Chinese Science Bulletin

© 2007 Science in Press Springer-Verlag

Genetic diversity of Tricholoma matsutake in Province

SHA Tao1, ZHANG HanBo1,2, DING HuaSun2, LI ZongJu2, CHENG LiZhong2, ZHAO ZhiWei1 & ZHANG YaPing1,3†

1 Laboratory for Conservation and Utilization of Bio-resource, Yunnan University, 650091, China; 2 School of Life Sciences, Yunnan University, Kunming 650091, China; 3 Laboratory of Cellular and Molecular Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China To investigate the genetic diversity of Tricholoma matsutake, we studied ITS and IGS1 sequences and PCR polymorphism of a retrotransposon in 56 fruit bodies collected from 13 counties of 9 regions in Yunnan Province. We found one and three haplotypes based on ITS and IGS1 sequences, respectively. Moreover, there was no significant difference in PCR polymorphism of the retrotransposon among different populations. Compared with Jilin Province (China) and Japanese populations, although Yun- nan was highly homogenous to Japanese populations, low genetic diversity of T. matsutake in Yunnan did not support the view that this species originated from Yunnan.

Tricholoma matsutake, ITS, IGS1, inter-retrotransposon-amplified polymorphism (IRAP)

Tricholoma matsutake (S. Ito et S. Imai) Singer, or past some researchers assumed that southwest China Song-rong in Chinese, is an ectomycorrhizal fungus[1,2]. was the abundance centre, diversity centre, and modern This species is mainly distributed in eastern Asia, in- distribution centre for T. matsutake[4]. Thus Yunnan is cluding populations in Japan, Korea and China. With its viewed as an appropriate region for studying the species nutritious and delicious fruit body, T. matsutake has been origin and genetic diversity. To provide suggestions to a traditional and famous edible mushroom in China. Due the government towards formulating a protection strat- to a recent increase in international market demand, egy for this mushroom resource, it is necessary to un- nearly 50 million US dollars per year could be earned by derstand the phylogenetic relationships and genetic dif- exporting this mushroom. To date, this mushroom can- ferences between geographically distinct populations in not be cultivated artificially and all trade depends upon Yunnan. natural mushroom production. Poor harvest management Currently, ITS (Internal transcribed spacer) has been widely used in phylogenetic studies at different taxo- and increasing trade demands have greatly impaired [5] ― mushroom resources and T. matsutake is currently listed nomic levels . Horton reported a 99% 100% sequence as a category II national endangered species. similarity of ITS regions between T. matsutake and T. nauseosum[6]. IGS (Intergenic Spacer) is also believed to As an important production region, each year Yunnan be a rapidly evolving region among rRNA genes, and is Province exports 25% of the total amount of the ex- ported T. matsutake in China, earning 320 million yuan Received October 20, 2006; accepted February 26, 2007 doi: 10.1007/s11434-007-0194-0 (RMB) each year. This mushroom has therefore become † [3] Corresponding author (email: [email protected]) the principal agricultural export in Yunnan . Due to its Supported by the 10th Five-year Chinese National Programms for Science and diversified climate and vegetation, Yunnan provides a Technology Development (Grant No. 2001BA707B01), National Natural Science Foundation of China (Grant No. 30621092), and Natural Science Foundation of variety of ecological niches for T. matsutake, and in the Yunnan Province (Grant No. 2005PG11) www.scichina.com www.springerlink.com Chinese Science Bulletin | May 2007 | vol. 52 | no. 9 | 1212-1216

often used in comparisons of intraspecific variation[7]. 3′)/ITS5(5′-GGAAGTAAAAGTCGTAACAAGG-3′)[11] [8]

