molecular / sequence study of mushroom / mycology / microbiology by dr. ahmed imtiaj
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8/3/2019 Molecular / Sequence study of Mushroom / mycology / Microbiology by Dr. Ahmed IMTIAJ
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Micol. Apl. int., 23(1), 2011, pp. 1-10
1PleurotussPeciesfrom eastern asia
INTRODUCTION
The oyster mushroom and its related spe-
cies are prominent fungi causing wood de-
sequencevariationofpleurotussPecies
collectedfrom 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.2 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 ofPleurotus
mushrooms is still unclear. Because of that, 20 strains ofPleurotus 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 make-
up. In this study, we used DNA sequences of the ITS (Internal Transcribed Spacer)
region to analyze the genetic diversity ofPleurotus strains. A few differences were
found in the sequences implying that all strains belonged toPleurotus regardless of
the geographical origin and species. This is also supported by phylogenetic analysis,
which revealed thatPleurotus 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.
* Corresponding author: Phone: +81-929483118. Fax: +81-929483116. E-mail: ohga@forest.kyushu-u.ac.jp
cay in terrestrial ecosystems worldwide,
and are widely collected and cultivated as
edible fungi. Because of its good avor,
culinary status and medicinal properties,
MicologiA AplicAdA internAtionAl, 23(1), 2011, pp. 1-10 2011, Berkeley, CA, U.S.A.
www.micaplint.com
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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 andgenetic relationships among these species
is still unclear. Phylogenetic analysis using
molecular sequences is useful for resolv-
ing relationships and understanding spe-
ciation in many problematic species com-
plexes ofBasidiomycetes1,13,18,21. With the
accumulation of ecological knowledge and
the development of phylogenetic analysis
based on DNA techniques, the traditional
taxonomy ofPleurotus spp.has come into
question. Mating compatibility studieshave demonstrated the existence of sepa-
rate biological species in Pleurotus, many
of which are largely distributed over one
or more continents19. Evidences revealed
that oyster mushrooms collected from dif-
ferent countries show a little genetic dif-
ference. Therefore, traditional taxonomy
of Pleurotus spp. may not be correct.
Characterization of relationships may clar-
ify the taxonomy ofPleurotus spp.isolat-
ed from different countries. The ability ofrDNA sequences to resolve phylogenetic
relationships among geographically isolat-
ed populations within intersterility groups
illustrated the importance of biogeographi-
cal studies for understanding speciation in
Pleurotus19. Mating tests are often used to
study the variation ofBasidiomycetes, but it
is difcult to apply to slow-growing species
due to poor spore germination and clamp-
connection frequency. DNA sequencing is
accordingly useful for elucidating taxonom-ic relationships among Pleurotus species
growing in different environments10,14,17.
Different DNA techniques have been used
to determine the genetic differences within
and amongBasidiomycetes.
In this study, 20 strains of different species
belonging to the genusPleurotus were col-
lected 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 ofPleurotusstrains derived from different places.
MATERIALS AND METHODS
The cultures of 20 strains ofPleurotus spp.
studied were obtained from the Culture
Collection and DNA Bank of Mushrooms
(CCDBM), University of Incheon, Korea
(Table 1).
DNA extraction. Mycelia ofPleurotusspp. 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 (Oper-
on, 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 in-
cubated at 65 C for 30 min in a steam waterbath. 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 removedand the residual alcohol allowed to evap-
orate. Then, 500 l of sterilized distilled
water was added and vortexed 1-2 min (it
is called stock solution). DNA concentra-
tion was assessed using spectrophotometer
(2120UV, Optizen, Korea), 20 l of the
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3PleurotussPeciesfrom eastern asia
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 con-trol, concentration of 800 l SDDW was
measured. Finally, exact concentration of
DNA solution was determined4. Polymerase chain reaction (PCR). TheDNA of all samples were amplied by PCR
(PTC-100TM, MJ Research Inc., U.S.A.)
using universal primers ITS1 forward
(5-TCCGTAGGTGAACCTGCG-3)
and ITS4 reverse (5- TCCTCCGCTTAT-
TGATATGC-3). Amplication reactions
were performed in a total volume of 20 lcontaining 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 ge-
nomic DNA, and 14.2 l of sterilized dis-
tilled water. PCR amplication was carried
out in 30 cycles at 94 C for 30 s denaturing,
Table 1.Pleurotus strains used in this study.
No. Species Code GenBank accession C
number
1 P. cornucopiae (Paulet) Rolland IUM1307 HM770899 Taiwan
2 P. cornucopiae (Paulet) Rolland IUM2652 HM770890 China
3 P. cystidiosus O. K. Miller IUM1309 HM770891 Taiwan
4 P. djamor(Rumph. ex Fr.) Boedijn IUM1794 HM770895 Korea
5 P. djamor(Rumph. ex Fr.) Boedijn IUM3705 HM770892 Korea
6 P. eryngii (De Cand.) Qul. IUM1659 HM770889 Korea
7 P. eryngii (De Cand.) Qul. IUM3568 HM770888 China
8 P. ferulae (Lanzi) X. L. Mao IUM0556 HM770903 China
9 P. ferulae (Lanzi) X. L. Mao IUM1635 HM770902 China
10 P. nebrodensis (Inzenga) Qul. IUM3511 HM770900 China11 P. ostreatus (Jacq.) P. Kumm. IUM1306 HM770897 Taiwan
12 P. ostreatus (Jacq.) P. Kumm. IUM1313 HM770884 Korea
13 P. ostreatus (Jacq.) P. Kumm. IUM1320 HM770887 Taiwan
14 P. ostreatus (Jacq.) P. Kumm. IUM1376 HM770898 Korea
15 P. ostreatus (Jacq.) P. Kumm. IUM2022 HM770901 China
16 P. ostreatus (Jacq.) P. Kumm. IUM2131 HM770894 Taiwan
17 P. ostreatus (Jacq.) P. Kumm. IUM3527 HM770885 China
18 P. ostreatus (Jacq.) P. Kumm. IUM3573 HM770893 China
19 P. pulmonarius (Fr.) Qul. IUM1271 HM770896 Korea
20 P. pulmonarius (Fr.) Qul. IUM2362 HM770886 Korea
All GenBank accession numbers belong to submission ID BankIt1372390 (NCBI). IUM: Incheon
University Mushroom.
