Environment  Health  Techniques Genetic diversity and core collections of Chinese shiitake

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Full Paper Evaluating genetic diversity and constructing core collections of Chinese Lentinula edodes cultivars using ISSR and SRAP markers Jun Liu1,2, Zhuo-Ren Wang1,2, Chuang Li1,2, Yin-Bing Bian1,2 and Yang Xiao1,2 1

2

Key Laboratory of Agro-Microbial Resource and Development (Ministry of Agriculture), Huazhong Agricultural University, Wuhan, Hubei Province, P.R. China Institute of Applied Mycology, Huazhong Agricultural University, Wuhan, Hubei Province, P.R. China

Genetic diversity among 89 Chinese Lentinula edodes cultivars was analyzed by inter-simple sequence repeat (ISSR) and sequence-related amplified polymorphism (SRAP) markers. A 123 out of 126 ISSR loci (97.62%) and 108 out of 129 SRAP loci (83.73%) were polymorphic between two or more strains. A dendrogram constructed by cluster analysis based on the ISSR and SRAP markers separated the L. edodes strains into two major groups, of which group B was further divided into five subgroups. Clustering results also showed a positive correlation with the main agronomic traits of the strains, and that strains with similar traits clustered together into the same groups or subgroups in most cases. The average coefficient of pairwise genetic similarity was 0.820 (range: 0.576–0.988). Compared to the wild strains, Chinese L. edodes cultivars indicated a lower level of genetic diversity. Two preliminary core collections of L. edodes, Core1 and Core2, were established based on the ISSR and SRAP data, respectively. Core1 was constructed by the advanced M (maximization) strategy using the PowerCore version 1.0 software and contained 21 strains, whereas Core2 was created by the allele preferred sampling strategy using the cluster method and contained 18 strains. Both core collections were highly representative of the genetic diversity of the original germplasm, as confirmed by the values of Na (observed number of alleles), Ne (effective number of alleles), H (Nei’s gene diversity) and I (Shannon’s information index), as well as results of principal coordinate analysis. The loci retention ratio of Core1 (99.61%) was higher than that of Core2 (97.65%). Moreover, Core1 contained strains with more types of agronomic traits than those in Core2. This study builds the basis for further effective protection, management and use of L. edodes germplasm resource. Keywords: Shiitake mushroom / Germplasm resource / Genetic relationship / Molecular markers / Core collection Received: October 6, 2014; accepted: December 7, 2014 DOI 10.1002/jobm.201400774

Introduction Lentinula edodes (Berk.) Pegler, also known as Shiitake mushroom or Xianggu, is the second most popular edible mushroom in the global mushroom market [1]. L. edodes contains numerous bioactive ingredients, such as sterols, lipids, polysaccharides and terpenoids, which have shown Jun Liu and Zhuo-Ren Wang contributed equally to this work. Correspondence: Yang Xiao, Key Laboratory of Agro-Microbial Resource and Development (Ministry of Agriculture), Huazhong Agricultural University, Wuhan, Hubei Province 430070, P.R. China E-mail: [email protected], [email protected] Phone: þ86-27-87282221 Fax: þ86-27-87287442 ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim

to be effective in treating various tumors and infections [2]. Due to its great edible and medicinal values, L. edodes is widely cultivated around the world, particularly in East Asia (such as China, Japan and Korea, etc.). At present, China is the largest producer of L. edodes, contributing to roughly 70% of the total world production [3]. More than 100 commercial shiitake strains have been developed in China. Estimating the genetic diversity of these germplasm resources is the prerequisite for effective protection and rational utilization. Several kinds of molecular markers have been successfully applied to examine the genetic relationships of L. edodes strains, such as random amplified polymorphic DNA (RAPD) [4],

