全 文 ：菌 物 系 统 21（3）：356~362, 2002
Mycosystema
PCR-BASED RESTRICTION ANALYSIS OF INTERNAL TRANSCRIBED
SPACERS OF NUCLEAR RIBOSOMAL DNA IN THE GENUS PLEUROTUS*
MA Fu-Ying LUO Xin-Chang
(Key Laboratory of Agricultural Microbiology, Ministry of Agriculture, Wuhan 430070)
(Department of Plant Protection, Huazhong Agricultural University, Wuhan 430070)

ABSTRACT: The internal transcribed spacers of the nuclear ribosomal DNA of eighteen Pleurotus taxa (52
isolates), together with outgroups A. bisporus, L. edodes, H. serotina, were amplified using PCR and then digested
with seven restriction endonucleases. PCR-RFLP results showed that AluI, Hae III, HinfI, TaqI, HhaI, MspI could
divide 52 isolates into 6, 5, 5, 4, 2, 2 groups respectively and distinguish P. citrinopileatus from the other Pleurotus.
No restriction site was observed for BamHI in all isolates. Dendrogram based on PCR-RFLP suggested that seven
groups were distinguishable for Pleurotus on 93% similarity coefficient, i.e. P. ostreatus complex
(including P. ostreatus, P. florida, P. sapidus, P. corticatus, P. cornucopiae, P. columbinus, P. spodoleucus,
P. ferulae, P. nebrodensis and P. sp .) , P. eryngii , P. pulmonarius-P. sajor - caju, P. tuber-regium,
P. abalonus-P. cystiodisus, P. djamor- P. salmoneostramineus and P. citrinopileatus. The phylogenetic
relationships between P.citrinopileatus and the other Pleurotus were more distant than L. edodes, A. bisporus and the
other Pleurotus.
KEYWORDS: Oyster mushroom, phylogeny, polymerase chain reaction, RFLP

The genus Pleurotus (Fr.) Quél. is widespread rot fungus of economical importance. Some species are
cultivated as edible mushrooms and some as industrial fungi for food production, a source of enzymes and
secondary metabolites. Although Pleurotus species account for nearly a quarter of world wide mushroom
production, little information exists about the evolution and relatedness of species within this genus and the
taxonomic position of some species are confused. Further studies on the breeding strategies and other aspects
depend on the exact taxonomic position.
The internal transcribed spacer (ITS) located between the small (18S) and the large (28S) ribosomal subunit
genes shows variability at an intraspecific level. PCR-amplification of this region followed by restriction analysis
or DNA sequencing, has been used to determine the relationships between mushroom species and explore
variability in mushroom (Gardes et al., 1990; Molina et al., 1992; Vilgalys & Sun, 1994; Moncalvo et al., 1995;
Nicholson et al., 1997).
The goal of this study was to utilize PCR-RFLP of ITS to construct the dendrogram of phylogenetic
relationships of Pleurotus species, including some commercial strains.

* Supported by the National Natural Science Foundation of China (国家自然科学基金资助项目)
Received:2002-03-25, accepted: 2002-04-22
DOI:10.13346/j.mycosystema.2002.03.011
3期 马富英等： 侧耳属核 rDNA转录间区扩增产物的限制性酶切分析 357
1 MATERIALS AND METHODS
1.1 Isolates and total DNA extraction
Fifty-two isolates of Pleurotus and three isolates of other genera as outgroups (L. edodes, A. bisporus
and H. serotina) were analyzed in this study (Table 1). They were grown in complete yeast liquid medium. After
7-10 days, mycelium was harvested by rinse, dried, ground in liquid nitrogen to a fine powder. After the nitrogen
had evaporated, powder was transferred to a 1.5 mL microcentrifuge tube, 700µL of lysis buffer (100 mmol/L
Tris .HCl pH 8.0, 20mmol/L EDTA, 2% CTAB, 1.4mol/L NaCl, 2% PVP) was added to the powder. The DNA was
extracted with an equal volume of chloroform and isoamylalcohol (24:1) for three times, in the last two times,
1/10 volume of 10% CTAB was added to supertanant before adding to chloroform and isoamylalcohol. It then was
precipitated with twice volumes of absolute ethanol and resuspended in 100 µL TE buffer (10 mmol/L Tris .HCl
pH 8.0, 1mmol/L EDTA).

