Development of Specific Primer for Tricholoma matsutake.

In this study, in an effort to develop a method for the molecular detection of Tricholoma matsutake in Korea from other closely related Tricholomataceae, a species-specific PCR primer pair, TmF and TmR, was designed using nuclear ribosomal intertranscribed spacer (ITS) sequences. The DTmF and DTmR sequences were 5'-CCTGACGCCAATCTTTTCA-3' and 5'-GGAGAGCAGACTTGTGAGCA-3', respectively. The PCR primers reliably amplified only the ITS sequences of T. matsutake, and not those of other species used in this study.

Tricholoma matsutake, an agriculturally important mushroom due to its strong volatile flavor and taste, is an ectomycorrhizal fungus belonging to the Tricholomataceae, Agaricales, Hymenomycetes, and Basidiomycota. Molecular techniques including DNA hybridization and polymerase chain reaction (PCR) have been extensively utilized to confirm the genotypes of mushrooms. Ribosomal repeat units, composed of 18S-ITS1-5.8S-ITS2-28S (Srivastava and Schlessinger, 1991), have already proven very informative in differentiating species and genera (Fig. 1). The internal transcribed spacer (ITS) is of particular usefulness, because it is accessible with universal primers and its DNA sequence is variable at both inter- and intra-specific levels. Many molecular studies have been conducted on T. matsutake (Chapela and Garbelotto, 2004; Dunstan et al., 2000; Kikuch et al., 2000; Lee et al., 1999, 2004; Lee and Hong, 1998; Lian et al., 2003). However, only a minimal amount of information is currently available for the differentiation of T. matsutake from the closely related Tricholomataceae. The principal objective of this study was to develop a method for the molecular detection of Tricholoma matsutake in Korea using the specific primers, DTmF and DTmR.

Fig. 1

Schemtic illustration of nuclear ITS region and species-specific primer sites for T. matsutake.

The mycelia of strains (Table 1) were grown at 24℃ for 30 days in a shaking incubator with DTM broth (Min and Han, 2000). DNA was extracted from the cultured mycelia via a modified version of the method of Raeder and Broda (1985). The mycelia were collected by filtration and then ground to fine powder in liquid N2-after which 50 mg of powder was placed in a micro-centrifugal tube and mixed with 700 µl of modified Carlson lysis buffer (2% CTAB, 100 mM Tris-HCl (pH 8.0), 20 mM EDTA, 1.4M NaCl, 1% PEG 8000, 1% β-mercaptoethanol). DNA was precipitated with an equal volume of cold isopropanol and centrifuged for 10 min at 12,000 rpm. The supernatant was discarded and the pellet was washed with 1 ml of 70% ethanol and then re-dissolved in 100 µl of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). The remaining RNA was degraded via RNase treatment (20 µg/ml).

Table 1

The strains of mushroom used in this study

aKCTC; Korean Collection for Type Cultures, Daejeon, DGUM; Dongguk University Microbiology Lab., IMSNU; Institute of Microbiology, Seoul National University.

For the amplification of the ITS sequence, ITS5 and ITS4 primers were utilized and PCR was conducted using a Mastercycler (Eppendorf, Germany) in a 50 µl reaction mixture containing 50 ng of genomic DNA, 0.2 µM of each primer, 5 µl of 10 × buffer, 200 µM of each dNTP and 1.25 unit of Taq DNA polymerase (Takara, Japan). PCR was conducted as follows: initial denaturation at 95℃ for 2 min, followed by 24 cycles of denaturation at 95℃ for 30 sec, annealing at 58℃ for 30 sec, extension at 72℃ for 1 min, and a final 5-minute extension at 72℃. The PCR products were electrophoresed for 40 min in 0.8% agarose gel at 50 V with TAE buffer, visualized via ethidium bromide staining, and photographed under a UV transilluminator. The DNA sequence analyses and primer preparation procedures were conducted using a CoreBio system (Korea). The consensus sequence using BLAST 2 SEQUENCES (NCBI) and CLUSTAL W (v1.81; Thompson et al., 1994) was used to design the specific primers of T. matsutake, and deposited into the GenBank database (accession no.; AY736068, Table 1).

Based on the sequence of the T. matsutake ITS region, a specific primer set (DTmF-DTmR) was designed using a multiple sequence alignment program (http://prodes.toulouse.inra.fr/multalin/multalin.html, Table 2). When the DTmF-DTmR primer was used, a ca. 400 bp PCR product of ITS region was detected only in T. matsutake, and not in the other species utilized herein (Fig. 2). The primer binding regions of DTmF and DTmR were confirmed using the Primer Show program on the web site (www.bioinformatics.org/sms/primer_show.html) (Fig. 3). These results demonstrate that the DTmF-DTmR primer pair is species-specific for T. matsutake in Korea, thus suggesting that the primer may prove useful in differentiating T. matsutake from other mushrooms related to the Tricholomataceae.

Table 2

The sequences of primers used in this study and other reports for T. matsutake

Fig. 2

Amplification of the ITS region of T. matsutake with the primer of DTmF and DTmR. No PCR sequence was shown in other mushrooms. M; size marker, Lane 1; T. matsutake KCTC6468, Lane 2; T. matsutake KCTC26249, Lane 3; T. matsutake DGUM26002, Lane 4; T. matsutake DGUM26003, Lane 5; R. botrytis DGUM 29001, Lane 6; C. pyridata DGUM29005, Lane 7; L. edodes IMSNU31010, Lane 8; L. lepideus DGUM25050, Lane 9; L. nuda DGUM26501.

Fig. 3

Sequence of the ITS region of T. matsutake KCTC26249 binding with the specific primer of DTmF (upper; bold type) and DTmR (lower; bold type underlined), respectively.