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Pleurotus sPecies from eastern 2011, MicologiA AplicAdA internAtionAl, 23(1),asia pp. 1-10 1

sequence variation of pleurotus sPecies collected from eastern asia

a. imtiaj1, t. s. lee2 and s. ohga1*
1

Division of Forest Environmental Sciences, Department of Agro-environmental Sciences, Kyushu University, Fukuoka 811-2415, Japan. Department of Biology, University of Incheon, Incheon 402-749, Korea.

Accepted for publication December 21, 2010

ABSTRACT The systematic and genetic relationship among different species of Pleurotus mushrooms is still unclear. Because of that, 20 strains of Pleurotus spp. collected from differing regions, such as Korea (P. djamor, P. eryngii, P. ostreatus, P. pulmonarius), China (P. cornucopiae, P. eryngii, P. ferulae, P. nebrodensis, P. ostreatus), and Taiwan (P. cornucopiae, P. cystidiosus, P. ostreatus) were used to study their genetic makeup. In this study, we used DNA sequences of the ITS (Internal Transcribed Spacer) region to analyze the genetic diversity of Pleurotus strains. A few differences were found in the sequences implying that all strains belonged to Pleurotus regardless of the geographical origin and species. This is also supported by phylogenetic analysis, which revealed that Pleurotus strains collected from different environments have a little genetic variation in case of differing species. Some strains belonging to the same species showed 100% similarities, even those collected from different regions, suggesting that strains studied might be distributed from a common ancestor. Key words: DNA sequences, ITS region, phylogeny, Pleurotus spp.

INTRODUCTION The oyster mushroom and its related species are prominent fungi causing wood de-

cay in terrestrial ecosystems worldwide, and are widely collected and cultivated as edible fungi. Because of its good flavor, culinary status and medicinal properties,

MCorrespondingnt., 23(1), 2011, pp. 1-10 Fax: +81-929483116. E-mail: ohga@forest.kyushu-u.ac.jp * icol. Apl. i author: Phone: +81-929483118.

a. imtiaj et Al.
belonging to the genus Pleurotus were collected from Korea, China and Taiwan. We used the sequence of the internal transcribed spacer (ITS1-5.8S rDNA-ITS2) region to analyze the genetic diversity of Pleurotus strains derived from different places. MATERIALS AND METHODS The cultures of 20 strains of Pleurotus spp. studied were obtained from the Culture Collection and DNA Bank of Mushrooms (CCDBM), University of Incheon, Korea (Table 1). DNA extraction. Mycelia of Pleurotus spp. were grown either on potato dextrose agar (PDA) or malt extract agar (MEA), harvested using a spatula, transferred into 1.5 ml Eppendorf tubes, freeze-dried (Operon, Korea), and ground into powder with a pestle using liquid nitrogen. As extraction buffer, equal amount of 50 mM Tris-HCl (pH 7.5), 50 mM EDTA (pH 8) and 1% sarkosyl were added to Eppendorf tube, vortexed (Barnstead Int., U.S.A.), and incubated at 65 C for 30 min in a steam water bath. After incubation, PCI (25 ml phenol : 24 ml chloroform : 1 ml isoamyl-alcohol) was added, vortexed and centrifuged at 4 C, 10 min, 12,000 rpm. The supernatant was put into 1.5 ml Eppendorf tubes, 1,000 l of 99.9% ethyl alcohol was added and centrifuged at 4 C, 5 min, 12,000 rpm. The supernatant was decanted, 500 l of 70% alcohol was added to precipitated DNA, and again centrifuged at 4 C, 5 min, 12,000 rpm. Finally, the supernatant was removed and the residual alcohol allowed to evaporate. Then, 500 l of sterilized distilled water was added and vortexed 1-2 min (it is called stock solution). DNA concentration was assessed using spectrophotometer (2120UV, Optizen, Korea), 20 l of the

the production and consumption of the oyster mushroom has increased at rapid rate during the last few years throughout the world. Oyster mushrooms have a high commercial value, but the systematic and genetic relationships among these species is still unclear. Phylogenetic analysis using molecular sequences is useful for resolving relationships and understanding speciation in many problematic species complexes of Basidiomycetes1,13,18,21. With the accumulation of ecological knowledge and the development of phylogenetic analysis based on DNA techniques, the traditional taxonomy of Pleurotus spp. has come into question. Mating compatibility studies have demonstrated the existence of separate biological species in Pleurotus, many of which are largely distributed over one or more continents19. Evidences revealed that oyster mushrooms collected from different countries show a little genetic difference. Therefore, traditional taxonomy of Pleurotus spp. may not be correct. Characterization of relationships may clarify the taxonomy of Pleurotus spp. isolated from different countries. The ability of rDNA sequences to resolve phylogenetic relationships among geographically isolated populations within intersterility groups illustrated the importance of biogeographical studies for understanding speciation in Pleurotus19. Mating tests are often used to study the variation of Basidiomycetes, but it is difficult to apply to slow-growing species due to poor spore germination and clampconnection frequency. DNA sequencing is accordingly useful for elucidating taxonomic relationships among Pleurotus species growing in different environments10,14,17. Different DNA techniques have been used to determine the genetic differences within and among Basidiomycetes. In this study, 20 strains of different species