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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 nal extension
was at 72 C for 10 min.
Gel electrophoresis and sequencing.Amplied 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 amplied
products were visualized by ethidium bro-
mide staining under UV light. The length of
amplied products was estimated by com-
paring to DNA size marker. The PCR prod-
uct was added to 100 l of direct purica-
tion buffer in Eppendorf tube, and puriedusing the Wizard PCR Preps DNA purica-
tion 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 se-
quences was performed with the basic
sequence alignment BLAST program run
against the NCBI database (www.ncbi.nlm.
nih.gov). Sequence alignment and prepara-
tion of the phylogenetic tree were carriedout 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) amplied with
primers ITS1 and ITS4 were visualized asa single band in agarose gels. The size of
the PCR fragments was about 600-800 bp
(ITS1, 200-230 bp; 5.8S rRNA gene, 120-
150 bp; ITS2, 280-320 bp) in length for
all taxa (data not shown). Sequence align-
ment compared to previously published se-
quences (P. ostreatus, strain CGMCC23, ac-
cession 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 to70% 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 andIUM1307 (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 givenin Table 2 and Fig. 1.
There are many mushroom species dis-
tributed 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 dif-
ferent parts of the world and found at least
eight intersterility groups and gene phylog-enies for two different regions of the nucle-
ar rDNA locus representing 38 individuals.
Their results demonstrated the utility of
rDNA phylogenies for understanding pat-
terns of relationship, distribution, evolu-
tion and speciation in basidiomycete fungi.
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5PleurotussPeciesfrom eastern asia
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 se-
quence 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 simi-
larities 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 phyloge-
netic tree (Fig. 2), seven sister pairs, includ-
ing the reference strain, were found withfour types of homologies. Among them,
four branches in different clades showed
high homology (100%), while the remain-
ing three sister pairs showed low bootstrap
value or less homology (56%, 52%, 50%).
Results suggested that the distribution of
Table 2. Statistics of nucleotide sequences of the ITS region fromPleurotus strains studied.
Item Incheon University Mushroom (IUM) strain code
1307 2652 1309 1794 3705 1659 3568 0556 1635 3511
Length (nucleotide) 545 507 545 625 616 599 473 607 591 607
Weight (kDa) 175.3 163.1 175.2 201.5 197.7 192.6 152.1 195.4 190.1 195.11C + G 256 231 250 252 251 267 206 273 265 2691A + T 289 276 295 373 365 332 267 334 326 3382C + G 0.47 0.456 0.459 0.403 0.407 0.446 0.436 0.45 0.448 0.4432A + T 0.53 0.544 0.541 0.597 0.593 0.554 0.564 0.55 0.552 0.557
1306 1313 1320 1376 2022 2131 3527 3573 1271 2362
Length (nuc) 555 600 535 563 587 591 584 592 596 512Weight (kDa) 178.4 192.9 171.9 181.2 189.0 190.0 187.6 190.4 192.2 164.81C + G 256 265 236 256 266 259 275 259 264 2271A + T 299 335 299 307 321 332 309 333 332 2852C + G 0.461 0.442 0.441 0.455 0.453 0.438 0.471 0.438 0.443 0.4432A + T 0.539 0.558 0.559 0.545 0.547 0.562 0.529 0.562 0.557 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)}.
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Fig. 1. Alignment of sequences from the ITS region of differentPleurotus species studied.
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7PleurotussPeciesfrom eastern asia
Fig. 1 (continued)
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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 ge-
ographically distributed. Further studies are
needed to know if these eight strains are allinterbreedable. 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.
Fig. 2. Phylogenetic tree generated by neighbor-joining analysis (standard, bootstrap repli-
cates: 100) of ITS sequences from different Pleurotus species. The reference strain was: P.
ostreatus, CGMCC, accession no.: EF514247. C: China, K: Korea, T: Taiwan.
Huerta et al.12 studied the genetic rela-
tionships 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 re-
vealed that Pleurotus strains collectedfrom different ecological environments
may have a little genetic variation in case
of differing species. Some strains belong-
ing to the same species showed 100% sim-
ilarities, even those collected from unlike
environments, indicating that the strains
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22. Xu, J., Q. Zhang, X. Xu, Z. Wang and J. Qi. 2009.
Intragenomic variability and pseudogenes
of ribosomal DNA in stone ounderKareius
bicoloratus. Molecular Phylogenetics and
Evolution 52: 157-166.
23. Yang, Z. H., J. X. Huang and Y. J. Yao. 2007.
Autoscreening of restriction endonucleases
for PCR-restriction fragment length polymor-
phism identication of fungal species, with
Pleurotus spp. as an example. Applied and
Environmental Microbiology 73: 7947-7958.
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