Table 1 The culture numbers and sources of isolates used in this study
No. Species Source No. Species Source
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15

16
17
18
19
20
21
22
23
24
25
26
27
28
P. ostreatus (Jacq.:Fr.)Quél.
P. ostreatus
P. ostreatus*
P. ostreatus*
Pleurotus sp.
Pleurotus sp.
Pleurotus sp.
P. eryngii (DC.:Fr) Quél.
P. eryngii*
P. eryngii*
P. pulmonarius (Fr.) Quél.*
P. pulmonarius
P. florida Eger.
P. florida
P. sapidus
(Schulz.&Kalchbr.)Sacc
P. corticatus (Fr.) Quél*
P. cornucopiae (Paul:Pers.) Roll
P. columbinus (Quél) Quél
P. tuber-regium (Fr.) Sing.
P. spodoleucus Fr.
P. djamor (Fr.) Boedijn
P. salmoneostramineus Vassiljeva
P. nebrodensis Inzenga
P. ferulae Lanzi
P. citrinopileatus Sing.
P. citrinopileatus
P. citrinopileatus
Pleurotus sp.
Japan
Germany
Spain
Greece
Germany
Germany
Germany
Shanghai
France
Spain
Greece
Europe
Beijing
Beijing
Beijing

Shanxi
Kunming
Hongkong
Fujian
Jilin
Germany
Guanxi
Beijing
Fujian
Jilin
Dongbei
Germany
Shanghai
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44

45
46
47
48
49
50
51
52
53
54
55
Pleurotus sp.
Pleurotus sp.
Pleurotus sp.
Pleurotus sp.
Pleurotus sp.
Pleurotus sp.
Pleurotus sp.
P. sajor-caju (Fr.) Sing.
Pleurotus sp.*
Pleurotus sp.
Pleurotus sp.*
P. sajor-caju
H. serotina (Schrad.:Fr.) Sing.
P. abalones O.K.Mill.
P. abalonus
P.cystidiosus Han, KM Chen & S
Chen
P. cystidiosus
P. cystidiosus
Pleurotus sp.
P. cystidiosus
Pleurotus sp.
Pleurotus sp.
Pleurotus sp.
Pleurotus sp.
Pleurotus sp.
L. edodes (Berk.) Pegler
A. bisporus (Lange) Imbach
* Wild isolate
Wuhan
Hebei
Hebei
Fujian
Fujian
Fujian
Wuhan
Wuhan
Wuhan
American
Fujian
Wuhan
Jilin
Fujian
Fujian
Philippines

