Journal of Basic Microbiology 2010, 50, 475 – 483
475
© 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.jbm-journal.com
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
476 Y.
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Journal of Basic Microbiology 2010, 50, 475 – 483
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Figure 1. Geographical distribution of the 14 Chinese provinces from which the 56 shiitake strains were collected.
sessment of genetic diversity. Hitherto, well-established
molecular marker techniques for classifying and evalu-
ating genetic diversity in edible fungi include restric-
tion fragment length polymorphism (RFLP) [2], random-
amplified polymorphic DNA (RAPD) [3], amplified
fragment length polymorphism (AFLP) [4], inter-simple
sequence repeats (ISSR) [5], simple sequence repeats
(SSR) [6], and sequence-related amplified polymorphism
(SRAP) [7, 8].
Target region amplification polymorphism (TRAP) is
a fairly new marker technique that uses a fixed primer
coupled with arbitrary primers to generate polymor-
phic DNA fragments. A fixed primer of about 18 nu-
cleotides is designed from the target sequence of an
expressed sequence tag (EST) or gene, and an arbitrary
primer of about the same length is designed with either
an AT- or GC-rich core to anneal with an intron or
exon, respectively [9, 10]. So, TRAP markers could be
easily connected with target candidate ESTs or genes.
Currently, more than ten thousands of EST sequences
of L. edodes in GenBank can provide an abundant selec-
tion for designing the fixed-primer.
To date, the TRAP technique has been used success-
fully for fingerprinting lettuce and Porphyra [11, 12],
revealing the genetic diversity of sugarcane and spin-
ach germplasm [13, 14], and constructing genetic link-
age maps in wheat and sunflower [15, 16]. However, it
has not yet been employed in fungal species. The first
goal of the present study was to evaluate the usefulness
of the TRAP technique in analyzing the genetic diver-
sity of L. edodes in China, and thereby to establish a new
marker technique for fungal research. The second goal
was to infer the genetic relationship among natural
germplasms of L. edodes in China.
Materials and methods
Strains
Fifty-five wild strains of L. edodes and one non-wild
strain, LE271, cultivated in Taiwan for a long time,
were surveyed in this study. The tested strains of
L. edodes originated from 14 Chinese provinces covering
a wide range of geographical and ecological conditions
(Fig. 1). They were divided into eight populations ac-
cording to different floristic regions (Table 1) [17]. The
56 strains were either collected from natural reserves
and remote mountainous areas that are far from culti-
vation sites or were generously provided by profes-
sional research institutes.
DNA extraction
The mycelia of each tested strain of L. edodes were cul-
tured in potato dextrose (PD) broth at 25 °C for 2 weeks
and were then collected by filtration. Genomic DNA
was extracted from the mycelia via the method of Zhang
Journal of Basic Microbiology 2010, 50, 475 – 483
Biodiversity of Lentinula edodes in China
477
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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
4
1
LNL076
Shenyang
2
LNL081
Shenyang
3
LNL075
Shenyang
4
LNL083
Shenyang
Northwest China
Shanxi
9
5
SHX002
Liuba
6
SHX007
Liuba
7
SHX012
Liuba
8
SHX029
Liuba
9
SHX044
Liuba
14
SHX020
Liuba
15
SHX021
Liuba
16
SHX039
Lvyang
17
SHX041
Lvyang
Gansu
8
20
GAN046
Kangxian
10
GAN052
Kangxian
18
GAN057
Kangxian
19
GAN059
Kangxian
13
GAN062
Kangxian
11
GAN067
Kangxian
12
GAN072
Kangxian
21
GAN074
Kangxian
East China
Anhui
2
22
ACCC50624
Anhui
a
23
ACCC50786
Huangshang
Mountain
Zhejiang
2
24
EFISAAS0340
Qingyuan
25
EFISAAS0342
Longquan
Central China
Hubei
12
28
HUB004
Hefeng
29
HUB016
Hefeng
35
HUB028
Shenlongjia
36
HUB037
Shenlongjia
37
HUB049
Shenlongjia
32
HUB071
Shenlongjia
30
HUB084
Xingshan
31
HUB086
Xingshan
38
HUB087
Xiangfen
33
HUB090
Zhuxi
34
HUB091
Chanyang
39
HUB021
Hefeng
Hunan
2
26
HUN001
Mangshan
Mountain
27
HUN002
Mangshan
Mountain
South China
Guangxi
1
42
ACCC50741
Guangxi
a
Guangdong
1
41
ACCC50726
Guangdong
a
Fujian
1
40
SMI96025
Yangshan
Yunnan Plateau
Yunnan
8
47
YUN039
Wuding
43
YUN001
Wuding
44
YUN105
Wuding
45
YUN106
Wuding
46
YUN107
Wuding
48
YUN109
Wuding
51
EFISAAS0350
Lijiang
52
EFISAAS0351
Jindong
Guizhou
2
49
ACCC50784
Zunyi
50
EFISAAS0229
Chaisan
Hengduanshan Mountains
Sichuan
2
53
SC002
Dechang
54
SC010
Qingchuan
Taiwan Taiwan
2
55
WU9807-41
Nantou
56
LE271
Main
cultivar
a
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
m
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
2
, 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
i
, where k
i
is the
Table 2. Fixed primers used in this study.
