Korea, consisting of approximately 32% of the entire
J. Microbiol. Biotechnol. (2009), 19(2), 000–000
First published online 25 February 2009
Development and Characterization of New Microsatellite Markers for the
Oyster Mushroom (Pleurotus ostreatus)
Ma, Kyung-Ho1, Gi-An Lee1, Sok-Young Lee1, Jae-Gyun Gwag1, Tae-San Kim1, Won-Sik Kong2,
Kyoung-In Seo2, Gang-Seob Lee1, and Yong-Jin Park3*
1Genetic Resources Division, National Institute of Agricultural Biotechnology, Suwon 441-707, Republic of Korea
2Applied Microbiology Division, National Institute of Agricultural Science and Technology, Suwon 441-707, Republic of Korea
3Department of Plant Resoures, College of Industrial Science, National Kongju University, Yesan 340-802, Republic of Korea
Received: November 4, 2008 / Accepted: February 3, 2009
We developed and characterized 36 polymorphic microsatellite
markers for the oyster mushroom (Pleurotus ostreatus). In
total, 169 alleles were identified with an average of 4.7
alleles per locus. Values for observed (HO) and expected
(HE) heterozygosities ranged from 0.027 to 0.946 and from
0.027 to 0.810, respectively. Nineteen loci deviated from
Hardy-Weinberg equilibrium. Significant (P<0.05) excess
heterozygosity was observed at nine loci. Linkage
disequilibrium (LD) was significant (P<0.05) between pairs
of locus alleles. Cluster analysis revealed that five species
of genus Pleurotus made a distinct group, and the individual
cultivars were grouped into five major groups from G-1 to
G-5. The diverse cultivars of P. ostreatus were discriminated
and the other four species revealed a different section in
the UPGMA tree. These microsatellite markers proved to
be very useful tools for genetic studies, including assessment
of the diversity and population structure of P. ostreatus.
Keywords: Oyster mushroom, genetic diversity, heterozygosity,
microsatellite markers, Pleurotus ostreatus
The genus Pleurotus is a cosmopolitan group, including
several cultivated species such as P. ostreatus, P. cornucopiae,
P. sajor-caju, P. eryngiim, P. cystidiosus, and P. pulmonarius
. The oyster mushroom (Pleurotus ostreatus) is commercially
important in the world mushroom market, particularly in
East Asia. It is the most popular edible mushroom in
mushroom production in Korea . Besides its importance
for food production, P. ostreatus is important in applications
such as paper pulp bleaching, cosmetics, and other potential
industrial uses [1, 3, 14, 15]. Pleurotus ostreatus also plays
a role in increasing macrophage and lymphocyte activities
, reducing cholesterol levels , enhancing the anti-
complementary properties of polysaccharides , and
increasing antihepatoma and antisarcoma activities .
These applications have stimulated research on specific
aspects of the molecular biology of the organism.
Cultivars of the oyster mushroom P. ostreatus are readily
affected by environmental conditions, making them difficult
to differentiate. Disputes between farmers and spawn suppliers
related to cultivated strains are becoming more frequent.
As Pleurotus is the most commonly cultivated edible
mushroom, and its consumption is continuously increasing,
many attempts have been made to standardize the distribution
of various Pleurotus cultivars for mushroom farming [5,10,11].
Identical strains with different commercial names or different
strains with the same name often occur in the cultivation
and spawn market. Incorrectly designated strains can result
in huge economic losses for farmers. Therefore, precise
identification and classification of commercial lines of
edible Pleurotus spp. strains are of major importance. The
aim of this work was to develop a rapid and accurate strain
discrimination system for commercial Pleurotus strains
using simple sequence repeat (SSR) markers.
A microsatellite-enriched library was constructed using
a modification of the biotin-streptavidin capture method of
Dixit et al. . Briefly, total genomic DNA of oyster
mushroom was digested with seven restriction enzymes
(EcoRV, DraI, SmaI, PvuI, AluI, HaeIII, and RsaI) in
separate reactions. The pooled digest was size-fractionated
on a 1.5% agarose gel. Fragments ranging from 300 to
1,500 bp were eluted from the gel followed by purification
using a gel extraction kit (QIAGEN). DNA fragments
(1 µg) were ligated with 1 µg of double-stranded adaptor
molecules (AP11-5'-CTCTTGCTTAGATCTGGACTA-3' and
adaptor-ligated DNA was hybridized with a mixture of biotin-
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312535 P. ostreatusByeongneutari1
P. ostreatus Heukjinju
2 Ma et al.
labeled SSR probes [(GA)20, (AGC)15, (GGC)15, (AAG)15,
(AAC)15, (AGG)15]. The hybridized DNA fragments were
captured with streptavidin-coated magnetic beads (Promega).