Guerin-Laguette et al. have revealed high variation in was used to amplify the ITS region, 5SA-Anderson ARTICLES IGS1 restriction fragment length polymorphism (RFLP) (5′-CAGAGTCCTATGGCCGTGGAT-3′)/CNL12 (5′- from T. matsutake collected from different regions of CTGAACGCCTCTAAGTCAG-3′)[8] was used to am- Japan. In addition, retrotransposons generally present plify the IGS1 region, and pS48 (5′-GAGGTGGGGA- multiple copies in fungal genomes and can transfer be- AAAATATGGGACGAAC-3′)/pL281 (5′-CTTCACAT- tween species or across generations through lateral and [10] ATACTGGGCATCAGCAAGGG-3′) was used to vertical transfer, respectively. Consequently, high se- examine retrotransposon PCR polymorphism. quence heterogeneity can be found even in the same family of retrotransposons. Because of these qualities, Table 1 Geographic distribution of T. matsutake in Yunnan, China IRAP (inter-retrotransposon-amplified polymorphism) is No. of fruit body Region County Location TF 31－35 Diqing Weixi Northwest also widely used to analyze the genetic diversity of or- TF 41, TF 43－46 Diqing Zhongdian [9] [10] ganisms such as barley . Murata et al. found a high TF 100, TF 102－106 Diqing Deqin degree of polymorphism in retrotransposons in T. ma- TF 51, TF 53－56 tsutake between Asian and American regions. TF 61, 62, TF 65, TF 66 Dali Yongping West Using the three types of genetic markers described TF 84, TF 87－89 Dali Jianchuan TF 127 Baoshan Longling above, we compared genetic variation between Yunnan TF 110, TF 112－114, TF 116 Nujiang Lanping and Japanese populations, and here we provided pre- TF 134－136 Lincang liminary results that will aid in further exploration of the TF 74－76, TF 78, TF 79 Chuxiong Nanhua symbiosis between mushroom and host plant, restoration TF 154, TF 156－159 Chuxiong Lufeng Central of the damaged habitat, and improvement of artificial TF 94, TF 97－99 Kunming Luquan TF 140－142, TF 153 Yimen reproduction techniques.

1 Materials and methods 1.3 Extraction of genomic DNA The CTAB method was used to extract genomic DNA. 1.1 Sample sources 1.4 PCR amplification Samples were collected from 13 counties located in 9 regions of Yunnan Province (Table 1 and Figure 1). Fruit All PCRs were performed in a 50-μL reaction mixture body TF9 was collected from , Kun- containing 5 μL 10× buffer, 2 μL dNTP (25 mmol/L ming. each), 0.25 μL Taq enzyme (Taq Ex enzyme for ampli- fication of retrotransponson, 5 U/μL) (TaKaRa, Dalian, China), 1 μL DNA template, 1 μL of each primer, and a layer of mineral oil. PCR amplification of the ITS region was included the following steps: initial denaturation at 94℃ for 2 min; 40 cycles of 94℃ for 40 s, 52℃ for 60 s, 72℃ for 60 s; and a final extension step at 72℃ for 5 min. IGS1 region amplification included: initial denatu- GENETICS ration at 94℃ for 5 min; 35 cycles of 94℃for 40 s, 63℃ for 60 s, 72℃ for 180 s; and a final extension step at 72℃ for 10 min. Retrotransponson PCR was conducted as follows: initial denaturation at 94℃ for 2 min; 25 cycles of 94℃ for 40 s, 62℃ for 30 s, 72℃ for 300 s; and a final extension step at 72℃ for 10 min. All PCR products were visualized with electrophoresis (1.8% of Figure 1 Geographic distribution of samples in Yunnan, China. agar) and EB staining. 1.2 PCR primers 1.5 Transformation and cloning Primer pair ITS4 (5′-TCCTCCGCTTATTGATATGC- Purified PCR products were ligated into the pMD18-T

