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Micología Aplicada International
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Colegio de Postgraduados
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Imtiaj, A.; Lee, T. S.; Ohga, S.
Sequence variation of Pleurotus species collected from Eastern Asia
Micología Aplicada International, vol. 23, núm. 1, enero, 2011, pp. 1-10
Colegio de Postgraduados
Puebla, México
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Pleurotus
from Eastern
sia pp. 1-10
Micologia
Aplicadaspecies
International
, 23(1),A2011,
1
© 2011, Berkeley, CA, U.S.A.
www.micaplint.com
Sequence variation of Pleurotus species
collected from Eastern Asia
A. Imtiaj1, T. S. Lee2 and S. Ohga1*
1
2
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,
M
icol. Apl. Int
., 23(1),
2011,
pp. 1-10 Fax: +81-929483116. E-mail: [email protected]
* Corresponding
author:
Phone:
+81-929483118.
2
A. Imtiaj et al.
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
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
μl of 99.9% ethyl alcohol was added and
centrifuged at 4 C, 5 min, 12,000 rpm. The
supernatant was decanted, 500 μl 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 μl 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 μl of the
Micol. Apl. Int., 23(1), 2011, pp. 1-10
Pleurotus species from Eastern Asia
3
Table 1. Pleurotus strains used in this study.
No. Species
Code
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.) Quél.
P. eryngii (De Cand.) Quél.
P. ferulae (Lanzi) X. L. Mao
P. ferulae (Lanzi) X. L. Mao
P. nebrodensis (Inzenga) Quél.
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.) Quél.
P. pulmonarius (Fr.) Quél.
IUM1307 IUM2652 IUM1309 IUM1794
IUM3705 IUM1659 IUM3568 IUM0556 IUM1635 IUM3511 IUM1306 IUM1313
IUM1320 IUM1376 IUM2022 IUM2131 IUM3527
IUM3573 IUM1271 IUM2362 GenBank accession
Country
number
HM770899
HM770890
HM770891
HM770895
HM770892
HM770889
HM770888
HM770903
HM770902
HM770900
HM770897
HM770884
HM770887
HM770898
HM770901
HM770894
HM770885
HM770893
HM770896
HM770886
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 μl of
SDDW (sterilized double distilled water),
and then 800 μl of DNA mixture was taken
into the cuvette, and the concentration was
measured at 260 nm and 280 nm. For control, concentration of 800 μl 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.)
Micol. Apl. Int., 23(1), 2011, pp. 1-10
using universal primers ITS1 forward
(5′-TCCGTAGGTGAACCTGCG-3′)
and ITS4 reverse (5′- TCCTCCGCTTATTGATATGC-3′). Amplification reactions
were performed in a total volume of 20 μl
containing 10x PCR buffer 2 μl, dNTP mix
1.6 μl, 0.5 μl of each primer, 0.2 μl of Taq
polymerase (Cosmo, Korea), 1 μl of genomic DNA, and 14.2 μl of sterilized distilled water. PCR amplification was carried
out in 30 cycles at 94 C for 30 s denaturing,
4
A. Imtiaj et al.
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 μl 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-
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.
Micol. Apl. Int., 23(1), 2011, pp. 1-10
Pleurotus species from Eastern Asia
5
Table 2. Statistics of nucleotide sequences of the ITS region from Pleurotus strains studied.
Item
Incheon University Mushroom (IUM) strain code
1307
2652
1309
1794
3705
1659
3568
0556
1635
3511
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
507
163.1
231
276
0.456
0.544
545
175.2
250
295
0.459
0.541
625
201.5
252
373
0.403
0.597
616
197.7
251
365
0.407
0.593
599
192.6
267
332
0.446
0.554
473
152.1
206
267
0.436
0.564
607
195.4
273
334
0.45
0.55
591
190.1
265
326
0.448
0.552
607
195.1
269
338
0.443
0.557
1306
1313
1320
1376
2022
2131
3527
3573
1271
2362
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
600
192.9
265
335
0.442
0.558
535
171.9
236
299
0.441
0.559
563
181.2
256
307
0.455
0.545
587
189.0
266
321
0.453
0.547
591
190.0
259
332
0.438
0.562
584
187.6
275
309
0.471
0.529
592
190.4
259
333
0.438
0.562
596
192.2
264
332
0.443
0.557
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
Micol. Apl. Int., 23(1), 2011, pp. 1-10
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
6
A. Imtiaj et al.
Fig. 1. Alignment of sequences from the ITS region of different Pleurotus species studied.
Micol. Apl. Int., 23(1), 2011, pp. 1-10
Pleurotus species from Eastern Asia
Fig. 1 (continued)
Micol. Apl. Int., 23(1), 2011, pp. 1-10
7
8
A. Imtiaj et al.
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
Micol. Apl. Int., 23(1), 2011, pp. 1-10
Pleurotus species from Eastern Asia
used in this study might be distributed
from common ancestor.
12.
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