Molecular / Sequence study of Mushroom / mycology / Microbiology by Dr. Ahmed IMTIAJ

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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: [email protected]

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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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



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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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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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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Molecular / Sequence study of Mushroom / mycology / Microbiology by Dr. Ahmed IMTIAJ