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amplified fragment length polymorphism (AFLP) [5], intersimple sequence repeats (ISSR) [6], sequence-related amplified polymorphism (SRAP) [4] and sequence characterized amplified region (SCAR) [7]. Previously, the genetic diversity of about 20 L. edodes cultivars in China was analyzed by RAPD, ISSR and SRAP markers. Results revealed the suitability of those markers for genetic diversity research and a low level of genetic diversity of Chinese shiitake cultivars [4, 7]. However, the number of strains examined was not sufficient to cover the whole genetic diversity of L. edodes cultivars in China. It remains urgent to reveal the genetic relationships of Chinese shiitake cultivars in a comprehensive perspective by analyzing more strains. To increase the efficiency of characterization and utilization of germplasm resources, the concept of core collection was introduced and defined as a subset of accessions presenting the maximum possible genetic diversity contained in the entire collection with minimum repetition [8]. A good core collection should minimize redundant entries and be sufficiently large so as to provide reliable conclusions for the whole germplasm collection [9]. The core collection was traditionally constructed by morphological characters. With the advantages of stability, accuracy and independence of environmental impacts, molecular markers were increasingly utilized for this purpose in plants [10, 11]. On the ground of the same dataset, assessment of the genetic diversity of germplasm collections could also been accompanied by the construction of core collection using proper sampling methods and bioinformatic tools [12, 13]. The development of core collection in edible mushrooms is still in its early stage. Until now, there is only one core collection of Pleurotus ostreatus which included 25 cultivars [14]. The collection was built by an allele preferred sampling strategy using the UPGMA (unweighted pair group method with arithmetic averaging algorithm) cluster method based on 11 SSR markers. Results showed that the allele preferred sampling strategy could be used to establish the most representative core collection with the minimum samples when the SSR allele retention ratio was maintained at the 95% level [14]. In this study, the genetic diversity and relationships of 89 L. edodes cultivars from the major production areas of China were analyzed by using ISSR and SRAP markers. Two preliminary core collections of Chinese L. edodes cultivars were established by the advanced M (maximization) strategy using the PowerCore program [15] and the allele preferred sampling strategy using the UPGMA cluster method [16, 17]. ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim

The objectives of this work were to (1) investigate the genetic diversity and relationships of Chinese L. edodes cultivars in a large scale; (2) create core collections of L. edodes in China; (3) compare and evaluate the feasibility of the two different methods in generating core collections of L. edodes.

Materials and methods L. edodes strains Eighty-nine shiitake strains currently cultivated in the main production areas of China were surveyed in this study. All the tested strains were either provided by professional research institutes or collected from different mushroom growing farms. All the tested strains used in this study were cultivated in our experimental mushroom farms, and their morphological characteristics of fruiting bodies indicated that all the tested strains belonged to L. edodes. Characteristics of each strain were listed in Table 1. Main agronomic traits of the tested strains were assessed through years of cultivation test. Genomic DNA extraction The mycelia from the tested strains of L. edodes were cultured in potato dextrose broth (PDB) at 25 °C for 2 weeks and were then collected by filtration. Genomic DNA was extracted from 100 mg of wet mycelia using the cetyl trimethyl ammonium bromide (CTAB) method described by Murray and Thompson [18]. DNA concentration and purity were determined with a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA), and DNA samples were diluted to 50 ng ml1. ISSR and SRAP analyses A total of 28 ISSR primers and 56 SRAP primer combinations (seven forward primers and eight reverse primers) were initially screened using DNA samples from six L. edodes strains that were collected from five different provinces and the Shanghai Municipality. Nine ISSR primers and nine SRAP primer combinations produced highly polymorphic bands in the six strains (Table 2). Each ISSR-PCR reaction mixture contained 20 ml of 1 PCR buffer, 100 ng of template DNA, 0.2 mM of dNTPs, 2.5 mM of MgCl2, 0.75 mM of each primer and 0.5 U Taq polymerase (Biocolors, Shanghai, China). Amplification program was: 5 min of denaturing at 94 °C, 35 cycles of 35 s at 94 °C, 45 s at 50 °C or 53 °C, 90 s at 72 °C and followed by a final extension of 7 min at 72 °C. Each SRAP–PCR reaction mixture (20 ml final volume) consisted of 50 ng of genomic DNA, 0.2 mM of dNTPs,

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Table 1. Cultivated strains of L. edodes used in this study. Strain No. 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 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 56 57 58 59 60

Strain name a,b

S605 S606b 241–4 No.9 Cr04a Cr62 L087 L12a,b L135a,b L205 L236 L26 L305 L307–1 L605 L607b L7025 L808 L856a L9319 L952 S602a,b Dizai-1 Ganxiang-1 Guhuang-1 Guangxiang-51a,b Hunong-1a,b Hunong-3 Huaxiang-5b Huaxiang-8a Jiayou-1 Jindixianggu Jiuxiang-4 Junxing-8 Biyang-4 Biyang-2 Biyang-6 Minfeng-1 Qin02 Qin06 Qingke-20a,b Qingyuan9015 Qiu-2 Qiu-3 Qiu-7 Qiu-6a Rifen-34a,b Senyuan-10a,b Senyuan-1a Senyuan-2a Senyuan-8404 Senyuan-8 Shandong-1 Shandong-2 Shenxiang-10 Shenxiang-12 Shenxiang-2 Shenxiang-4 Shenxiang-6b Shenxiang-8