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Table 1. Pleurotus strains used in this study. No. Species Code GenBank accession number HM770899 HM770890 HM770891 HM770895 HM770892 HM770889 HM770888 HM770903 HM770902 HM770900 HM770897 HM770884 HM770887 HM770898 HM770901 HM770894 HM770885 HM770893 HM770896 HM770886

Country

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

P. cornucopiae (Paulet) Rolland P. cornucopiae (Paulet) Rolland P. cystidiosus O. K. Miller P. djamor (Rumph. ex Fr.) Boedijn P. djamor (Rumph. ex Fr.) Boedijn P. eryngii (De Cand.) Qul. P. eryngii (De Cand.) Qul. P. ferulae (Lanzi) X. L. Mao P. ferulae (Lanzi) X. L. Mao P. nebrodensis (Inzenga) Qul. P. ostreatus (Jacq.) P. Kumm. P. ostreatus (Jacq.) P. Kumm. P. ostreatus (Jacq.) P. Kumm. P. ostreatus (Jacq.) P. Kumm. P. ostreatus (Jacq.) P. Kumm. P. ostreatus (Jacq.) P. Kumm. P. ostreatus (Jacq.) P. Kumm. P. ostreatus (Jacq.) P. Kumm. P. pulmonarius (Fr.) Qul. P. pulmonarius (Fr.) Qul.

IUM1307 IUM2652 IUM1309 IUM1794 IUM3705 IUM1659 IUM3568 IUM0556 IUM1635 IUM3511 IUM1306 IUM1313 IUM1320 IUM1376 IUM2022 IUM2131 IUM3527 IUM3573 IUM1271 IUM2362

Taiwan China Taiwan Korea Korea Korea China China China China Taiwan Korea Taiwan Korea China Taiwan China China Korea Korea

All GenBank accession numbers belong to submission ID BankIt1372390 (NCBI). IUM: Incheon University Mushroom.

DNA stock solution was added to 780 l of SDDW (sterilized double distilled water), and then 800 l of DNA mixture was taken into the cuvette, and the concentration was measured at 260 nm and 280 nm. For control, concentration of 800 l SDDW was measured. Finally, exact concentration of DNA solution was determined4. Polymerase chain reaction (PCR). The DNA of all samples were amplified by PCR (PTC-100TM, MJ Research Inc., U.S.A.)

using universal primers ITS1 forward (5-TCCGTAGGTGAACCTGCG-3) and ITS4 reverse (5- TCCTCCGCTTATTGATATGC-3). Amplification reactions were performed in a total volume of 20 l containing 10x PCR buffer 2 l, dNTP mix 1.6 l, 0.5 l of each primer, 0.2 l of Taq polymerase (Cosmo, Korea), 1 l of genomic DNA, and 14.2 l of sterilized distilled water. PCR amplification was carried out in 30 cycles at 94 C for 30 s denaturing,

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quences (P. ostreatus, strain CGMCC23, accession no.: EF514247, China) revealed up to 90% homology at the 3 end of the 18S gene (bases 1 to 52); 80% homology with the 5.8S gene (bases 214 to 383); and up to 70% homology with the 5 end of the 28S gene (bases 526 to 600). Among our studied sequences, maximum similarity was found between the bp 400 to 600, and similarity was gradually less going from either 5 or 3 end. Furthermore, some minor deletions and insertions were found in different locations compared to the published sequence. In the range of 201-350 bp and 651-800 bp, there were a few deletions occurring in strains IUM3568 (201-234 bp), IUM2652 and IUM1307 (253-260 bp). Simultaneously, there were some insertions also found for the strains IUM2652, IUM1307, IUM3705, and IUM1794 (211-213 bp). For the range of 351-650 bp, no insertion or deletion was found. Although several minor insertions or deletions were found during sequences alignment, it was possible to edit sequences manually and reconstruct a nearly complete sequence for each strain. Details about the statistics of nucleotide sequences are given in Table 2 and Fig. 1. There are many mushroom species distributed worldwide; they may be quite recent or ancient species with diverse biological relationships. Evidences from molecular systematics help to understand these patterns. Vilgalys and Sun19 studied mating compatibility relationships among Pleurotus mushrooms collected from different parts of the world and found at least eight intersterility groups and gene phylogenies for two different regions of the nuclear rDNA locus representing 38 individuals. Their results demonstrated the utility of rDNA phylogenies for understanding patterns of relationship, distribution, evolution and speciation in basidiomycete fungi.

