Applying target region amplification polymorphism markers for analyzing genetic diversity of Lentinula edodes in China

Journal of Basic Microbiology 2010, 50, 475 – 483

475

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Research Paper

Applying target region amplification polymorphism markers
for analyzing genetic diversity of Lentinula edodes in China

Yang Xiao, Wei Liu, Ying-Ying Lu, Wen-Bing Gong and Yin-Bing Bian

Institute of Applied Mycology, Huazhong Agricultural University, Wuhan, China

The target region amplification polymorphism (TRAP) technique was utilized for assessing the
genetic diversity of 55 wild strains and one cultivated strain of Lentinula edodes in China. From
these strains, 932 DNA fragments were amplified using 12 primer combinations, 929 fragments
(99.68%) of which were polymorphic between two or more strains. The average coefficient of
pairwise genetic similarity was 0.696, within a range from 0.503 to 0.947. Cluster analysis and
principal coordinate analysis separated the tested strains of L. edodes into two major groups.
Group A was further divided into seven subgroups. In most cases, the strains from the same or
adjoining regions could be preferentially clustered into small groups. The results from the
average genetic similarity and the weighted average value of Shannon’s Information Index
among the tested strains of L. edodes from the same region revealed a vast genetic diversity in
the natural germplasm found in China. Compared with the L. edodes strains from other regions,
those found on the Yunnan Plateau, in the Hengduanshan Mountains, in Taiwan, South China,
and Northeast China showed greater genetic diversity. The results of the present study
indicated that the wild strains of L. edodes in China possessed abundant genetic variation, and
the genetic relationships among them were highly associated with the geographic distribution.
This is the first report demonstrating that TRAP markers were powerful for analyzing the
genetic diversity of L. edodes, and the study lays the foundation for a further application of this
remarkable technique to other fungi.

Abbreviations: Target region amplification polymorphism (TRAP); Restriction fragment length polymor-

phism (RFLP); Random-amplified polymorphic DNA (RAPD), Amplified fragment length polymorphism

(AFLP), Inter-simple sequence repeats (ISSR), Simple sequence repeats (SSR); Sequence-related amplified

polymorphism (SRAP); Expressed sequence tag (EST); Quantitative trait loci (QTL); Potato dextrose (PD)

Keywords: Shiitake / DNA polymorphism / Genetic relationship

Received: January 14, 2010; accepted: May 25, 2010

DOI 10.1002/jobm.201000018

Introduction

Lentinula edodes, widely known as shiitake or xianggu
mushroom, is the second most cultivated edible mush-
room (after Agaricus bisporus) in terms of total world
production. In 2003, the production of L. edodes in
China was 2.227 million tons. This accounts for two-
thirds of the total production in the world [1].

Correspondence: Prof. Yin-Bing Bian, Institute of Applied Mycology,
Huazhong Agricultural University, Wuhan 430070, China
E-mail: bybpaper@gmail.com; bianyinbing@mail.hzau.edu.cn
Phone: +86-27-87282221
Fax: +86-27-87287442

China stretches across vast tropical and subtropical
areas with complex geographic environment and cli-
matic conditions, thus forming abundant natural
germplasm of L. edodes. The rich germplasm creates
favorable conditions for further breeding of superior
varieties of L. edodes, ensuring the steady development
of the L. edodes industry. An analysis of the genetic
variation among the natural germplasms of L. edodes
can play a crucial role in the effective utilization and
protection of the natural resources.
Among the fungal genetic markers, DNA-based mo-
lecular markers are stable, accurate and independent of
environmental impacts. They have been widely applied
in DNA fingerprinting, strain identification and as

Journal of Basic Microbiology 2010, 50, 475 – 483

Biodiversity of Lentinula edodes in China

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Table 1. Strains of L. edodes used in this study.

Floristic region

Province

Number of strains Strain no.

