Development and characterization of new microsatellite markers for the oyster mushroom (Pleurotus ostreatus).
ABSTRACT 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 major five 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.
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ABSTRACT: In this study, microsatellite markers were utilized to reveal the genetic diversity of 56 strains of Lentinula edodes grown in China. A total of 224 DNA bands were detected through 25 primer pairs, of which, 223 bands (99.6%) were polymorphic between two or more strains. The variation in SSR (Simple Sequence Repeat) DNA band patterns and average genetic similarity among the wild strains of L. edodes obtained from the same region uncovered a vast genetic diversity in the natural germplasm found in China. Compared with L. edodes strains from other areas, the genetic diversity of those from the Yunnan Plateau, Hengduanshan mountains, Taiwan, and south China was significantly greater. Based on cluster analysis and principal coordinate analysis, the results indicated that all L. edodes strains could be divided into three major groups. These results effectively displayed the differences between the strains from North and South China, and those from the same or adjoining regions could cluster preferentially into small groups in most cases, suggesting the positive correlation between the cluster results and the geographical origin for the natural germplasm of Chinese L. edodes. To our knowledge, this is a pioneering report on the utilization of the SSR marker technique in analyzing L. edodes’ genetic diversity. KeywordsShiitake mushroom-Microsatellite-DNA polymorphism-UPGMA-PCoAWorld Journal of Microbiology and Biotechnology 26(3):527-536. · 1.26 Impact Factor
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ABSTRACT: Identification of edible mushrooms particularly Pleurotus genus has been restricted due to various obstacles. The present study attempted to use the combination of two variable regions of IGS1 and ITS for classifying the economically cultivated Pleurotus species. Integration of the two regions proved a high ability that not only could clearly distinguish the species but also served sufficient intraspecies variation. Phylogenetic tree (IGS1 + ITS) showed seven distinct clades, each clade belonging to a separate species group. Moreover, the species differentiation was tested by AMOVA and the results were reconfirmed by presenting appropriate amounts of divergence (91.82% among and 8.18% within the species). In spite of achieving a proper classification of species by combination of IGS1 and ITS sequences, the phylogenetic tree showed the misclassification of the species of P. nebrodensis and P. eryngii var. ferulae with other strains of P. eryngii. However, the constructed median joining (MJ) network could not only differentiate between these species but also offer a profound perception of the species' evolutionary process. Eventually, due to the sufficient variation among and within species, distinct sequences, simple amplification, and location between ideal conserved ribosomal genes, the integration of IGS1 and ITS sequences is recommended as a desirable DNA barcode.The Scientific World Journal 01/2014; 2014:793414. · 1.73 Impact Factor
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ABSTRACT: Identification of edible mushrooms particularly Pleurotus genus has been restricted due to various obstacles. The present study attempted to use the combination of two variable regions of IGS1 and ITS for classifying the economically cultivated Pleurotus species. Integration of the two regions proved a high ability that not only could clearly distinguish the species but also served sufficient intraspecies variation. Phylogenetic tree (IGS1 + ITS) showed seven distinct clades, each clade belonging to a separate species group. Moreover, the species differentiation was tested by AMOVA and the results were reconfirmed by presenting appropriate amounts of divergence (91.82% among and 8.18% within the species). In spite of achieving a proper classification of species by combination of IGS1 and ITS sequences, the phylogenetic tree showed the misclassification of the species of P. nebrodensis and P. eryngii var. ferulae with other strains of P. eryngii. However, the constructed median joining (MJ) network could not only differentiate between these species but also offer a profound perception of the species' evolutionary process. Eventually, due to the sufficient variation among and within species, distinct sequences, simple amplification, and location between ideal conserved ribosomal genes, the integration of IGS1 and ITS sequences is recommended as a desirable DNA barcode.