Fujian
Fujian
Shandong
Taiwan
Sichang
Sichang
Hunan
Fujian
Fujian
Germany
Guangxi
358 菌 物 系 统 21卷
1.2 Amplification of ITS region of rDNA
Known fungal ribosomal DNA primers conserved among fungal taxa were used to amplify ITS region for the
ultimate purpose of RFLP analysis. The sequences of the primers are as follows: ITS1 is 5’-TCCGTAGGTGAA
CCTGCGG and ITS4 is 5’-TCCTCCGCTTATTGATATGC. DNA was diluted to optimal concentrations for each
isolate and combined with 200 μmol/L each dNTP, 1×PCR buffer, 1.5mmol/L MgCl2, 1µmol/L each primer,
1.25u TaqTM polymerase (TaKaRa), and water to final volume 50µL. Amplification conditions were 94℃ 5 min,
following 94 ℃ 1min, 50℃ 1min, 72℃ 1.5min for 35cycles, with a final extention of 10min in PTC-200
Peltier Thermal Cycler (MJ Research, USA). Products were detected electrophoretically on 1.4% agarose gel in
Tris-acetic acid-EDTA (TAE) buffer and visualized with ethidium bromide. No further purification of the
amplified products was necessary.
1.3 Restriction analysis
PCR-amplified products were digested with seven restriction endonucleases AluI, HinfI, HaeIII, TaqI, HhaI,
MspI，BamHI respectively. Restriction fragments were electrophoresed on 2% agarose in 1× TAE buffer with
DNA Marker DL 2000 (TaKaRa).
1.4 Phylogenetic analysis
The presence or absence of individual restriction fragments derived from endonucleases digestion was scored,
and the similarity coefficient (Sc) between species was calculated by SIMQUAL program of NTSYS-pc software.
Dendrogram was constructed by unweighted pair group method arithmetic clustering (UPGMA) analysis.
2 RESULTS
2.1 PCR amplification of ITS region
Primers ITS1 and ITS4 produced 680bp PCR products of Pleurotus, 700bp of H. serotina, 720bp
of L. edodes and A. bisporus, estimated by comparing with the DNA Marker DL 2000.
2.2 Restriction analysis of PCR products
Polymorphisms were observed when ITS regions from all isolates were digested with different endonucleases
except BamHⅠ. The banding patterns of some species showed interspecifically identical. All tested enzymes
except BamHⅠcould distinguish P.citrinopileatus from the other species. No restriction site was observed for
BamHⅠin all isolates. Only P. citrinopileatus and L. edodes had an MspⅠrestriction site.
There were nine genotypes observed for all isolates digested with AluⅠ(Table 2). Genotype I, III included
one morphotaxa with two fragments respectively, P.eryngii, P. tuber-regium, P.citrinopileatus. Genotype IV with
three fragments, consisted of two morphotaxa P.djamor and P.salmoneostramineus. GenotypeV consisted of two
morphotaxa with three fragments too, P.abalonus and P.cystidiosus. GenotypeVI is comprised of the other isolate
belonging to II morphotaxa: P. ostreatus, P.pulmonarius, P.sajor-caju, P. florida, P. sapidus, P. corticatus,
P. cornucopiae, P. columbinus, P.spodoleucus, P. ferulae and P. nebrodensis. The genotypes of No.41, 54 and 55
were difference from any isolate of Pleurotus.





3期 马富英等： 侧耳属核 rDNA转录间区扩增产物的限制性酶切分析 359
AluⅠ-RFLP:
Table 2 Genotype of ITS digested with AluI
Genotype Isolate no. Species Fragment size (bp)
I
II
III
IV
V
VI


VII
VIII
IX
8-10
19
25-27
21,22
42-46,48
1-7,11-18,20,23,
24,28-33,35-40,
47,49-53
41
54
55
P. eryngii
P. tuber-regium
P. citrinopileatus
P. djamor, P. salmoneostramineus
P. abalonus, P. cystidiosus
P. ostreatus, P. florida, P. corticatus, P. spodoleucus,
P. columbinus, P. pulmonarius, P. sajor-caju, P. sp.
P. cornucopiae, P. sapidus, P. ferulae, P. nebrodensis
H. serotina,
L. edodes
A. bisporus
512, 177
435, 95
572, 60,47
415,125, 95
400,140, 95
585,95


345,288, 95
334,160, 95
457,160, 95
Hae Ⅲ-RFLP:
Hae III could divide 52 isolates into 5 groups. Group 1 included isolate 25,26,27 and yielded two fragments
of 458bp and 137bp. Group 2 included isolate 11,12,19,21,22,36,40 and 49, with DNA size 458bp and 198bp.
Group 3 with DNA size 326, 241and 120bp, included isolate 42,43,44,45,48. Group 4 with DNA size 458, 326,
241and 120bp, had only one isolate 46. Group 5 consisted of the other isolates, with DNA size 241 and 198bp (the
latter fragment is probably a doublet) (Fig. 1). Fragments of isolate 41 and 54 were 556 and 145bp, 310 and 210bp
(the latter fragment is probably a doublet) respectively. No restriction site was observed in isolate 55.