After stringent washing, the captured DNA fragments
were eluted in 50 ml of distilled water. Final eluates were
amplified with AP11 primers and cloned into a pGEM-T
easy vector (Promega). In total, 638 recombinant clones
were randomly picked from primary transformation plates
containing ampicillin (100 µg/ml), X-gal (2 mg), and IPTG
(8 µM). Plasmid DNA was isolated using a QIAprep Spin
Miniprep Kit (QIAGEN) and sequenced using an ABI
3100 DNA sequencer with a BigDye terminator kit (Applied
SSR identification within cloned sequences and primer
design were carried out using the SSR MANAGER program
. Among 638 sequenced clones, 144 (22.6%) were
redundant. Of the remaining 494 unique clones, 201
(40.7%) were suitable for the design of SSR primer pairs
for microsatellite sequences, whereas the other clones’
SSR motif leaned to the end side of the sequence, which
made it difficult to design primer sets. In total, 201 primer
pairs were designed and evaluated for polymorphism
Table 1. Thirty-seven strains of genus Pleurotus used in the study.
No. ASI No. SpeciesCommercial Name SourceYear
1 2830P. ostreatusBaekdu1 Korea 2005
2 2001 P. ostreatusNonggi2-1 Korea (MKACC52243) 1971
3 2072 P. ostreatusNonggi202Korea (MKACC51410)1980
4 2180 P. ostreatusWonhyeong Korea (MKACC51493) 1990
5 2183P. ostreatusWonhyeong2 Korea (MKACC51496)1990
6 2240 P. ostreatus Wonhyeong3Korea (MKACC52342) 1994
7 2706 P. ostreatusHeukpyeong Korea (MKACC52328) 2001
8 2506 P. ostreatus Gyunhyeop1 Korea (KACC360)2000
9 2488 P. ostreatusMyeongweol Korea (MKACC51732) 1999
102549 P. ostreatusSinnong94Korea (MKACC52282) 2000
11 2595 P. ostreatus Suhan2 Korea (MKACC51818)2001
12 2593P. ostreatus JanganPKKorea (MKACC52326)2001
13 2707P. ostreatus Kimjae9 Korea (MKACC52311) 2001
142708 P. ostreatusKimjae10Korea (MKACC52312) 2001
152710P. ostreatus Heungrim1 Korea (MKACC52314)2001
16 2722 P. ostreatusJangan6 Korea (MKACC52324)2002
172724 P. ostreatus Nongong99Korea2003
18 2727 P. ostreatusSinnong12Korea2003
202790 P. ostreatus SamguPJKorea 2004
21 2793P. ostreatus Hanra2 Korea2004
232851P. ostreatus Nongmin59 Korea 2006
242228 P. ostreatusChunchu1 China (MKACC51529)1994
252344P. ostreatus Chunchu2 Netherland (MKACC51632)1995
262333 P. sajor-cajuYeoreum2 Korea (MKACC51621)1995
272394 P. eryngiiKeunneutari3 Japan (MKACC52327)1997
282720 P. nebrodensisBaeksongi Korea (MKACC52323)2002
292825P. ostreatus Sinnong14 Korea2005
302018 P. ostreatusNonggi201Korea (MKACC51362) 1978
332594 P. ostreatusIlseong2 Korea (MKACC51817)2001
342721 P. ostreatusJangan5 Korea2002
352728 P. ostreatusSinnong13 Korea2003
362791 P. ostreatusSamgu01 Korea2004
37 2070P. sajor-caju Yeoreum India (MKACC52247)1982
ASI, Agricultural Sciences Institute, Suwon, Korea; KACC, Korea Agricultural Culture Collection; MKACC, Mushroom Korea Agricultural Culture Collection;
Year, Collected year.
NEWLY DEVELOPED POLYMORPHIC MICROSATELLITE MARKERS OF OYSTER MUSHROOM
against a panel of 10 oyster mushroom samples using a
procedure described earlier . Thirty-six primer pairs
produced reproducible polymorphic bands and were further
characterized using a diverse set of 20 accessions. The
M13-tail PCR method of Schuelke was used to measure
the size of PCR products . The method involves three
primers; the forward SSR-specific primer with the M13
(5'-TGTAAAACGACGGCCAGT-3') tail at the 5' end, the
reverse SSR-specific primer, and a phosphoramidite fluorescent
dye-labeled (FAM, HEX, or NED) M13 (5'-TGTAAAACG
ACGGCCAGT-3') universal primer. The amount of forward
primer was adjusted to less than half of the reverse primer.
