Applying target region amplification polymorphism markers for analyzing genetic diversity of...

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: [email protected]; [email protected] 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. Xiao et al. Journal of Basic Microbiology 2010, 50, 475–483


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


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 Anhuia 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 Guangxia Guangdong 1 41 ACCC50726 Guangdonga 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.



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 Tm 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 MgCl2, 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 ki, where ki is the

Table 2. Fixed primers used in this study.

Name GenBank accession no.

Tm [°C]

Sequence (5′–3′) Homologue by BLAST search

Acta AB195375 54.1 TACCCTTTTCACCACCAC Actin (Schizophyllum commune) Alda AB195326 52.8 GTCAAATCCCAGGAAGAG Alcohol dehydrogenase Ⅲ (Aspergillus nidulans) Elna AB195388 53.5 AATCCTCTGGCACGATAG Hypothetical protein elns (Coprinus cinereus) Gnba AB195334 52.8 AGCCTAATCCGATACTGC Guanine nucleotide binding protein γ-subunit (Lentinula

edodes) CP450

a AB195349 52.9 CCATTAGCCCTGGTAGTC Cytochrome P450 2 Le.CYP2 (Lentinula edodes) Gpia AB195404 52.4 GACTCCCTTGGAGAAGAA Putative glucose-6-phosphate isomerase (Agaricus bisporus) Phda AB195324 53.4 TGATCCAAGCTTCTACCC Phosphatidylserine decarboxylase 2 (Neurospora crassa) Pyda AB195364 53.2 GGGGTGCTACTGACAACT Pyruvate decarboxylase (Hanseniaspora uvarum) Sppa AB195355 53.1 CGACCAAGAGGAAATAGG Sporulation protein SPS19 (Saccharomyces cerevisiae) Ubia AB195403 53.4 GAACCCTTTCCGACTACA Ubiquitin (Saccharomyces cerevisiae) Pri X60956 52.8 CCCTTCAGGAAAGTCTTG Developmentally regulated gene (Lentinula edodes) STE3b 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.



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 A1 consisted of three strains from Northeast China. Subgroup A2 included eleven strains from Northwest China and two strains from Yunnan and Hubei. Subgroup A3 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 A4, and three strains from Central China and one strain from North-west China were included in Subgroup A5. Two Taiwan strains (LE271 and WU9807-41) were distributed into Subgroup A6. Subgroup A7 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 CP450 + 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



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 A6 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



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 A1 and A2 in the TRAP analysis. However, every strain of the major Group B in the SSR analysis could be distributed into Subgroups A2, A3, A4, A5, A6 and A7 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 A3 and all the Taiwan strains formed Subgroup A6. 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



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))

Applying target region amplification polymorphism markers for analyzing genetic diversity of...

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