Five groups were distinguishable by polymorphisms in the HinfⅠpattern. The first group included No.25, 26
and 27, yielded fragments of 221,118,115and 106bp. The second group included No.21 and 22, yielded fragments
of 353 and 326bp. The third group consisted of No.42, 43,44,45,46 and 48 with DNA size 326,235 and 115bp.
The fourth group had only one isolate (No.19) with one broader DNA band (size about 189 to 159bp). The last
group contained the other isolates and yielded fragments of 353,221 and 106bp(Fig.2).
HinfⅠ-RFLP:
22 23 24 25 26 27 M 28 29 30 31 32 33 34 35 36 37 M 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 M
Fig. 1 Restriction fragment length polymorphisms from Hae III digestion
Lane No. indicated the same isolates as in table 1; M indicated DNA Marker DL 2000,
size (bp): 2000, 1000,750, 500, 250, 100 (from up to down)
360 菌 物 系 统 21卷







HhaⅠ-RFLP:
All Pleurotus isolates except No.25,26 and 27 had the same HhaⅠpattern with two fragments (about 378
and 150bp, the latter fragment is probably a doublet). No.25, 26 and 27 had the same pattern with fragments of
353 and 314bp. The patterns of H. serotina, A. bisporus and L. edodes were different from Pleurotus isolates.
H. serotina had two fragments of 306 and 194bp(the latter fragment is probably a doublet), A. bisporus had
fragments of 435and 300bp, L. edodes had two very close fragments with each other(about 360bp).
TaqⅠ-RFLP:
Four groups were obtained in the TaqⅠpattern. The first group included No.25, 26 and 27, yielded fragments
of 343bp and 286bp. The second group included No.21 and 22, with DNA size 320bp (probably a doublet). The
third had No.11, 12, 21, 22, 36 and 40, with one band (about 310bp, probably a doublet). The last contained the
other isolates with two fragments (320bp and 310bp).
2.3 Phylogenetic analysis
Cluster analysis showed that Sc was between 0.558-1.000. Fifty-two isolates of Pleurotus were divided into
seven groups on 93% Sc, i.e. I. P.ostreatus complex (including P.ostreatus, P. florida, P. sapidus, P. corticatus,
P. cornucopiae, P. columbinus, P. spodoleucus, P. nebrodensis, P. ferulae and P. sp.); II. P. eryngii;
III. P. pulmonarius-P. sajor-caju; IV. P.tuber-regium; V. P. abalonus-P. cystiodisus; VI. P. djamor-
P. salmoneostramineus and Ⅶ. P. citrinopileatus (Fig. 3).
3 DISCUSSION
PCR-amplification of ITS followed by restriction analysis was useful for fungi taxonomy and phylogenetic
relationships. This method was utilized to study the phylogeny of Pleurotus species.
Primers ITS1 and ITS4 successfully amplified ITS region from 55 isolates. The size of ITS region within
Pleurotus was uniform, but it differed from that of the other genera. Gardes et al.(1991) got length uniformity of
amplified products used the same primers in Laccaria species. But Molina et al.(1992) found length mutation of
this region between strains of the same species P. levis.
The taxonomy of Pleurotus is ambiguous, especially some commercial strains. To resolve this confusion, we
have used rDNA ITS PCR-RFLP to detect genotypic difference in 52 isolates of Pleurotus and 3 isolates of related
genera. Our results showed that:
1) P. citrinopileatus could be easily distinguished from the other species because of its unique patterns for all
tested enzymes except BamHⅠ.This revealed that P.citrinopileatus was distantly related to the other species.
Dendrogram showed that phylogenetic relationships between P.citrinopileatus and the other Pleurotus were more
23 24 25 26 27 M 28 29 30 31 32 33 34 35 36 37 38 39 M 40 41 42 43 44 M 45 46 47 48 49 50 51 52 53 54
Fig. 2 Restriction fragment length polymorphisms from HinfI digestion
Lane No. and M indicated the same isolates and marker as in Fig.1
3期 马富英等： 侧耳属核 rDNA转录间区扩增产物的限制性酶切分析 361
