Microsatellite alleles were resolved on an ABI PRISM 3100
DNA sequencer (Applied Biosystems) using GENESCAN 3.7
software and sized using GeneScan 500 ROX (6-carbon-X-
rhodamine) molecular size standards (35 to 500bp) with
GENOTYPER 3.7 software (Applied Biosystems) (Fig. 1).
A total of 37 cultivars belonging to genus Pleurotus,
collected from the Agricultural Sciences Institute, were
chosen for variablilty test and cluster analysis (Table 1).
The variability at each locus was measured in terms of
the number of alleles, observed heterozygosity (HO), and
expected heterozygosity (HE), using the genetic analysis
Fig. 2. UPGMA dendrogram showing phylogenetic relationships among different oyster mushroom (Pleurotus ostreatus) including other
Fig. 1. An example of the M13-tail PCR method.
The PCR products were resolved on an ABI PRISM 3130 DNA sequencer.
Ma et al.
Table 2. Characteristics of 36 microsatellite loci developed from an enriched library of oyster mushroom (Pleurotus ostreatus).
GB-PO-001 EU502619 CGCAAGCTACAAACGGAC AGCAGCAAGCACAAGAGC (GCA)3(ATTGGC)(GCA)1
522 307-313 0.182 0.298 0.402
GB-PO-006 EU502620 TGTGGCAAACCCAAGTTC CCCAAAGGATGAGGAAGG (GGC)4(GAC)(GGC)1
523 203-227 0.324 0.517 0.387
GB-PO-011aEU502622 TCCCATACCCTGACATCG ATCATCAAGCGCCACAAC(CTG)4(TA)(CTG)1
527 166-262 0.265 0.783 0.670
GB-PO-025 EU502624 TGATCATGGCGAGTAGGGGGAACTGTCAGCAGACGC (GGA)10
523 211-217 0.314 0.269 -0.154
GB-PO-026 EU502625 AATCGCATGGGCTCTGCTGTCCCTCCGTGTACCA(TTG)3(TTC)(TTG)3
522 293-296 0.027 0.027 0.000
GB-PO-028aEU502626 CTGGAGAATCGTAGCCCC ACAAGCGCTCGGAATACA (GTC)13(CAT)5
528 285-327 0.622 0.805 0.240
GB-PO-039 EU502627 TGTGGATGTGATGTGATGTG ACGTCCAGCGTCGAGTTA(GGT)2(GGC)(GGT)5
525 191-236 0.568 0.594 0.058
GB-PO-050aEU502628 CATCCGATACAGACCCGA AGGCATCCCACAACACTG(GTT)5
528 145-175 0.351 0.810 0.576
GB-PO-051aEU502629 CATAGGGACGACAGCGAG ACTGAGCCTTCAGCACCA (GCT)6
524 269-278 0.568 0.653 0.144
GB-PO-061aEU502631 TAACTTGGGCGCTTGAAATGGAACGCGTAGACTTGG (GCA)2(CAGTAC)(GCA)3
523 252-270 0.206 0.355 0.432
GB-PO-064aEU502632 GTTCTGAGGGTTGAGGGG CCAACCACACTCTTCCCA(GTT)5(ATT)(GTT)3
525 209-230 0.086 0.236 0.645
GB-PO-076 EU502633 TCGATTGTCAGATTGTTGGA CGGAGAAGCAGTTGGTTG (GGC)6
525 233-251 0.405 0.663 0.400
GB-PO-079aEU502634 ACCCAGACGATTTGGGAG AGGCTGGCGTGGAATACT (GGA)4(AGA)(GGA)2
529 221-329 0.541 0.739 0.281
GB-PO-080 EU502635 CACCCATGTGCCTCAGTC TGTCTATGGGTTACGGCG (GGC)6
522 230-233 0.324 0.394 0.191
524 376-446 0.081 0.242 0.673
524303-324 0.243 0.349 0.316
525 241-253 0.486 0.679 0.296
GB-PO-102aEU502638 TGTCTATGGGTTACGGCGTGCAAAGCAAATCGGAAC (GCC)4(GC)(GCC)1
522266-269 0.222 0.494 0.560
GB-PO-113aEU502639 GTTCATCTGAACGCCGTCCCTATGACGAGGGGAAGG (CGC)5
522 270-375 0.108 0.339 0.688
GB-PO-115 EU502641 TGGTAGCAGGTTGTTGGGCCGCTAAGCCACTGTTTG (TGC)5(GCTGGC)(TGC)4
526 213-234 0.811 0.665 -0.205
GB-PO-117EU502642 TCAAACTCACGTGGTACGC TCACATATCCGCCGGTAG (TGC)7
524 218-227 0.622 0.623 0.017
GB-PO-124 EU502643 TGCGTTTGCTCGGTTAAT CGCTACTACGTCGATCCG(CG)5
528264-300 0.919 0.732 -0.243
aLoci deviated from the Hardy-Weinberg equilibrium (HWE); TA, annealing temperature; NA, number of alleles; HO, observed heterozygosity; HE, expected heterozygosity; FIS, Wright’s fixation index.