Strain name

Origin

Northeast China

Liaoning

LNL076

Shenyang

LNL081

Shenyang

LNL075

Shenyang

LNL083

Shenyang

Northwest China

Shanxi

SHX002

Liuba

SHX007

Liuba

SHX012

Liuba

SHX029

Liuba

SHX044

Liuba

SHX020

Liuba

SHX021

Liuba

SHX039

Lvyang

SHX041

Lvyang

Gansu

GAN046

Kangxian

GAN052

Kangxian

GAN057

Kangxian

GAN059

Kangxian

GAN062

Kangxian

GAN067

Kangxian

GAN072

Kangxian

GAN074

Kangxian

East China

Anhui

ACCC50624

Anhui

ACCC50786

Huangshang

Mountain

Zhejiang

EFISAAS0340

Qingyuan

EFISAAS0342

Longquan

Central China

Hubei

HUB004

Hefeng

HUB016

Hefeng

HUB028

Shenlongjia

HUB037

Shenlongjia

HUB049

Shenlongjia

HUB071

Shenlongjia

HUB084

Xingshan

HUB086

Xingshan

HUB087

Xiangfen

HUB090

Zhuxi

HUB091

Chanyang

HUB021

Hefeng

Hunan

HUN001

Mangshan

Mountain

HUN002

Mangshan

Mountain

South China

Guangxi

ACCC50741

Guangxi

Guangdong

ACCC50726

Guangdong

Fujian

SMI96025

Yangshan

Yunnan Plateau

Yunnan

YUN039

Wuding

YUN001

Wuding

YUN105

Wuding

YUN106

Wuding

YUN107

Wuding

YUN109

Wuding

EFISAAS0350

Lijiang

EFISAAS0351

Jindong

Guizhou

ACCC50784

Zunyi

EFISAAS0229

Chaisan

Hengduanshan Mountains

Sichuan

SC002

Dechang

SC010

Qingchuan

Taiwan Taiwan

WU9807-41

Nantou

LE271

Main

cultivar

The exact location of collection is unknown.

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and Molina [3]. The concentration and purity of the
DNA samples were determined with a UV-1700 spectro-
photometer (Shimadzu, Japan) and diluted to 50 ng/μl.

Primer design
Twelve fixed primers were used in the current study,
including 11 primers derived from shiitake ESTs or
genes related to the development of the fruit body [18,
19], and an additional one designed from the mating
type gene of L. edodes [20] (Table 2). All the primers were
designed by the web-derived software “Primer

3”

(http://frodo.wi.mit.edu/primer3/). The characteristics of
the fixed primers are shown in Table 2.
The sequences of the arbitrary primers with an AT-
rich core sequence were as follows [21]: 5′-GACTGCGTA
CGAAATTGC-3′ (ab2); 5′-GACTGCGTACGAAATGAC-3′
(ab3); 5′-GACTGCGTACGAAATTGA-3′ (ab4). The T

val-

ues of the three arbitrary primers were 55, 55 and
52.7 °C, respectively.

TRAP PCR protocol
Polymerase chain reaction (PCR) was carried out in a
MyCycler thermal cycler (Bio-Rad, USA). Each PCR reac-
tion mixture (20 μl final volume) contained 1 × PCR
buffer, 50 ng genomic DNA, 0.20 mM of each dNTP,
2.0 mM MgCl

, 0.625 μM fixed primer, 0.15 μM arbi-

trary primer, and 1.75 U Taq DNA polymerase (TaKaRa,
Japan). The PCR amplification program consisted of the
following conditions: 5 min initial denaturation at
94 °C, 5 cycles of 1 min at 94 °C, 1 min at 35 °C, and
1 min at 72 °C, followed by 35 cycles of 1 min at 94 °C,
1 min at 50 °C, and 1 min at 72 °C and, finally, elonga-
tion at 72 °C for 7 min. The PCR products were run on a
6% denaturing acrylamide gel at 60 W for about 2 h;

then, the DNA fragments were visualized by silver ni-
trate staining. The large gels contain very many ampli-
fied DNA fragments. In order to well record the geno-
types of the tested strains, it would be better to analyze
the gels directly instead of analyzing the photographs
of the gels. A GeneRuler 50 bp DNA ladder (Fermentas,
China) was used as molecular size marker.

Data analysis
The DNA fragments on the TRAP gel were manually
scored with “1” for the presence and “0” for the ab-
sence of each genotype; then, the binary matrix was
recorded as an Excel file for further computer analysis.
The data were analyzed with the Numerical Taxonomy
Multivariate Analysis System (NTSYS-pc), version 2.10e
(Exeter Software, Setauket, NY, USA) software package
[22]. The simple matching (SM) coefficient was used to
calculate the pairwise genetic similarity (GS) matrix
with the SIMQUAL option [23]. A dendrogram was then
constructed based on the GS matrix by the unweighted
pair group method with the arithmetic averaging algo-
rithm (UPGMA), employing the SAHN option. The co-
phenetic values were calculated to test the goodness-of-
fit between clusters and the data matrix, using the
COPH and MXCOMP options. Principal coordinate ana-
lysis (PCoA) was likewise performed based on the vari-
ance-covariance matrix calculated from the TRAP data,
using the DCENTER and EIGEN options. In addition, the
POPGENE (1.32) software was used to calculate Shan-
non’s Information Index (h) [24]. Considering the differ-
ence in the number of strains from the eight regions,
the weighted average value of Shannon’s Information
Index (H) for each population was calculated according
to the following formula: H = h/ln k

, where k

is the

Table 2. Fixed primers used in this study.

