真菌检测鉴定通用引物-Fungal Primers

ITS1: 5’-CCGTAGGTGAACCTGCGG-3’

ITS4: 5’-TCCTCCGCTTATTGATATGC-3’ Tm 55℃ NS17: CATGTCTAAGTTTAAGCAA NS3: GCAAGTCTGGTGCCAGCAGCC NS4: CTTCCGTCAATTCCTTTAAG NS22: AATTAAGCAGACAAATCACT NS24: AAACCTTgTTACgACTTTTA

LR0R: 5’-GTACCCGCTGAACTTAAGC-3’ LR3: 5’-CCGTGTTTCAAGACGGG

LR3R: 5’-GTCTTGAAACACGGACC (complementary to RLR3R: GGTCCGTGTTTCAAGAC) LR5: 5’-TTAAAAAGCTCGTAGTTGAAC-3’ LR7: 5’-TACTACCACCAAGATCT LR12: 5’-GACTTAGAGGCGTTCAG Lr0R/LR5: Tm 50-52℃

NL1: 5’-GCATATCAATAAGCGGAGGAAAAG

NL1: 5′-TGCGTTGATTACGTCCCTGC (also called V9: TGCGTTGATTACGTCCCTGC) NL1: 5’-TGCTGGAGCCATGGATC-3

NL2: 5’-CTCTCTTTTCAAAGTTCTTTTCATCT NL2: 5’-AACGGCTTCGACAACAGC-3

NL2: 5’-CTTGTTCGCTATCGGTCTC (also NL2A: 5′-CTTGTTCGCTATCGGTCTC) NL2: 5’-TACTTGTTCGCTATCGGTCT-3'

NL3: 5’-GAGACCGATAGCGAACAAG (also NL3A: 5’-GAGACCGATAGCGAACAAG) NL3: 5’-AGACCGATAGCGAACAAGTA NL3: 5’-GCTGTTGTCGAAGCCGTT-3 NL4: 5’-GGTCCGTGTTTCAAGACGG （similar to RLR3R：5′-GGTCCGTGTTTCAAGAC) NL4: 5’-TAGATACATGGCGCAGTC-3

Conserved primer sequences for PCR amplification and sequencing from nuclear ribosomal RNA (http://www.biology.duke.edu/fungi/mycolab/primers.htm) 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 \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 (SR) primers:

Primer name Sequence (5'-->3') BMB-'A' BMB-'B' BMB-'C' BMB-BR BMB-CR SR1R SR1 SR2 SR3 SR4 SR5 SR6 SR6R SR7 SR7R SR8R SR9R SR10R NS1 NS2 NS3 NS4 NS5 NS6 NS7 NS8 GRATTACCGCGGCWGCTG CCGTCAATTCVTTTPAGTTT ACGGGCGGTGTGTPC CTTAAAGGAATTGACGGAA GTACACACCGCCCGTCG TACCTGGTTGATQCTGCCAGT ATTACCGCGGCTGCT CGGCCATGCACCACC GAAAGTTGATAGGGCT AAACCAACAAAATAGAA GTGCCCTTCCGTCAATT TGTTACGACTTTTACTT AAGWAAAAGTCGTAACAAGG GTTCAACTACGAGCTTTTTAA AGTTAAAAAGCTCGTAGTTG GAACCAGGACTTTTACCTT QAGAGGTGAAATTCT TTTGACTCAACACGGG GTAGTCATATGCTTGTCTC GGCTGCTGGCACCAGACTTGC GCAAGTCTGGTGCCAGCAGCC CTTCCGTCAATTCCTTTAAG AACTTAAAGGAATTGACGGAAG Position within S. cereviseae 17S RNA 580-558 1146-1127 1638-1624 1130-1148 1624-1640 1-21 578-564 1277-1263 318-302 838-820 1146-1130 1760-1744 1744-1763 617-637 637-617 732-749 896-910 1181-1196 (similar to BMB-B) (is similar to BMB-BR) GCATCACAGACCTGTTATTGCCTC GAGGCAATAACAGGTCTGTGATGC TCCGCAGGTTCACCTACGGA BMB = \SR = primers developed by Vilgalys lab

NS = primers described by White et al., 1990

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 Sequence (5'-->3') 5.8S 5.8SR LR0R LR1 LR2 LR2R LR3 LR3R LR4 LR5 LR6 LR7 LR7R LR8 LR8R LR9 LR10 LR10R LR11 LR12 LR12R LR13 LR14 LR15 LR16 LR17R LR20R LR21 LR22 CGCTGCGTTCTTCATCG TCGATGAAGAACGCAGCG ACCCGCTGAACTTAAGC GGTTGGTTTCTTTTCCT TTTTCAAAGTTCTTTTC AAGAACTTTGAAAAGAG CCGTGTTTCAAGACGGG GTCTTGAAACACGGACC ACCAGAGTTTCCTCTGG TCCTGAGGGAAACTTCG CGCCAGTTCTGCTTACC TACTACCACCAAGATCT GCAGATCTTGGTGGTAG CACCTTGGAGACCTGCT AGCAGGTCTCCAAGGTG AGAGCACTGGGCAGAAA AGTCAAGCTCAACAGGG GACCCTGTTGAGCTTGA GCCAGTTATCCCTGTGGTAA GACTTAGAGGCGTTCAG CTGAACGCCTCTAAGTCAGAA CGTAACAACAAGGCTACT AGCCAAACTCCCCACCTG TAAATTACAACTCGGAC TTCCACCCAAACACTCG TAACCTATTCTCAAACTT ACTTCAAGCGTTTCCCTTT CCTCACGGTACTTGTTCGCT Position within S. cereviseae rRNA comments 51-35 (5.8S RNA) 34-51 (5.8S RNA) 26-42 73-57 385-370 374-389 651-635 638-654 854-838 964-948 1141-1125 1448-1432 1430-1446 1861-1845 1845-1861 2204-2188 2420-2404 2402-2418 2821-2802 3124-3106 3106-3126 3357-3340 2616-2599 154-138 1081-1065 1033-1050 424-393 364-344 contains EcoRI site contains EcoRI site contains BglII site contains BglII site GTGAGACAGGTTAGTTTTACCCT 2959-2982 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 sequence (5'->3') comments (is similar to 5.8S below) (is similar to 5.8SR below) reference White et al, 1990 White et al, 1990 White et al, 1990 White et al, 1990 White et al, 1990 Gardes & Bruns, 1993 Gardes & Bruns, 1993 Vilgalys lab Vilgalys lab Vilgalys lab ITS1 ITS2 ITS3 ITS4 ITS5 ITS1-F ITS4-B 5.8S 5.8SR SR6R TCCGTAGGTGAACCTGCGG GCTGCGTTCTTCATCGATGC GCATCGATGAAGAACGCAGC TCCTCCGCTTATTGATATGC CTTGGTCATTTAGAGGAAGTAA CGCTGCGTTCTTCATCG TCGATGAAGAACGCAGCG AAGWAAAAGTCGTAACAAGG GGAAGTAAAAGTCGTAACAAGG (is similar to SR6R) CAGGAGACTTGTACACGGTCCAG 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 sequence (5'->3') LR12R 5SRNA 5SRNAR GAACGCCTCTAAGTCAGAATCC ATCAGACGGGATGCGGT ACQGCATCCCGTCTGAT invSR1R ACTGGCAGAATCAACCAGGTA comments located within the LSU RNA (see above) reference Vilgalys lab located within the SSU RNA (positions 21-1) Vilgalys lab (complementary to 5S RNA positions 46-26) Vilgalys lab (5S RNA positions 26-46) Vilgalys lab 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.

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