NEWLY DEVELOPED POLYMORPHIC MICROSATELLITE MARKERS OF OYSTER MUSHROOM
Table 2. Continued
GB-PO-128 EU502644 TGATTGGTTTGAATGGGC GCACGATGAGGATGCAGT (GTT)10
52 11 214-247 0.568 0.801 0.304
GB-PO-131 EU502645 CTCCCTCCTCCGTGTACCCGTAACGTTCGCTTCCTG (CCT)2(CCCC)(CCT)2
523 200-212 0.730 0.528 -0.371
GB-PO-134 EU502647 GAGTGTGAAGAATCGGCG GTGCACTCTGCCTATCGC(GA)2(GT)(GA)3
523 115-285 0.757 0.561 -0.337
GB-PO-135 EU502646 AGGAGGGGGTGCTTGATATCCTCCGCCTTCTCTACC(GGA)2(GGGA)(GGA)2
523 217-271 0.162 0.151 -0.061
GB-PO-138aEU502648 TATGGAACGGTGCGAAGTGCCGTCAAAAGGGAACTC (CCG)4, (TTC)3
524 191-209 0.946 0.723 -0.296
527 193-277 0.297 0.746 0.610
GB-PO-152 EU502650 ACTGAGCCTTCAGCACCACATAGGGACGACAGCGAG (AGC)5
524 272-395 0.500 0.615 0.201
525 254-269 0.649 0.698 0.084
GB-PO-157aEU502652 ATGGACGTGGTGTTCTGCAAACCAAGCCTACCCAGC (GCT)4
526 281-305 0.514 0.656 0.231
GB-PO-171aEU502654 TCTCGGGCATCATTCTTGACGTCAGGGTGTCAAACG(TTG)3, (TA)4, (TA)4
524 295-310 0.194 0.674 0.718
GB-PO-172 EU502655 GCAGAAGTTGCCCAAAGA ATGTCCAGCGGAAGACCT(TGC)2(TAC)(TGC)5
523 169-229 0.306 0.422 0.290
GB-PO-173aEU502656 ATGAAGTGTGAGCCGTGGTGTCCATTCATGCGTTCA (GT)8
525 294-309 0.114 0.748 0.851
GB-PO-181aEU502657 TTATTGTGAAGCCCCCG GACATCGGCAGAAGGTCA(CAG)1(CAA)(CAG)5
524 240-249 0.054 0.595 0.911
GB-PO-190 EU502658 TTTCCATTTCCGTTGGTGCAGGGGGTGATTATGCAA (TGC)3(TGT)(TGC)2
526 186-222 0.278 0.579 0.531
Mean 4.70.398 0.549
SD 2.160.25 0.21
ACCEPTED Download full-text
3. Curreli, N., F. Sollai, L. Massa, O. Comandini, A. Rufo, E.
6 Ma et al.
package POPGENE Version 1.31 . The same program
was used to test Hardy-Weinberg equilibrium (HWE) and
pair-wise linkage disequilibrium (LD). In total, 169 alleles
were detected with an average of 4.7 alleles per locus.
Values for HO and HE ranged from 0.027 to 0.946
(mean=0.398) and from 0.027 to 0.810 (mean=0.549),
respectively (Table 2). Nineteen loci deviated from HWE
(P<0.001). The analysis also revealed significant (P<0.05)
excess heterozygosity for nine loci. LD was significant
(P<0.05) between pairs of locus alleles. Cluster analysis
was performed in PowerMarker version 3.23 software with
the SharedAllele method to calculate genetic distance and
was checked for discriminative power of the microsatellite
markers . The individual cultivars were grouped to
mainly five groups from G-1 to G-5. All four cultivars in
G-1 were P. ostreatus, and G-2 included one distinct
cultivar of P. sajor-caju. G-3 included 12 cultivars; one P.
eryngii and 11 P. ostreatus. G-4 included 14 cultivars; one
P. cornucopiae and 13 P. ostreatus. G-5 included seven
cultivars; one P. nebrodensis, one P. sajor-caju, and five
P. ostreatus (Fig. 2). The diverse cultivars of P. ostreatus
were discriminated, and the other four species revealed a
different section in the UPGMA tree. The microsatellite
markers reported here provide very useful tools for several
genetic studies, including assessment of the diversity and
population structure of P. ostreatus.