Source

Main agronomic traits

Shanghai Shanghai Qingyuan Suizhou-1 Sanming-1 Sanming-1 Sanming-2 Sanming-1 Sanming-1 Wuhan Sanming-1 Sanming-1 Wuhan Wuhan Wuhan Wuhan Wuhan Lishui Sanming-1 Lishui Wuhan Shanghai Fujian-1 Nanchang Biyang Guangdong Shanghai Shanghai Wuhan Wuhan Fujian-2 Chengdu Suizhou-2 Zhejiang-1 Biyang Biyang Biyang Sanming-1 Suizhou-3 Suizhou-3 Qingyuan Qingyuan Wuhan Wuhan Wuhan Wuhan Henan-1 Yichang-1 Yichang-1 Yichang-1 Yichang-1 Yichang-1 Shandong Shandong Shanghai Shanghai Shanghai Shanghai Shanghai Shanghai

S, F, M and MLa S, F, M and Ea S, D, ML and La S, F, ML and MLa S, F, MH and Ea S, F, M and Ea S, F or D, ML and Ea W, D, ML S, F or D, L and La S, D, ML and Ea S, F, M and Ea S, F, M and Ea S, D, ML and Ea S, D, M and Ea S, D, ML and Ea S, D, ML and Ea W or S, D, ML and MLa S, F, M and MLa S, F or D, ML and Ea S, F, H and MLa W, D, ML S, F, M and La S, F, H and Ea S, F, M and Ea S, D, ML and Ea W, D, ML W, D, M W, D, ML S, D, M and MLa S, F, M and Ea S, F, M and Ea S, F, ML and MLa S, D, ML and Ea S, F, H and Ea S, D, ML and Ea S, D, M and Ea S, D, M and Ea S, F, M and Ea S, D, ML and Ea S, D, ML and Ea S, D, ML and MLa S, F or D, ML and MLa S, D, M and Ea S, D, M and Ea S, D, ML and Ea S, D, M and Ea W, D, L W or S, D, L and MLa W, D, L W or S, D, L and La W, D, L W, D, ML S, F, MH and MLa S, F, MH and MLa S, F, M and Ea S, F, MH and Ea S, F, MH and Ea S, F, M and Ea S, F, M and Ea S, F, MH and Ea (Continued)

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Table 1. (Continued) Strain No.

Strain name

Source

Main agronomic traits

61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89

Suxiang-1 Wuxiang-1 430a 7401 903 908a,b 9207 939 945 9508 Xiang952b 9608 9908 Cr02 K95–1 L109–1b L236-m L241 Baihua-2 66 868a Xiagu-18 Huanong-2 Xiangjiua,b Xiangza-26 Yuhua-2a Yuhua-4a,b Yuhua-7 Zhumadian-3

Jiangsu Wuyi Wuhan Wuhan Wuhan Henan-2 Yichang-2 Zhejiang-2 Hubei Suizhou-4 Wuhan Henan-2 Henan-2 Sanming-1 Henan-1 Wuhan Fujian-2 Sanming-1 Wuhan Sanming-1 Zhejiang-1 Wuhan Wuhan Guangdong Guangdong Biyang Biyang Biyang Biyang

S, F, M and Ea S, F, H and Ea S, D, ML and Ea W, D, ML S, D, ML and MLa S, D, ML and MLa W, D, L S, D, ML and MLa S, F, M and Ea S, D, ML and MLa W, D, ML S, D, ML and MLa S, D, ML and MLa S, F, ML and Ea W or S, D, ML and MLa S, F, M and Ea S, F, M and Ea W, D, ML S, D, ML and Ea S, F, M and Ea S, F, ML and Ea S, F, H and Ea W, D, ML W, D, M S, F, H and Ea S, D, ML and Ea S, D, ML and Ea S, D, ML and Ea S, D, ML and Ea