51 C for 30 s annealing and 72 C for 1 min extension. Initial denaturing at 95 C was extended to 5 min and the final extension was at 72 C for 10 min. Gel electrophoresis and sequencing. Amplified PCR products were separated by gel electrophoresis containing 1.5% (w/v) agarose (Blue marine 200, Serva Electrophoresis). The electrophoresis was run in 1x TAE buffer and the amplified products were visualized by ethidium bromide staining under UV light. The length of amplified products was estimated by comparing to DNA size marker. The PCR product was added to 100 l of direct purification buffer in Eppendorf tube, and purified using the Wizard PCR Preps DNA purification system. The sequencing was done by SolGent Co., Ltd., Daejeon 350-380, Korea. Analysis of DNA sequences. To make DNA sequences, two universal primers (ITS1, ITS4) were used. Analysis of sequences was performed with the basic sequence alignment BLAST program run against the NCBI database (www.ncbi.nlm. nih.gov). Sequence alignment and preparation of the phylogenetic tree were carried out using CLC sequence viewer software. Regions showing ambiguous alignments were removed from the analysis. RESULTS AND DISCUSSION Characterization of DNA sequences and their alignment. PCR products of the ITS region (ITS1 + 5.8S + ITS2) amplified with primers ITS1 and ITS4 were visualized as a single band in agarose gels. The size of the PCR fragments was about 600-800 bp (ITS1, 200-230 bp; 5.8S rRNA gene, 120150 bp; ITS2, 280-320 bp) in length for all taxa (data not shown). Sequence alignment compared to previously published se-

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Table 2. Statistics of nucleotide sequences of the ITS region from Pleurotus strains studied. Item
1307 Length (nucleotide) 545 Weight (kDa) 175.3 1 C+G 256 1 A+T 289 2 C+G 0.47 2 A+T 0.53 1306 Length (nuc) Weight (kDa) 1 C+G 1 A+T 2 C+G 2 A+T 555 178.4 256 299 0.461 0.539 2652 507 163.1 231 276 0.456 0.544 1313 600 192.9 265 335 0.442 0.558

Incheon University Mushroom (IUM) strain code

1309 545 175.2 250 295 0.459 0.541 1320 535 171.9 236 299 0.441 0.559 1794 625 201.5 252 373 0.403 0.597 1376 563 181.2 256 307 0.455 0.545 3705 616 197.7 251 365 0.407 0.593 2022 587 189.0 266 321 0.453 0.547 1659 599 192.6 267 332 0.446 0.554 2131 591 190.0 259 332 0.438 0.562 3568 473 152.1 206 267 0.436 0.564 3527 584 187.6 275 309 0.471 0.529 0556 607 195.4 273 334 0.45 0.55 3573 592 190.4 259 333 0.438 0.562 1635 591 190.1 265 326 0.448 0.552 1271 596 192.2 264 332 0.443 0.557 3511 607 195.1 269 338 0.443 0.557 2362 512 164.8 227 285 0.443 0.557

Count of cytosine-guanine (1C + G) and adenine-thymine (1A + T). Frequency of cytosine-guanine (2C + G) and adenine-thymine (2A + T). Frequency was calculated as (C+G) or (A+T) / {(A+T) + (C+G)}.

It is worth mentioning that the 5.8S gene has a slow rate of evolutionary change, but the level of sequence dissimilarity of the spacers is high11 and they can be used to conclude phylogenetic relationships from populations to families and even higher taxonomic levels3,7,20. As members of a sequence family, the multiple copies of the ITS do not progress independently. They tend to change in a concerted fashion, which means that in a species the repeats evolve together, maintaining high similarities among themselves, as they diverge from repeats in other species2,5. Unequal crossing-over and gene conversion are the prominent mechanisms responsible

for the homogenization of sequences. Nevertheless, variation among repeats within genomes has been documented in a range of taxa6,8,9,15,16,22, showing that the level of intra-individual variation should be considered to interpret ITS information accurately. Phylogenetic analysis. From the phylogenetic tree (Fig. 2), seven sister pairs, including the reference strain, were found with four types of homologies. Among them, four branches in different clades showed high homology (100%), while the remaining three sister pairs showed low bootstrap value or less homology (56%, 52%, 50%). Results suggested that the distribution of

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Fig. 1. Alignment of sequences from the ITS region of different Pleurotus species studied.

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Pleurotus sPecies from eastern asia

Fig. 1 (continued)

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Fig. 2. Phylogenetic tree generated by neighbor-joining analysis (standard, bootstrap replicates: 100) of ITS sequences from different Pleurotus species. The reference strain was: P. ostreatus, CGMCC, accession no.: EF514247. C: China, K: Korea, T: Taiwan.

species studied took place gradually from IUM3573 (China), IUM2131 (Taiwan), and IUM1313 (Korea) giving rise to the other branches, as these strains are placed near the root node of phylogenetic tree. Strains belonging to Pleurotus ostreatus showed differing homologies, although they are geographically distributed. Further studies are needed to know if these eight strains are all interbreedable. From the sample studied, it could also be thought that the Chinese and Taiwanese strains IUM3573 and IUM2131, directly linked to the root node, are close to the most recent origin or ancestor from which distribution took place.

Huerta et al.12 studied the genetic relationships among 25 Mexican strains of Pleurotus species analyzing the ITS region from rDNA. They discussed that most of the sequences were clearly separated from reference strains of European and North American origin in the consensus tree. In this study, the phylogenetic analysis revealed that Pleurotus strains collected from different ecological environments may have a little genetic variation in case of differing species. Some strains belonging to the same species showed 100% similarities, even those collected from unlike environments, indicating that the strains

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