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vector (TaKaRa) and transformed into competent cells structed based on these haplotypes and 10 haplotypes (Escherichia coli HB101). Positive clones were screened from Japan T. matsutake (Figure 2). The results showed as white colonies in selective solid LB medium contain- that all T. matsutake formed a branch distinct from TF9, ing 100 μg/mL Amp, 80 μg/mL X-Gal and 0.5 mmol/L and T. matsutake from Yunnan and Japan were highly isopropylthiogalactoside (IPTG). The insert DNA frag- homogenous. Moreover, comparing our results with ment was then sequenced. IGS1 Cfr13I digestion patterns revealed by a Japan population, we found that all fruit bodies belonged to 1.6 DNA sequencing and analysis type A, with the exception of TF89, which belonged to ITS and IGS1 regions were sequenced with an ABI type C[8]. DNA3700 sequencer. The sequences were carefully checked and primer sequences were deleted using Edit- seq and Seqman of the DNAstar package. The closest relatives were searched for and downloaded using a BLAST search of the NCBI database. A phylogenetic tree was constructed using the Mega3 package, using the Kimura two-parameter evolutionary model and the neighbor-joining method. Tricholoma bakamatsutake TF9 was used as an out-group and 1000 bootstrap repli- cates were included in the analysis. 1.7 Analysis of retrotransposon Based on electrophoresis patterns, the presence (1) or absence (0) of major bands was recorded in a binary matrix for each sample. These patterns were combined Figure 2 IGS neighbor-joining tree of T. matsutake from Yunnan and and the genetic distances were calculated using the Japan. AF280439, AF280443, AF280445, AF2804446, AF280449, Popgene program. The phylogenetic tree was con- AF280450, AF280451, AF280453, AF280454 and AF356188 are T. ma- structed using a UPGMA method in Mega3. tsutake from Japan; TF49, TF89c3 and TF89c7 are T. matsutake from Yunnan Province, China; TF9 is Tricholoma bakamatsutake and is used as out-group. 2 Results 2.2 Analysis of IRAP 2.1 Analysis of ITS and IGS1 The phylogenetic tree suggested that there was little We sequenced ITS and IGS1 fragments from 56 fruit IGS1 variation between geographically close popula- bodies collected from 13 counties of Yunnan province. tions (Figures 3 and 4). For example, Weixi, Zhongdian All ITS sequences presented as one haplotype, TF46 and Deqin counties from Diqing region grouped into a (DQ323063), identical to those from Japan. Among clade at D=0.0001, Lijiang and Dali region (Yongping IGS1 sequences, 55 presented one haplotype and Jianchuan counties) formed a clade at D=0.0000, (DQ323058). TF89 was heterozygous and separated into and Baoshan (), Linchang (Lichang- two haplotypes TF89c3 (DQ323060) and TF89c7 County), Chuxiong region (Nanhua and Lufeng coun- (DQ323062) by cloning. A phylogenetic tree was con- ties), Kunming (Luquan County) and Yuxi region-

Figure 3 PCR amplification of retrotransposon from Yunnan T. matsutake.

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pecies and intrapopulation genetic diversity estimated

with the Shannon index (I) was 0.0769 and 0.0741, re- ARTICLES spectively. These data showed that, of the total genetic variation, 96.4% was intrapopulation variation whereas only 3.6% was interpopulation variation. The total in- traspecies and intrapopulation gene diversity estimated

with the Nei index (Ht) was 0.0554 and 0.0527, respec- tively. Further, the gene differentiation coefficient (Gst) was 0.0464, suggesting that 95.36% of the genetic di- versity was found within populations, while only 4.64% was found between populations. Additionally, migration of each generation among populations was 10.2778, in- Figure 4 Genetic relationships among 13 T. matsutake populations. dicating significant gene flow among populations. grouped into a clade at D=0.0000. This phylogeographic We concluded that total genetic variation among these pattern was most likely related to differences in altitude populations was very low and that most variation was or latitude. For example, Diqun and Lanping from Nu- found within populations. This was also supported by jiang region (with an altitude of 2500―3500 m) formed the 99% similarity coefficient in Table 3. a clade at D=0.0002. This clade then clustered with Li- 3 Discussion jiang and Dali region (2500―2900 m) at D=0.0005. Several subpopulations from relatively low altitude, for We detected only one haplotype from the ITS sequences example, Kunming (1700 ― 1900 m) and Chuxiong of Yunnan T. matsutake populations and this result was (1900―2100 m), formed a branch at D=0.0002, and congruent with that of a previous study[14]. For IGS1 [8] then clustered with Baoshan (2000―2500 m) and Lin- sequences, Guerin-Laguette et al. reported eight types chang (2100―2600 m) at D=0.0007. Finally, these two of Cfr13I restriction fragments. In 2005, Matsushita et [14] clades grouped together at D=0.0023 to form a larger al. found that there were three types (type A―C) in clade. four Jilin province fruit bodies, whereas only type A was Genetic variation between different populations from detected in 12 Yunnan fruit bodies. A previous report Yunnan was very low (Table 2). On average the propor- indicated that type A was the most common type, ac- tion of polymorphism bands (PPB) was 11.11%. For counting for 66.67% of Japanese T. matsutake. Our re- each gene locus, the observed allele number was 1.1111 sults revealed three haplotypes from IGS1 sequences of and the effective allele number was 1.1105. The intras- Yunnan T. matsutake; type A accounted for 98.21% of