This study was supported by the Biogreen 21 project
(No.20080401034058) of the Rural Development
Administration (RDA) and a grant (No.200803101010290)
from the National Institute of Agricultural Biotechnology,
RDA, Republic of Korea.
1. Bezalel, L., Y. Hadar, and C. E. Cerniglia. 1997. Enzymatic
mechanisms involved in phenanthrene degradation by the white
rot fungus Pleurotus ostreatus. Appl. Environ. Microbiol. 63:
2. Bobek, P., S. Galbavy, and L. Ozdin. 1998. Effect of oyster
mushroom (Pleurotus ostreatus) on pathological changes in
dimethylhydrazine-induced rat colon cancer. Oncol. Rep. 5:
Sanjust, A. Rinaldi, and A. C. Rinaldi. 2001. Effects of plant-
derived naphthoquinones on the growth of Pleurotus sajor-caju
and degradation of the compounds by fungal cultures. J. Basic
Microbiol. 41: 253-259.
4. Dixit, A., M. H. Jin, J. W. Chung, J. W. Yu, H. K. Chung, K. H.
Ma, Y. J. Park, and E. G. Cho. 2005. Development of polymorphic
microsatellite markers in sesame (Sesamum indicum L.). Mol.
Ecol. Notes 5: 736-738.
5. Gonzalez, P. and J. Labarere. 2000. Phylogenetic relationships
of Pleurotus species according to the sequence and secondary
structure of the mitochondrial small-subunit rRNA V4, V6 and
V9 domains. Microbiology 146: 209-221.
6. Kim, K. Y. 2004. Developing one-step program (SSR MANAGER)
for rapid identification of clones with SSRs and primer designing.
Thesis (MSc), Department of Plant Science, the Graduate School
of Seoul National University, Seoul, Republic of Korea.
7. Kurashige, S., Y. Akuzawa, and F. Endo. 1997. Effects of Lentinus
edodes, Grifola frondosa and Pleurotus ostreatus administration
on cancer outbreak, and activities of macrophages and lymphocytes
in mice treated with a carcinogen, N-butyl-N-butanolnitrosoamine.
Immunopharmacol. Immunotoxicol. 19: 175-183.
8. Kweon, M. H., H. Jang, W. J. Lim, H. I. Chang, C. W. Kim, H. C.
Yang, H. J. Hwang, and H. C. Sung. 1999. Anti-complementary
properties of polysaccharides isolated from fruit bodies of
mushroom Pleurotus ostreatus. J. Microbiol. Biotechnol. 9: 450-
9. Liu, K. and S. V. Muse. 2005. PowerMarker: An integrated
analysis environment for genetic marker analysis. Bioinformatics
10. Matsumoto, T. and Y. Fukumasa-Nakai. 1995. Mitochondrial
DNA restriction fragment length polymorphisms and phenetic
relationships in natural populations of the oyster mushroom
Pleurotus ostreatus. Mycol. Res. 99: 562-566.
11. Matsumoto, T., K. Mimura, and Y. Fukumasa-Nakai. 1995. Isozyme
variation and genetic relatedness among natural populations of
Pleurotus ostreatus. J. Gen. Appl. Microbiol. 41: 487-497.
12. Ministry of Agriculture and Forestry. 2007. An Actual Yield of
Cash Crops in 2006. Ministry of Agriculture and Forestry,
Seoul, The Republic of Korea.
13. Schuelke, M. 2000. An economic method for the fluorescent
labeling of PCR fragments. Nat. Biotechnol. 18: 233-234.
14. Shin, K., I. Oh, and C. Kim. 1997. Production and purification
of remazol brilliant blue R decolorizing peroxidase from the
culture filtrate of Pleurotus ostreatus. Appl. Environ. Microbiol.
15. Sigoillot, C., S. Camarero, T. Vidal, E. Record, M. Asther, M.
Perez-Boada, et al. 2005. Comparison of different fungal enzymes
for bleaching high-quality paper pulps. J. Biotechnol. 115: 333-
16. Yeh, F. C., R. C. Yang, and T. Boyle. 1999. POPGENE Version
1.31. Microsoft Windows-based freeware for population genetic
analysis. University of Alberta and Centre for International
Forestry Research, Edmonton, AB, Canada.
17. Wang, H., J. Gao, and T. B. Ng. 2000. A new lectin with highly
potent antihepatoma and antisarcoma activities from the oyster
mushroom Pleurotus ostreatus. Biochem. Biophys. Res. Commun.