Name GenBank
accession no.
T
m
[°C]
Sequence (5′–3′)
Homologue by BLAST search
Act
a
AB195375 54.1
TACCCTTTTCACCACCAC
Actin
(Schizophyllum commune)
Ald
a
AB195326 52.8
GTCAAATCCCAGGAAGAG
Alcohol
dehydrogenase
Ⅲ
(Aspergillus nidulans)
Eln
a
AB195388
53.5
AATCCTCTGGCACGATAG
Hypothetical protein elns (Coprinus cinereus)
Gnb
a
AB195334
52.8
AGCCTAATCCGATACTGC
Guanine nucleotide binding protein γ-subunit (Lentinula
edodes)
CP
450
a
AB195349
52.9
CCATTAGCCCTGGTAGTC
Cytochrome P450 2 Le.CYP2 (Lentinula edodes)
Gpi
a
AB195404 52.4
GACTCCCTTGGAGAAGAA
Putative glucose-6-phosphate isomerase (Agaricus bisporus)
Phd
a
AB195324
53.4
TGATCCAAGCTTCTACCC
Phosphatidylserine decarboxylase 2 (Neurospora crassa)
Pyd
a
AB195364
53.2
GGGGTGCTACTGACAACT
Pyruvate decarboxylase (Hanseniaspora uvarum)
Spp
a
AB195355
53.1
CGACCAAGAGGAAATAGG
Sporulation protein SPS19 (Saccharomyces cerevisiae)
Ubi
a
AB195403 53.4
GAACCCTTTCCGACTACA
Ubiquitin
(Saccharomyces cerevisiae)
Pri
X60956
52.8
CCCTTCAGGAAAGTCTTG
Developmentally regulated gene (Lentinula edodes)
STE3
b
DQ480719
53.3
GACAGCTTCCCAAATCAT
STE3 pheromone receptor gene (Lentinula edodes)
a
BLAST results quoted from Miyazaki et al. [18].
b
The sole mating type gene used in this study [20] and 11 other genes associated with the development of the fruit body.
Journal of Basic Microbiology 2010, 50, 475 – 483
Biodiversity of Lentinula edodes in China
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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
1
consisted of three strains
from Northeast China. Subgroup A
2
included eleven
strains from Northwest China and two strains from
Yunnan and Hubei. Subgroup A
3
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
4
, and three
strains from Central China and one strain from North-
west China were included in Subgroup A
5
. Two Taiwan
strains (LE271 and WU9807-41) were distributed into
Subgroup A
6
. Subgroup A
7
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
76
76
100
Ald + ab2
73
73
100
Eln + ab3
62
62
100
Gnb + ab2
97
97
100
CP
450
+ ab2
41
41
100
Gpi + ab4
81
81
100
Phd + ab3
79
79
100
Pyd + ab4
86
85
98.84
Spp + ab4
115
114
99.13
Ubi + ab2
67
67
100
Pri + ab4
65
65
100
STE3 + ab2
90
89
98.89
Average
77.67
77.42
99.68
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Figure 2. Dendrogram derived using UPGMA cluster analysis based on the SM genetic similarity coefficient of 56 strains. The strain names
refer to those listed in Table 1.
vealed that the classification of the tested strains of
L. edodes was similar to that of the UPGMA analysis (Fig.
3). However, PCoA was unable to subdivide Group A
into small groups. The first and second coordinates
explained 7.4 and 5.8% of the total variation, respec-
tively. The first three Eigen vectors accounted for
18.5% of the cumulative variation.
Figure 3. PCoA of 56 strains of L. edodes using TRAP data. The
strain numbers are shown in Table 1. The first, second and third
PCoA axis explained 7.4, 5.8 and 5.3% of the total variation,
respectively.
Discussion
The average coefficient of pairwise GS was 0.696, vary-
ing from 0.503 to 0.947 and indicating the rich genetic
variation among the strains of L. edodes from different
regions of China. In the study, most strains were clearly
distributed into two major groups (Fig. 2), while Group
A was divided into seven subgroups according to the
geographical origins of the tested strains. The cluster
results demonstrated the tightly positive correlation
with the geographical origins of the tested strains. It
is worth mentioning that the two Taiwan strains
(WU9807-41 and LE271) were clustered into Subgroup
A
6
in the UPGMA dendrogram, suggesting that these
strains were possibly derived from the same origin. The
smallest GS occurred between two strains: ACCC50741
from Guangxi in Southern China and LNL076 from
Liaoning in Northern China. The two provinces are
situated within a very long geographical distance. In
contrast, the largest GS occurred between two strains
that both grew in Liuba County, Shanxi Province. Sun
and Lin [17] explained that the disparity between natu-
ral geography and ecological environment was one of
the factors impacting on the genetic divergence among
strains from different regions across China. The abun-
dant genetic diversity of natural germplasms of L. edodes
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Biodiversity of Lentinula edodes in China
481
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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
1
and A
2
in the TRAP analysis.
However, every strain of the major Group B in the SSR
analysis could be distributed into Subgroups A
2
, A
3
, A
4
,
A
5
, A
6
and A
7
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
3
and all the
Taiwan strains formed Subgroup A
6
. 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))