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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2Ma 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.Species Commercial NameSourceYear
1 2830P. ostreatus Baekdu1 Korea2005
22001P. ostreatusNonggi2-1 Korea (MKACC52243)1971
32072P. ostreatus Nonggi202 Korea (MKACC51410)1980
42180P. ostreatusWonhyeongKorea (MKACC51493) 1990
5 2183P. ostreatus Wonhyeong2Korea (MKACC51496)1990
62240 P. ostreatus Wonhyeong3Korea (MKACC52342) 1994
72706P. ostreatus HeukpyeongKorea (MKACC52328) 2001
82506P. ostreatusGyunhyeop1Korea (KACC360) 2000
92488 P. ostreatusMyeongweolKorea (MKACC51732)1999
102549P. ostreatusSinnong94 Korea (MKACC52282)2000
112595P. ostreatusSuhan2Korea (MKACC51818)2001
122593P. ostreatusJanganPKKorea (MKACC52326)2001
132707P. ostreatusKimjae9 Korea (MKACC52311)2001
142708P. ostreatusKimjae10Korea (MKACC52312)2001
152710P. ostreatusHeungrim1Korea (MKACC52314)2001
162722P. ostreatusJangan6Korea (MKACC52324)2002
17 2724P. ostreatusNongong99Korea2003
242228P. ostreatusChunchu1China (MKACC51529)1994
252344P. ostreatus Chunchu2Netherland (MKACC51632)1995
26 2333P. sajor-cajuYeoreum2Korea (MKACC51621)1995
27 2394P. eryngiiKeunneutari3Japan (MKACC52327)1997
282720P. nebrodensisBaeksongiKorea (MKACC52323)2002
302018P. ostreatusNonggi201 Korea (MKACC51362)1978
332594 P. ostreatusIlseong2Korea (MKACC51817) 2001
342721 P. ostreatusJangan5 Korea 2002
35 2728P. ostreatus Sinnong13Korea 2003
36 2791 P. ostreatusSamgu01Korea 2004
372070P. sajor-cajuYeoreumIndia (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
52 2307-313 0.182 0.298 0.402
GB-PO-006 EU502620 TGTGGCAAACCCAAGTTCCCCAAAGGATGAGGAAGG (GGC)4(GAC)(GGC)1
523203-227 0.324 0.517 0.387
527166-262 0.265 0.783 0.670
GB-PO-025 EU502624 TGATCATGGCGAGTAGGG GGAACTGTCAGCAGACGC (GGA)10
523 211-217 0.314 0.269 -0.154
GB-PO-026 EU502625 AATCGCATGGGCTCTGCTGTCCCTCCGTGTACCA (TTG)3(TTC)(TTG)3
522293-296 0.027 0.027 0.000
GB-PO-028aEU502626 CTGGAGAATCGTAGCCCCACAAGCGCTCGGAATACA (GTC)13(CAT)5
528285-327 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 CATCCGATACAGACCCGAAGGCATCCCACAACACTG (GTT)5
528145-175 0.351 0.810 0.576
GB-PO-051aEU502629 CATAGGGACGACAGCGAG ACTGAGCCTTCAGCACCA(GCT)6
524269-278 0.568 0.653 0.144
GB-PO-061aEU502631 TAACTTGGGCGCTTGAAA TGGAACGCGTAGACTTGG(GCA)2(CAGTAC)(GCA)3
523252-270 0.206 0.355 0.432
525209-230 0.086 0.236 0.645
GB-PO-076 EU502633 TCGATTGTCAGATTGTTGGA CGGAGAAGCAGTTGGTTG(GGC)6
525233-251 0.405 0.663 0.400
529221-329 0.541 0.739 0.281
GB-PO-080 EU502635 CACCCATGTGCCTCAGTCTGTCTATGGGTTACGGCG(GGC)6
522230-233 0.324 0.394 0.191
524 376-446 0.081 0.242 0.673
524303-324 0.243 0.349 0.316
525241-253 0.486 0.679 0.296
GB-PO-102aEU502638 TGTCTATGGGTTACGGCG TGCAAAGCAAATCGGAAC (GCC)4(GC)(GCC)1
522 266-269 0.222 0.494 0.560
GB-PO-113aEU502639 GTTCATCTGAACGCCGTCCCTATGACGAGGGGAAGG (CGC)5
522270-375 0.108 0.339 0.688
GB-PO-115EU502641 TGGTAGCAGGTTGTTGGG CCGCTAAGCCACTGTTTG (TGC)5(GCTGGC)(TGC)4
526213-234 0.811 0.665 -0.205
GB-PO-117EU502642 TCAAACTCACGTGGTACGC TCACATATCCGCCGGTAG(TGC)7
524218-227 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 TGATTGGTTTGAATGGGCGCACGATGAGGATGCAGT (GTT)10
5211214-247 0.568 0.801 0.304
GB-PO-131 EU502645 CTCCCTCCTCCGTGTACCCGTAACGTTCGCTTCCTG (CCT)2(CCCC)(CCT)2
523200-212 0.730 0.528 -0.371
GB-PO-134 EU502647 GAGTGTGAAGAATCGGCGGTGCACTCTGCCTATCGC(GA)2(GT)(GA)3
523115-285 0.757 0.561 -0.337
GB-PO-135 EU502646 AGGAGGGGGTGCTTGATATCCTCCGCCTTCTCTACC (GGA)2(GGGA)(GGA)2
523217-271 0.162 0.151 -0.061
GB-PO-138aEU502648 TATGGAACGGTGCGAAGT GCCGTCAAAAGGGAACTC (CCG)4, (TTC)3
524 191-209 0.946 0.723 -0.296
GB-PO-149aEU502649 AGTGCATATGCCCGACACCGTCGTAGATGCAGGCTC (TCC)8
527 193-277 0.297 0.746 0.610
GB-PO-152 EU502650 ACTGAGCCTTCAGCACCACATAGGGACGACAGCGAG (AGC)5
524272-395 0.500 0.615 0.201
GB-PO-154aEU502651 GTCGTAGCCAGCCATGAGAGGGTATCTCGGGTGCAT (CGA)7
525254-269 0.649 0.698 0.084
GB-PO-157aEU502652 ATGGACGTGGTGTTCTGC AAACCAAGCCTACCCAGC (GCT)4
526 281-305 0.514 0.656 0.231
GB-PO-171aEU502654 TCTCGGGCATCATTCTTGACGTCAGGGTGTCAAACG (TTG)3, (TA)4, (TA)4
524295-310 0.194 0.674 0.718
GB-PO-172 EU502655 GCAGAAGTTGCCCAAAGAATGTCCAGCGGAAGACCT(TGC)2(TAC)(TGC)5
523169-229 0.306 0.422 0.290
525294-309 0.114 0.748 0.851
524240-249 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
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.
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