Name GenBank

accession no.

[°C]

Sequence (5′–3′)

Homologue by BLAST search

Act

AB195375 54.1

TACCCTTTTCACCACCAC

Actin

(Schizophyllum commune)

Ald

AB195326 52.8

GTCAAATCCCAGGAAGAG

Alcohol

dehydrogenase

Ⅲ

(Aspergillus nidulans)

Eln

AB195388

53.5

AATCCTCTGGCACGATAG

Hypothetical protein elns (Coprinus cinereus)

Gnb

AB195334

52.8

AGCCTAATCCGATACTGC

Guanine nucleotide binding protein γ-subunit (Lentinula

edodes)

450

AB195349

52.9

CCATTAGCCCTGGTAGTC

Cytochrome P450 2 Le.CYP2 (Lentinula edodes)

Gpi

AB195404 52.4

GACTCCCTTGGAGAAGAA

Putative glucose-6-phosphate isomerase (Agaricus bisporus)

Phd

AB195324

53.4

TGATCCAAGCTTCTACCC

Phosphatidylserine decarboxylase 2 (Neurospora crassa)

Pyd

AB195364

53.2

GGGGTGCTACTGACAACT

Pyruvate decarboxylase (Hanseniaspora uvarum)

Spp

AB195355

53.1

CGACCAAGAGGAAATAGG

Sporulation protein SPS19 (Saccharomyces cerevisiae)

Ubi

AB195403 53.4

GAACCCTTTCCGACTACA

Ubiquitin

(Saccharomyces cerevisiae)

Pri

X60956

52.8

CCCTTCAGGAAAGTCTTG

Developmentally regulated gene (Lentinula edodes)

STE3

DQ480719

53.3

GACAGCTTCCCAAATCAT

STE3 pheromone receptor gene (Lentinula edodes)

BLAST results quoted from Miyazaki et al. [18].

The sole mating type gene used in this study [20] and 11 other genes associated with the development of the fruit body.

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number of strains in population i [25]. A lower H value
displayed a higher genetic similarity among strains
from different regions.

Results

Polymorphism of the TRAP markers
A total of 36 primer combinations (12 fixed primers in
combination with 3 arbitrary primers) were initially
screened using the DNA sample of 6 L. edodes strains, 12
of which turned out to produce highly polymorphic
fragments (Table 3). After two repetitive experiments,
the 12 primer combinations produced 932 unambiguous
and reproducible DNA fragments from the 56 strains, of
which 929 (99.68%) were polymorphic. The sizes of the
most amplified fragments ranged from 50 to 1000 bp,
and the number of fragments detected by each primer
combination varied from 41 to 115, with an average of
77.67 fragments per primer combination (Table 3).
From the 56 tested strains of L. edodes, GS estimated
by SM coefficient varied from 0.503 (ACCC50741 and
LNL076) to 0.947 (SHX020 and SHX021), with an overall
mean of 0.696. Based on the similarity coefficient ma-
trix, average GS values of the L. edodes strains for each
region were calculated as follows: Northeast China
(0.688), Northwest China (0.729), East China (0.747),
Central China (0.746), South China (0.677), Yunnan
Plateau (0.695), Hengduanshan Mountains (0.714), and
Taiwan (0.725).
From the eight natural populations of L. edodes in
China, the weighted average values of Shannon’s In-
formation Index were detected as follows: Northeast
China (0.220), Northwest China (0.125), East China
(0.175), Central China (0.123), South China (0.209),
Yunnan Plateau (0.155), Hengduanshan Mountains
(0.250), and Taiwan (0.240).