Fig 3. Dendrogram based on PCR-RFLP of ITS region by UPGMA

distant than A. bisporus, L. edodes and the other Pleurotus, the further studies will be done.
2) fifty-two isolates of Pleurotus were divided into seven groups on 93% Sc. P.ostreatus complex (including,
P. spodoleucus, P. florida, P. sapidus, P. corticatus, P. cornucopiae, P. columbinus, P. nebrodensis, P. ferulae) were
the same species, the similarity coefficient between these species was 1.000 (Fig.3). This result was not consistent
with previous report (Iracabal et al.,1995; Gonzalez & Labarère, 2000; Lee et al., 2000) and consistent with other
report, Bunyard et al. (1996) thought that P. ostreatus, P. florida, P. sapidus were synonyms and one
P. cornucopiae isolate was closely related to P. ostreatus by PCR-RFLP of 5’half and 3’ half of 26S rDNA and
IGR-2/5S rDNA. P. pulmonarius and P. sajor-caju was a single species (Gonzalez & Labarère, 2000), as well
as P. abalonus and P.cystiodisus, P.djamor and P. salmoneostramineus. Iracabal et al.(1995) thought P. abalonus,
despite morphological similarity and interfertility with P. cystiodisus, was a discrete taxon by rDNA-RFLP, but
P. pulmonarius and P. sajor-caju had the common genetic background.
3) The RFLP patterns within species were uniform. This showed that they were not related to geographical
origin of isolates. However, Bunyard et al. (1996) found more variation between geographically isolated
populations within a species than between different species of Pleurotus.
In conclusion, PCR-based restriction analysis of ITS region is reliable, useful and economic to study the
molecular taxonomy and phylogenetic relationships within Pleurotus species. If it combines with other method
such as PCR-RFLP of 28S rDNA and IGR (the data will be published), the result will be more convicing and
reliable.
ACKNOWLEDGEMENT: We would like to thank Prof. Labarère, Prof. Lelley, Dr. Nakaya, Prof. Huang
Nian-Lai, Prof. Zhang Jin-Xia Prof. Fu Wei-Jie and Mr. Wang Zuo-Ren for their kindly providing some isolates.

362 菌 物 系 统 21卷
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侧耳属核 rDNA转录间区扩增产物的限制性酶切分析
马富英 罗信昌
（农业部农业微生物重点实验室; 华中农业大学植物保护系 武汉 430070）
摘 要：对侧耳属 18个形态种 52个菌株和蘑菇属、香菇属、亚侧耳属各 1个菌株的核糖体 DNA
转录间区进行 PCR 特异性扩增后，用 7种限制性内切酶进行酶切分析。结果表明，BamHⅠ对
所有的菌株均无酶切位点；AluⅠ, Hae Ⅲ,HinfⅠ, TaqⅠ, HhaⅠ, MspⅠ可分别将 52个菌株分为
6, 5, 5, 4, 2, 2组，且可将金顶侧耳与其它侧耳区分开。基于限制性酶切多态性构建的聚类图表明，
在 93%相似水平，可将所有的侧耳分为七组，第一组：糙皮侧耳、佛罗里达侧耳、美味侧耳、
裂皮侧耳、黄白侧耳、哥伦比亚侧耳、灰白侧耳、白阿魏蘑、阿魏蘑及一些未定名的侧耳；第
二组：刺芹侧耳；第三组：肺形侧耳和凤尾菇；第四组：具核侧耳；第五组：鲍鱼菇和囊盖侧
耳；第六组：红平菇和桃红侧耳；第七组：金顶侧耳。聚类图结果进一步表明，金顶侧耳与其
它侧耳的关系较双孢蘑菇和香菇与其它侧耳的关系要远。
关键词：侧耳，系统发育，聚合酶链式反应, 限制性片段长度多态性
中图分类号：Q939.96 文献标识码：A 文章编号：1007-3515（2002）03-0356-0362