Shanghai, Shanghai Academy of Agricultural Sciences; Qingyuan, Qingyuan Edible Fungi Scientific Research Center, Zhejiang Province; Suizhou-1, mushroom growing farm in Sanligang Town, Hubei Province; Suizhou-2, Suizhou Changjiu Mushroom Company, Hubei Province; Suizhou-3, mushroom growing farm in Caodian Town, Hubei Province; Suizhou-4, mushroom growing farm in Hongshan Town, Hubei Province; Sanming-1, Sanming Mycological Institute, Fujian Province; Sanming-2, Sanming Food Industry Institute, Fujian Province; Wuhan, Huazhong Agricultural University, Hubei Province; Lishui, Lishui Dashan Mushroom Research and Development Company, Zhejiang Province; Fujian-1, mushroom growing farm in Changting County, Fujian Province; Fujian-2, mushroom growing farm in Gutian County, Fujian Province; Nanchang, Jiangxi Academy of Agricultural Sciences, Jiangxi Province; Biyang, mushroom growing farm in Biyang County, Henan Province; Guangdong, Guangdong Institute of Microbiology, Guangdong Province; Chengdu, Sichuan Academy of Agricultural Sciences, Sichuan Province; Zhejiang-1, Zhejiang Academy of Forestry Sciences, Zhejiang Province; Zhejiang-2, mushroom growing farm in Qinyuan County, Zhejiang Province; Henan-1, Chengguan mushroom growing farm in Lushan County, Henan Province; Henan-2, mushroom growing farm in Xixia County, Henan Province; Yichang-1, Hubei Senyuan Mushroom Company, Hubei Province; Yichang-2, mushroom growing farm in Yuanan County, Hubei Province; Shandong, mushroom growing farm in Zibo City, Shandong Province; Jiangsu, Jiangsu Institute of Microbiology, Jiangsu Province; Wuyi, Wuyi Mycological Institute, Zhejiang Province; Hubei, mushroom growing farm in Huangpi District, Hubei Province; S for sawdust cultivation; W for wood log cultivation; W or S both for sawdust and wood log cultivations; F (fresh fruiting body production type), fruiting body mostly sold in fresh form in the market; D (dried fruiting body production type), fruiting body mostly sold in dried form in the market; F or D, fruiting body sold both in fresh and dried forms in the market; H (high temperature type), fruiting body formation at 8–32 °C; MH (middle-high temperature type), fruiting body formation at 15–28 °C; M (medium-temperature type), fruiting body formation at 10–25 °C; ML (middle-low temperature type), fruiting body formation at 8–23 °C; L (low temperature type), fruiting body formation at 5–20 °C; Ea (early maturing type), duration from inoculation to fruiting body formation is 65–75 days; MLa (middle-late maturing type), duration from inoculation to fruiting body formation is 90–150 days; La (late maturing type), duration from inoculation to fruiting body formation is 160–200 days. a Strains included in Core1. b Strains included in Core2; the table is partly quoted from previous papers [4, 7].

2.0 mM of MgCl2, 0.75 mM of each primer, 1 PCR buffer and 1 U of Taq polymerase. PCR reaction was performed following Li and Quiros [19] with minor modifications: denaturing at 94 °C for 5 min, 5 cycles of 94 °C for 1 min, 35 °C for 1 min and 72 °C for 1 min; 35 cycles of 94 °C for ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim

1 min, 50°C for 1 min and 72 °C for 1 min; followed by a final extension of 7 min at 72 °C. Both ISSR and SRAP PCR products were separated on 6% denaturing PAGE gels, then were stained with silver nitrate solution.

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Table 2. Primer sequences used for ISSR and SRAP analyses in this study. SRAP primers ISSR primers(50 –30 ) P2 P4 P5 P6 P7 P8 P16 P20 P27

GTGACACACACACAC GGATGCAACACACACACAC CGTGTGTGTGTGTGT AGTGTGTGTGTGTGT CCAGTGGTGGTGGTG GGAGTGGTGGTGGTG TGTGTGTGTGTGTGTGGA ACACACACACACACACCTG GTATGTATGTATGTATGG

Forward primers(50 –30 ) me1 me3 me5 me6 me7

TGAGTCCAAACCGGATA TGAGTCCAAACCGGAAT TGAGTCCAAACCGGAAG TGAGTCCAAACCGGACA TGAGTCCAAACCGGACG

Data analysis for genetic diversity DNA bands on the gels were manually scored with “1” for presence and “0” for absence to generate a binary matrix. The data were analyzed with the Numerical Taxonomy Multivariate Analysis System (NTSYS-pc) version 2.10e (Exeter Software, Setauket, NY, USA) software package [20]. The simple matching (SM) coefficient was used to calculate the pairwise genetic similarity (GS) between any two L. edodes strains. The pairwise GS matrix was imported into Excel to calculate the average GS value. The average GS value of 19 strains for wood log cultivation and that of 70 strains for sawdust cultivation was compared by t-test using SPSS version 17.0 (SPSS Inc., Chicago, IL, USA). A UPGMA dendrogram was constructed based on the SM coefficients. The goodness of fit of clustering to the data matrix was calculated using the COPH and MAXCOMP options in NTSYS-pc. Both ISSR and SRAP are dominant genetic markers, and each DNA band produced is regarded as a genetic locus. Genetic parameters for each ISSR primer and SRAP primer combination, such as the number of polymorphic loci (NPL), percentage of polymorphic loci (PPL), observed number of alleles (Na), effective number of alleles (Ne), Nei’s gene diversity (H) and Shannon’s information index (I) were calculated using the POPGENE program (version 1.32) [21]. Construction of preliminary core collections The preliminary core collections of 89 L. edodes cultivars were constructed in two ways. The first one was based on the advanced M strategy in PowerCore version 1.0 [15], and the other was based on the allele preferred sampling strategy using the UPGMA cluster method [16, 17]. The latter is based on the following principles: when clustering a subgroup with two strains at the lowest level of sorting, strains with the maximum number of rare alleles (allele frequency 5%) will be preferably selected for the next cluster. If the two strains contain an equal ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim

Reverse primers(50 –30 ) em1 em2 em4 em6 em7 em8

GACTGCGTACGAATTAAT GACTGCGTACGAATTTGC GACTGCGTACGAATTTGA GACTGCGTACGAATTGCA GACTGCGTACGAATTCAA GACTGCGTACGAATTCAC

number of rare alleles, the one with the minimum frequency of rare allele is preferred. If both the number and the frequency of the rare allele are the same in the two strains, a strain will be selected randomly. If there is only one strain in a subgroup, it will be directly sampled for the next cluster [16, 17]. The preliminary core collections obtained by the PowerCore software and the UPGMA cluster method were respectively named as Core1 and Core2. To evaluate the representativeness of the core collections, t-test of Na, Ne, H and I were performed using SPSS. In addition, principal coordinate analysis (PCoA) was performed to obtain a graphical representation of the relationships between the core collections and the original germplasm [20].

Results Polymorphism of the ISSR and SRAP markers Both ISSR and SRAP PCR experiments were repeated twice. Only unambiguous and reproducible DNA bands were manually recorded. Characteristics of the ISSR and SRAP markers were listed in Table 3. For the ISSR analysis, a total of 126 bands were identified from the 89 L. edodes strains, of which 123 (97.62%) were polymorphic. The values of Ne, H and I were 1.311, 0.193 and 0.309, respectively. For the SRAP analysis, a total of 129 bands were scored from all the strains, of which 108 (83.73%) were polymorphic. The values of Ne, H and I were 1.312, 0.193 and 0.304, respectively. Genetic diversity and relatedness among Chinese shiitake cultivars based on ISSR and SRAP markers For the 89 L. edodes cultivars, the GS estimated by the SM coefficient varied from 0.576 (L12 and Xiangjiu) to 0.988 (Biyang-2 and Qin06), with an overall mean of 0.820. A dendrogram constructed from the SM coefficient matrix using the UPGMA method was shown in Fig. 1. Almost all

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Table 3. Characteristics of ISSR and SRAP markers used in this study. Primers

Na

NPL

P2 13 13 P4 10 10 P5 18 16 P6 12 11 P7 9 9 P8 19 19 P16 8 8 P20 16 16 P27 21 21 ME1þEM2 14 14 ME1þEM4 11 9 ME3þEM8 14 8 ME5þEM2 23 23 ME5þEM4 11 9 ME6þEM4 9 6 ME6þEM6 15 14 ME6þEM7 14 11 ME7þEM1 18 14 Total 255 231 Average 14.17 12.83 ISSR average 14 13.67 SRAP average 14.33 12 a

PPL (%)

Ne

H

I

100 100 88.89 91.67 100 100 100 100 100 100 81.82 57.14 100 81.82 66.67 93.33 78.57 77.78

1.422 1.438 1.347 1.254 1.356 1.375 1.561 1.200 1.098 1.483 1.467 1.202 1.206 1.402 1.446 1.332 1.248 1.219

0.242 0.261 0.204 0.165 0.219 0.228 0.325 0.140 0.082 0.296 0.266 0.117 0.151 0.237 0.255 0.206 0.159 0.141

0.372 0.401 0.317 0.268 0.348 0.359 0.487 0.242 0.164 0.457 0.399 0.180 0.260 0.359 0.376 0.303 0.260 0.228

90.59 97.62 83.73

1.312 0.193 0.307 1.311 0.193 0.309 1.312 0.193 0.304

Number of total loci.

the strains of L. edodes were clustered into two major groups at the similarity level of 0.78 (Fig. 1), with one distinct strain (Xiangjiu) that showed a smaller GS coefficient than other strains and was separated from group A and B at a similarity level of 0.66. Group A comprised 14 strains, and group B included 74 strains that were roughly delineated into five subgroups (B1–B5). The numbers of strains in subgroup B1, B2, B3, B4 and B5 were 8, 28, 28, 5 and 5, respectively. Grouping of the 89 L. edodes cultivars revealed by PCoA was highly similar to that of the UPGMA analysis (data not shown). The correlation index between the similarity coefficient matrix and the cophenetic matrix derived from the UPGMA tree was 0.88, implying a strong goodness of fit. The UPGMA clustering results are also consistent well with the agronomic traits of the tested strains in most cases (Table 1). Strains with similar traits tended to cluster into the same groups or subgroups. Strains in group A are suitable for sawdust cultivation except for Senyuan-8, and most of them belong to the M or ML temperature type and MLa or La maturing type. Subgroup B1 contained strains for wood log cultivation with the exception of 241–4. Twenty-five out of 28 strains in subgroup B2 are cultivated using sawdust. They are Ea or MLa maturing type and their fruiting bodies are mainly sold in fresh form in the market. Twenty-four out of 28 strains in subgroup B3 are cultivated using sawdust. They are M or ML temperature ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim

type and Ea maturing type and their fruiting bodies are mostly sold in dried form in the market. Strains in subgroup B4 are cultivated using wood log except for Huaxiang-5. Strains in subgroup B5 are suitable for sawdust cultivation with the MLa maturing type. Preliminary core collections of L. edodes cultivars in China A total of 21 L. edodes strains were retained as a core collection (Core1) with the loci retention ratio of 99.61% using the PowerCore program (Table 1). By UPGMA cluster method with allele preferred sampling strategy, information of alleles retained in the core collections with the allele preferred sampling strategy was listed in Table 4. According to previous research in P. ostreatus, the rare loci retention ratio should be larger than 80% in the selected core collection [14]. In addition, the most representative core collection with the minimum samples could be established by the UPGMA cluster method with the allele preferred sampling strategy, when the loci retention ratio was maintained at 95% in P. ostreatus [14]. At this cut-off threshold, 18 strains were retained in Core2 with a loci retention ratio of 97.65% and a rare loci retention ratio of 89.66%. The numbers of strains in Core1 and Core2 accounted for 23.60% and 20.22% of the original germplasm, respectively. The total number of loci retention and other genetic diversity parameters in the ISSR and SRAP analyses for the original germplasm and the two core collections were summarized in Table 5. t-Test showed that there were no significant differences of Na and Ne values between the original germplasm and the two core collections, indicating that the core collections retained the majority of allele loci of the original germplasm. The H and I values in the two core collections were significantly (p < 0.05) higher than those in the original germplasm, implying a higher polymorphism of these representatives of L. edodes cultivars. There were no significant differences in the Na, Ne, H and I values between Core1 and Core2 (p > 0.05), suggesting that there were no difference in the genetic diversity between the two preliminary core collections. Therefore, both Core1 and Core2 were good representatives of the original germplasm. PCoA were also used to validate the representativeness of the selected L. edodes strains in Core1 and Core2. As shown in Fig. 2, the selected strains distributed across the main plot in a scattered pattern, guaranteeing their representativeness. The numbers of L. edodes strains with varied traits in the two core collections were recorded in Table 6. Strains in the two core collections covered almost all kinds of the main agronomic traits, thus validating the excellent

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Figure 1. UPGMA dendrogram of 89 L. edodes strains constructed using genetic similarity coefficients based on ISSR and SRAP data. Except for Xiangjiu, all the L. edodes strains were clustered into two main groups (A and B) and group B was further delineated into five subgroups (B1– B5). The UPGMA clustering results indicated a positive correlation with the traits of the tested strains in most cases, and that the strains with similar traits tended to cluster together into the same groups or subgroups.

representativeness of the core collections. However, the H temperature type was absent in both Core1 and Core2, and the MH temperature type was absent in Core2.

Discussion Polymorphism of the ISSR and SRAP markers Numerous molecular marker techniques have been increasingly and successfully utilized in strain genotyping, ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim

genetic diversity analysis and genetic map construction in edible mushrooms, among which ISSR and SRAP are the two commonly used markers. Recently, ISSR and SRAP techniques have been proven to be highly efficient to evaluate the genetic diversity of L. edodes cultivars [4, 7]. In the previous studies, numbers of average polymorphic DNA bands amplified by ISSR primers were 11.2 and 4.89 [4, 7], and those amplified by SRAP primers were 3.5 and 7.17 [4, 7]. In this study, a higher efficiency of polymorphism detection was reported using the same two

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Table 4. Information of loci retained in core collections with different sample sizes using the UPGMA cluster method with the allele preferred sampling strategy. Number of samples

Number of loci

Loci retention ratio (%)

Number of rare loci

Rare loci retention ratio (%)