Genetic relationships among the natural germplasms
of L. edodes in China
A dendrogram constructed from the SM coefficient
matrix using the UPGMA method is shown in Fig. 2.
Almost all the L. edodes strains were divided into two
major groups at the 0.68 similarity value, with two
distinct strains (LNL081 and ACCC50741) that were
genetically distantly related to the others. Group B
contained seven strains from Yunnan. Group A, includ-
ing the remaining 47 strains of L. edodes, was further
divided into seven subgroups at the similarity level of
0.71 (Fig. 2). Subgroup A

consisted of three strains

from Northeast China. Subgroup A

included eleven

strains from Northwest China and two strains from
Yunnan and Hubei. Subgroup A

contained ten strains

from Hubei and five strains from Northwest China.
Four strains from East China and two strains from
South China were clustered in Subgroup A

, and three

strains from Central China and one strain from North-
west China were included in Subgroup A

. Two Taiwan

strains (LE271 and WU9807-41) were distributed into
Subgroup A

. Subgroup A

consisted of two Guizhou

and two Sichuan strains collected from Southwest
China.
  Thirty of the 56 tested strains demonstrated similar-
ity values of over 0.73. These were further divided into
15 small groups, each containing two strains originat-
ing from either the same province or from adjoining
provinces, with the exception of one small group
(HUB091 and GAN059) (Fig. 2).
  The correlation between the similarity coefficient
matrix and the cophenetic matrix derived from the tree
produced by UPGMA was 0.88, corresponding to a
strong goodness of fit.
  PCoA was likewise performed based on the TRAP
data matrix. According to the three principal axes of
variation, the distribution of the different strains re-

Table 3. Comparison of 12 primer combinations for TRAP analysis.

Primer combination

Total bands

Polymorphic bands

Percentage of polymorphism [%]

Act + ab4

100

Ald + ab2

100

Eln + ab3

100

Gnb + ab2

100

450

+ ab2

100

Gpi + ab4

100

Phd + ab3

100

Pyd + ab4

98.84

Spp + ab4

115

114

99.13

Ubi + ab2

100

Pri + ab4

100

STE3 + ab2

98.89

Average

77.67

77.42

99.68

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resulted from the long-term interaction between the
varied ecological environments and the genetic systems
of L. edodes [17]. It therefore supports the finding of this
study that the genetic diversity of L. edodes is closely
correlated with the geographical origin.
  The results of the present study revealed that three
areas are exceptionally rich in genetic diversity of
L. edodes. The first area is a plateau area in Southwest
China, including the Hengduanshan Mountains and the
Yunnan Plateau. The second area is a low-altitude coastal
area in Southeast China, covering Taiwan and South
China. The last area is high-latitude Northeast China. It
is therefore reasonable to assume that special geographi-
cal conditions and various climates in these three areas
could be favorable for the origin and evolution of
L. edodes, thus forming the rich genetic diversity for shii-
take natural germplasm. In comparison, the genetic
diversity of shiitake strains from Northwest, East and
Central China was relatively low. The results of the pre-
sent study were overall consistent with those of previous
RAPD and SSR studies [17, 26], which confirmed that the
natural germplasms of L. edodes grown on the Yunnan
Plateau, in the Hengduanshan Mountains, and in Tai-
wan and South China possess rich genetic diversity.
  In cross-breeding of mushrooms, excellent hybrids
derived from two parental strains with greater genetic
distance between them based on molecular markers
tend to show obvious heterosis in Stropharia rugoso-
annulata and L. edodes [27, 28]. Therefore, the genetic
relationship detected by the present TRAP analysis has
great value in further selecting hybrid parents for
breeding elite strains of L. edodes.
  All the strains of L. edodes used in the present TRAP
analysis were identical to those used in a previous SSR
analysis [26]. The results between the two analyses were
overall complementary, which verified the reliability of
the study on the genetic diversity of wild shiitake
strains in China. In both analyses, cluster results well
indicated the geographical distribution of the tested
strains. Almost all the strains from the Yunnan and
Liaoning Provinces could respectively cluster into small
groups, indicating relative independence of these
strains from the two provinces. The tested strains could
be divided into three or two major groups in either the
SSR or TRAP analyses. Every strain of the major Group
A in the SSR analysis, except for GAN072, could be di-
vided into Subgroups A

and A

in the TRAP analysis.

However, every strain of the major Group B in the SSR
analysis could be distributed into Subgroups A

, A

and A

in the TRAP analysis. In addition, the

major Group C in the SSR analysis corresponded to the
major Group B in the TRAP analysis, which mainly

included seven Yunnan strains. In contrast, the TRAP
technique was very efficient for rapidly generating a
large number of markers per PCR reaction, which was
more informative for analyzing the genetic diversity of
L. edodes. Therefore, the shiitake strains from the same
floristic region possessed a stronger tendency to prefer-
entially cluster in TRAP analysis. For example, most
Hubei strains were included in Subgroup A

