC
(is similar to 5.8SR
below)
White et al, 1990
ITS4
TCCTCCGCTTATTGATATGC
White et al, 1990
ITS5
GGAAGTAAAAGTCGTAACAAGG (is similar to SR6R)
White et al, 1990
ITS1-F
CTTGGTCATTTAGAGGAAGTAA
Gardes & Bruns,
1993
ITS4-B
CAGGAGACTTGTACACGGTCCAG
Gardes & Bruns,
1993
5.8S
CGCTGCGTTCTTCATCG
Vilgalys lab
5.8SR
TCGATGAAGAACGCAGCG
Vilgalys lab
SR6R
AAGWAAAAGTCGTAACAAGG
Vilgalys lab
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Intergenic spacer (IGS) primers (including 5S RNA primer sequences for
basidiomycete fungi)
The greatest amount sequence variation in rDNA exists within the IGS region (sometimes also
known as the non-transcribed spacer or NTS region). The size of the IGS region may vary from 2
kb upwards. It is not unusual to find hypervariability for this region (necessitating cloning of
individual repeat haplotypes). Several patterns of organization can be found in different groups of
fungi:
1. Most filamentous ascomycetes have a single uninterrupted IGS region (between the end of
the LSU and start of the next SSU sequence), which may vary in length from 2-5 kb or
more. Amplification of the entire IGS region requires using primers anchored in the 3' end
of the LSU gene (e.g., LR12R) and 5' end of the SSU RNA gene (e.g., invSR1R).
2. In many ascomycetous yeasts and nearly all basidiomycetes, the IGS also contains a single
coding region for the 5S RNA gene, which divides the IGS into two smaller regions that
may be more easily amplified using. Depending on the orientation and position of the 5S
RNA gene, the PCR may be used to sequentially amplify either aportion of the intergenic
spacer region (IGS) beyond the large subunit RNA coding region.
primer
name
sequence (5'->3')
comments
reference
LR12R
GAACGCCTCTAAGTCAGAATCC
located within the LSU RNA (see
above)
Vilgalys
lab
invSR1R
ACTGGCAGAATCAACCAGGTA
located within the SSU RNA
(positions 21-1)
Vilgalys
lab
5SRNA
ATCAGACGGGATGCGGT
(complementary to 5S RNA
positions 46-26)
Vilgalys
lab
5SRNAR
ACQGCATCCCGTCTGAT
(5S RNA positions 26-46)
Vilgalys
lab
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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, and M. L. Sogin. 1992. Evolutionary relationships within the fungi:
analyses of nuclear small subunit rRNA sequences. Molec. Phylog. Evol. 1: 231-241.
Bruns, T. D., T. J. White, and J. W. Taylor. 1991. Fungal molecular systematics. Ann. Rev. Ecol.
Syst. 22: 525-564.
DePriest, P. T., and M. D. Been. 1992. Numerous group I introns with variable distributions in the
ribosomal DNA of a lichen fungus. J. Mol. Biol. 228: 315-321.
Elwood, H. J., G. J. Olsen, and M. L. Sogin. 1985. The small subunit ribosomal RNA gene
sequences from the hypotrichous ciliates Oxytricha nova and Stylonychia pustula. Mol. Biol. Evol.
2: 399-410.
Gardes, M., and T. D. Bruns. 1993. ITS primers with enhanced specificity for basidiomycetes application to the identification of mycorrhizae and rusts. Mol. Ecol. 2: 113-118.
Gargas, A., and P.T. DePriest. 1996. A nomenclature for fungal PCR primers with examples from
intron-containing SSU rDNA.
Mycologia 88: 745-748
Gargas, A., and J.W. Taylor. 1992. Polymerase chain reaction (PCR) primers for amplifying and
sequencing 18S rDNA from
lichenized fungi. Mycologia 84: 589-592.
Gerbi, S. A. 1986. Chapter 7 - Evolution of ribosomal DNA. Pp. 419-517 In: Molecular evolution,
ed. McIntyre, R.
Hibbett, D. S. 1991. Phylogenetic relationships of the Basidiomycete genus Lentinus: evidence
from ribosomal RNA and morphology. Ph.D. Thesis, Duke University, 1991.
Hibbett, D. S. 1992. Ribosomal RNA and fungal systematics. Trans. Mycol. Soc. Jpn. 33: 533-556.
Hibbett, D. S., and R. Vilgalys. 1991. Evolutionary relationships of Lentinus to the Polyporaceae:
evidence from restriction analysis of enzymatically amplified ribosomal DNA. Mycologia 83: 425439.
Hibbett, D. S., and R. Vilgalys. 1993. Phylogenetic relationships of the Basidiomycete genus
Lentinus inferred from molecular and morphological characters. Syst. Bot. 18: 409-433.
Hillis, D. M., and M. T. Dixon. 1991. Ribosomal DNA: molecular evoluiton and phylogenetic
inference. Quart. Rev. Biol. 66: 411-453.
Hopple, J. S., Jr., and R. Vilgalys. 1994. Phylogenetic relationship among coprinoid taxa and allies
based on data from restriction site mapping of nuclear rDNA. Mycologia 86: 96-107.
Lane, D. J., B. Pace, G. J. Olsen, D. A. Stahl, M. L. Sogin, and N. R. Pace. 1985. Rapid
determination of 16S ribosomal RNA sequences for phylogenetic analyses. Proc. Natl. Acad. Sci.,
U. S. A. 82: 6955-6959.
Vilgalys, R., and D. Gonzalez. 1990. Organization of ribosomal DNA in the basidiomycete
Thanatephorus praticola. Curr. Genet. 18: 277-280.
Vilgalys, R., J. S. Hopple, Jr., and D. S. Hibbett. 1994. Phylogenetic implications of generic
concepts in fungal taxonomy: The impact of molecular systematic studies. Mycologica Helvetica 6:
73-91.
White, T. J., T. Bruns, S. Lee, and J. W. Taylor. 1990. Amplification and direct sequencing of
fungal ribosomal RNA genes for phylogenetics. Pp. 315-322 In: PCR Protocols: A Guide to
Methods and Applications, eds. Innis, M. A., D. H. Gelfand, J. J. Sninsky, and T. J. White.
Academic Press, Inc., New York. ©W°