43 32 25 18 16 12 8

254 251 250 249 239 238 232

99.61 98.43 98.04 97.65 93.73 93.33 90.98

57 54 53 52 46 45 43

98.28 93.10 91.38 89.66 79.31 77.59 74.14

marker systems (13.67 polymorphic bands per ISSR primer and 12 polymorphic bands per SRAP primer combination). This could be attributed to: (1) more L. edodes strains were used here, thus representing a higher genetic diversity; and (2) PAGE gels instead of agarose gels were employed here to separate the PCR amplicons, therefore more DNA bands were detected. The values of Ne, H and I were similar between ISSR and SRAP markers in this study, suggesting that the two markers could survey a similar genetic diversity of the 89 L. edodes strains. Different molecular marker techniques have different principles, and they are focused on different targets in the genome. ISSR detects polymorphisms in the interval DNA region between simple sequence repeats (SSRs or microsatellite DNA) [22]. On the other hand, SRAP preferentially amplifies open reading frames (ORFs), and identifies polymorphisms fundamentally originated from the variation of the length of introns, promoters, and spacers [19]. Therefore, integrating molecular markers derived from different amplification regions is more effective in detecting genomic variation and in realizing a more complete analysis of genetic diversity. Genetic diversity of L. edodes cultivars in China Almost all edible species inevitably undergo a drastic loss of diversity, resulting from man’s selection in cultivated lines [23, 24]. The same is also true for edible mushrooms [25, 26]. Indeed, Chinese L. edodes cultivars demonstrated a low level of genetic diversity, with a higher mean of GS (0. 820) than that of wild strains (0.696) [27]. Chinese shiitake cultivars are mainly developed by the introduction of exotic strains, systematic breeding and

cross breeding. Exotic strains of L. edodes were introduced into cultivation in the mainland China in the 1960s and since then have been widely cultivated [28]. Some of these introduced strains, such as the 7401–7405 series and the 79 series, have become main cultivars and provided useful gene resources for the late cross breeding programs of L. edodes in China [29]. Some elite strains of L. edodes widely cultivated in China, such as Cr02, Cr04 and Cr62, were developed by cross breeding based on the introduced strains [4, 7, 30]. In China, wood log cultivation of L. edodes was started in the 1960s, after that sawdust cultivation method was rapidly developed since the 1980s [31]. With the enforcement of effective measures to protect forest resources by the Chinese government, wood log cultivation has been gradually replaced by sawdust cultivation. It was conceivable that L. edodes strains for wood log cultivation could be directly introduced to sawdust cultivation by systematic breeding, or new strains for sawdust cultivation were developed by cross breeding based on those for wood log cultivation. Indeed, this was the case in Japan: Terashima et al. [32] considered that strains for sawdust cultivation were developed from strains for log wood cultivation under the fruiting body formation temperature of 15–25 °C. L. edodes strains with similar traits tended to cluster together in the dendrogram, in agreement with previous studies [6, 32]. Currently, several cultivation models of L. edodes have been developed in the main production areas in China, and cultivars with similar agronomic traits are prone to be cultivated in certain model. For example, the autumn sawdust cultivation on shelf is a popular cultivation model in the Hubei and Henan Provinces

Table 5. Comparisons of the two preliminary core collections with the original germplasm. Group

Number of samples

Number of loci retention

NPL

PPL (%)

Original germplasm Core1 Core2

89 21 18

255 254 249

231 230 217

90.59 90.55 87.15

a

Na

Ne

H

I

1.906 1.906 1.872

1.312 1.366 1.366

0.193 0.224a 0.226a

0.307 0.352a 0.354a

Indicates significant difference in genetic diversity index at the 0.05 level between the core collection and the original germplasm.

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Figure 2. Principal coordinate plots for the Core1 and Core2 collections based on ISSR and SRAP markers. Core1 was established by the advanced M strategy using the PowerCore program, whereas Core2 was constructed by the allele preferred sampling strategy using the UPGMA cluster method. The selected strains in both Core1 and Core2 were distributed across the main plot in a scattered pattern, showing good representativeness of the original germplasm.

located in central China. Under this cultivation model, the main cultivars are strains having the traits of the M or ML temperature type, Ea maturing type and fruiting body mostly sold in dried form in the market. Ramanata Rao and Hodgkin [33] deemed that a limited number of elite lines are often used to generate many cultivars, thus resulting in an increasingly narrow genetic base of the crop. Chiu et al. [28] also reported that many shiitake cultivars in China showed genetic homogeneity and some of them might be derived from a vegetative clone or different generations of the same cultivated strain. So we speculated that strains under the same cultivation model could be propagated from several elite ones by vegetative means or by cross breeding, thus causing marginally genetic variations among those strains. This could be the reason why strains with similar traits

clustered together into groups or subgroups in the UPGMA dendrogram. Breeding fine strains of high yield and high quality is a primary task to meet the demand of the rapid development of modern L. edodes industry. Germplasm is the basics of L. edodes breeding. However, L. edodes cultivars in China had a low level of genetic diversity, with the same limited gene pool [28]. Using such germplasm in breeding programs could cause severely genetic erosion and inbreeding depression [5]. Genetic erosion could in turn increase the genetic vulnerability and may lead to dramatic effects on the production yield [34]. As a reservoir of biodiversity, wild strains of L. edodes should be introduced into breeding programs to increase the genetic heterogeneity and to improve the commercial characters of the cultivated strains [35].