and all the

Taiwan strains formed Subgroup A

. However, those

strains could not cluster into subgroups in the SSR ana-
lysis. Like AFLP and ISSR markers, TRAP markers are
dominant [15], but SSR markers are co-dominant [6].
Currently, due to the lack of enough SSR primers in
L. edodes, TRAP markers would be more useful for a ge-
netic study on this economically important mushroom.
In both our SSR and TRAP analyses, strains
ACCC50741 and LNL081 were outside the major groups
in the UPGMA dendrograms. Strain ACCC50741 was
from Guangxi Province, a large tropic area with various
geographical conditions. In addition, the climate and
temperature are changeable. The specific environment
in which strain ACCC50741 can grow is very complex,
resulting in the observation that this strain is geneti-
cally different from the other strains. As is well known,
L. edodes is widely cultivated in China. Some shiitake
strains could be introduced from one place to another
for large-scale commercial cultivation. LNL081 may be
an introduced strain from another place outside Liaon-
ing Province. So, all the strains from Liaoning, except
for LNL081, were closely related with each other in
both SSR and TRAP analyses.
TRAP has several advantages over other marker
techniques: It is easy to operate (like RAPD), high in
polymorphism (like AFLP), and suitable for primer de-
sign from the known sequences of putative genes [12].
So it is worth establishing such an excellent marker
technique in fungal species. In this study, 41–115 DNA
fragments were generated by each of the 12 primer
combinations from the 56 strains. Compared with the
ISSR and SRAP techniques in the previous studies on
L. edodes [5, 8], the TRAP technique produced more DNA
fragments per primer combination. A previous AFLP
analysis generated 20–160 DNA fragments per primer
pair [29], which was similar to that of our TRAP analy-
sis. In contrast to the previous genetic diversity studies
on L. edodes natural germplasm using RAPD, SSR and
internal transcribed spacer (ITS) sequence analyses [17,
26, 30], which were only able to investigate 147 loci,
224 loci, and ITS genes of approximately 700 bp, respec-
tively, TRAP analysis attained 932 loci, which could
provide more genetic information for assessing the
genetic relationship among the tested strains of

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L. edodes. The present results demonstrated that TRAP is
a simple yet powerful technique for estimating the
genetic diversity of L. edodes. Recently, Fu et al. [8] com-
pared three molecular marker techniques used for
evaluating the genetic diversity of 23 main cultivars of
L. edodes in China; their findings indicated that the ISSR
technique is more sensitive than the RAPD and SRAP
techniques. It would be very interesting to compare the
TRAP technique with other molecular marker tech-
niques in a future study on the genetic diversity of
shiitake cultivars.
  We hypothesized that the high polymorphism pro-
duced by the TRAP markers could be attributed to three
factors. Firstly, the rich genetic differentiation among
L. edodes wild strains in China directly resulted in the
polymorphic amplified DNA fragments. Secondly, mis-
matching due to the low annealing temperature would
generate a lot of nonspecific amplified fragments [15].
Thirdly, by using the fixed-arbitrary primer pair, the
TRAP technique yielded more fragments per PCR reac-
tion than the marker techniques that used only a single
primer.
  Although the fixed primers were designed from se-
quences of interesting ESTs or genes, it was difficult to
distinguish which DNA fragments in TRAP-PCR ampli-
fication were associated with target sequences. Several
papers report that only a small number of TRAP frag-
ments were related to targeted DNA sequences [11, 12,
15]. As described by Hu et al. (2005), mismatching be-
tween the primers and the target sequences due to the
low annealing temperature (35 °C) during the first five
cycles of the amplification led to approximately 1%
TRAP fragments from the desired genes [11]. It is rea-
sonable to postulate that most of the polymorphic
TRAP fragments derived from nonspecific PCR amplifi-
cation. Anyway, by using the fixed-arbitrary primer
combination, TRAP was more specific than the tech-
niques using only arbitrary primers.
  In summary, TRAP provides an efficient and robust
tool for analyzing the genetic diversity of L. edodes.
Since the TRAP technique is simple, reliable, highly
polymorphic, and easily associated with target genes, it
could be attractive to apply the TRAP markers to fin-
gerprinting different strains, constructing a genetic
linkage map, locating quantitative trait loci (QTL), and
selecting good hybrids in fungi.

Acknowledgements

This work was financially supported by the National
Key Technology R&D Program in the 11th Five-Year
Plan of China (Grant No. 2008BADA1B02) and the In-

dustry (Agriculture), Science and Technology Plans of
China (Grant No. nyhyzx07-008).

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((Funded by
• National Key Technology R&D Program in the 11th
Five-Year Plan of China; grant number: 2008BADA1B02
• Industry (Agriculture), Science and Technology Plans
of China; grant number: nyhyzx07-008))