Table 6. Number of L. edodes strains with varied traits in the original germplasm and the core collections. Cultivation medium Core collection

S

Original germplasm 70 Core1 13 Percentage of reservation (%) 18.57 Core2 11 Percentage of reservation (%) 15.71

W 15 6 40.0 6 40.0

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W or S 4 2 50.0 1 25.0

Production type F 35 5 14.29 5 14.29

D

Temperature type

F or D H

50 4 14 2 28.0 50.0 12 1 24.0 25.0

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MH

M

ML

Maturing type L

Ea

MLa

La

6 6 28 42 7 51 19 4 0 1 6 9 5 8 4 3 0 16.67 21.43 21.43 71.43 15.69 21.05 75.0 0 0 8 7 3 5 5 2 0 0 28.57 16.67 42.86 9.80 26.32 50.0

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It is noteworthy that the average value of SM genetic similarity among the 19 L. edodes strains for wood log cultivation was 0.767, whereas that among the 70 strains for sawdust cultivation was 0.841. The significant difference between the values (t-test, p < 0.05) suggested that L. edodes strains for wood log cultivation possessed more abundant genetic variation than those for sawdust cultivation. Environmental conditions of wood log cultivation in edible mushrooms are very similar to natural conditions, especially in cultivation medium. Under this circumstance, a loss of genetic diversity could be mitigated. Therefore, L. edodes strains for wood log cultivation could be another potential genetic resources in planning effective breeding programs. Preliminary core collections of L. edodes cultivars in China Core collections are subsamples of large populations that include the maximum genetic diversity by selecting the minimum number of representative accessions [8]. Before constructing a core collection, two aspects should be considered in advance: (i) the optimal number of accessions needed to retain an acceptable proportion of alleles presented in a given collection, and (ii) the method used to select accessions for the core subset [11]. Generally speaking, the sample sizes of core collections commonly account for 5–30% of the entire collection [11, 36]. The sample sizes of Core1 and Core2 here respectively amounted for 23.60% and 20.22% of the original germplasm, thus being within this reasonable range. The M strategy is one of the most common approaches to develop core collections [37]. Compared to other strategies, it is clearly considered as the most appropriate approach for selecting entries with the most diverse alleles and eliminating redundancy [38]. PowerCore, a software program developed by Kim et al. [15], uses an advanced M strategy with a modified heuristic algorithm to establish core collection. It has been widely used in creating core collections in plants [13, 39, 40] and shown the highest allele (fragment) capturing efficiency than other strategies in core collection development of mungbean [10]. In edible mushrooms, the UPGMA cluster method using allele preferred sampling strategy was the only way to establish core collection till now. Li et al. [14] employed this method to establish P. ostreatus core collections according to genetic distances among different strains, thus affording reference basis for such research in edible mushrooms. In this study, the PowerCore program employing the advanced M strategy and the UPGMA cluster method using the allele preferred sampling strategy were utilized to construct ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim

core collections of L. edodes. By comparing the values of Na, Ne, H and I, both Core1 and Core2 were found to be good representatives of the original germplasm, thus verifying the feasibility of the two different methods in generating core collections of L. edodes. However, the loci retention ratio of Core1 was higher than that of Core2, and strains of the MH temperature type was missing in Core2. The loci retention ratio is critical in maintaining the genetic diversity of a population and the missed loci are likely involved in important phenotypes [41, 42]. Therefore, Core1 seems to be better than Core2. It was the first time to utilize the PowerCore program to establish core collection in edible mushrooms. Results showed that the advanced M strategy in the PowerCore program was a potentially powerful alternative in constructing core collection in edible mushrooms. PowerCore is a user-friendly program and is simple to operate, allowing any types of character for data input, including molecular marker data and quantitative data. By contrast, the UPGMA cluster method with the allele preferred sampling strategy needs to use several cluster analyses and select strains manually. However, the two methods have diversified principles and sampling strategies, and a combination of these methods could supply more comprehensive solution for core collection construction. Since cultivated germplasm of L. edodes in China had a low level of genetic diversity, a core collection was crucial to avoid redundancy in the collection and reduce the management cost. In a breeding plan of L. edodes, a core collection would be favored to efficiently explore novel variation and enhance the use of germplasm. Only 89 L. edodes strains were used in the present study, and both Core1 and Core2 preserved most of their genetic diversity. In the long term, more L. edodes strains, especially the wild ones, should be introduced to construct core collections that present higher genetic diversity. In summary, this study provides a solid foundation for the establishment of core collections of L. edodes and other edible mushrooms.

Acknowledgments This research was supported by the National Key Technology Support Program in the 12th Five-Year Plan of China (Grant No. 2013BAD16B02), the National Science Foundation of China (Grant No. 31372117), and the Science and Technology Plan of Hubei Province (Grant No. 2012DBA19001). The authors appreciate Mr. Man Kit Cheung from the Chinese University of Hong Kong for the linguistic revision.

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Genetic diversity and core collections of Chinese shiitake

Conflict of interest None of the authors has any conflict of interest to disclose.

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