Table 2 Genetic diversity and structure of 13 T. matsutake populations from Yunnana)

population PPB (%) Na Ne H I Ht Hs Gst Nm 11.11 1.1111 1.1105 0.0554 0.0769 0.0554 0.0527 0.0464 10.2779

Weixi 11.11 1.1111 1.1039 0.0537 0.0751 GENETICS Zhongdian 11.11 1.1111 1.1039 0.0537 0.0751 Lijiang 11.11 1.1111 1.1111 0.0556 0.0770 Yongping 11.11 1.1111 1.1111 0.0556 0.0770 Nanhua 11.11 1.1111 1.1059 0.0542 0.0757 Jianchuan 11.11 1.1111 1.1111 0.0556 0.0770 Luquan 11.11 1.1111 1.0965 0.0517 0.0731 Deqin 11.11 1.1111 1.0986 0.0522 0.0737 Lanping 11.11 1.1111 1.1087 0.0549 0.0764 Longling 11.11 1.1111 1.0786 0.0460 0.0672 Lincang 11.11 1.1111 1.0786 0.0460 0.0672 Yuxi 11.11 1.1111 1.0965 0.0517 0.0731 Lufeng 11.11 1.1111 1.1059 0.0542 0.0757 [12] [13] a) Na, The observed allele number; Ne, the effective allele number; H, Nei gene diversity ; I, Shannon diversity index ; Gst, Nei gene differentiation coefficient; Nm, estimate of gene flow from Gst.

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Table 3 Nei’s genetic similarity (upper triangle) and distance (down triangle) pop ID Weixi Zhongdian Lijiang Yongping Nanhua Jianchuan Luquan Deqin Lanping Longling Lincang Yuxi Lufeng Weixi **** 1.0000 0.9990 0.9990 0.9966 0.9990 0.9941 0.9999 0.9998 0.9896 0.9896 0.9941 0.9966 Zhongdian 0.0000 **** 0.9990 0.9990 0.9966 0.9990 0.9941 0.9999 0.9998 0.9896 0.9896 0.9941 0.9966 Lijiang 0.0010 0.0010 **** 1.0000 0.9993 1.0000 0.9979 0.9983 0.9997 0.9950 0.9950 0.9979 0.9993 Yongping 0.0010 0.0010 0.0000 **** 0.9993 1.0000 0.9979 0.9983 0.9997 0.9950 0.9950 0.9979 0.9993 Nanhua 0.0034 0.0034 0.0007 0.0007 **** 0.9993 0.9996 0.9953 0.9980 0.9980 0.9980 0.9996 1.0000 Jianchuan 0.0010 0.0010 0.0000 0.0000 0.0007 **** 0.9979 0.9983 0.9997 0.9950 0.9950 0.9979 0.9993 Luquan 0.0059 0.0059 0.0021 0.0021 0.0004 0.0021 **** 0.9924 0.9960 0.9994 0.9994 1.0000 0.9996 Deqin 0.0001 0.0001 0.0017 0.0017 0.0047 0.0017 0.0076 **** 0.9994 0.9873 0.9873 0.9924 0.9953 Lanping 0.0002 0.0002 0.0003 0.0003 0.0020 0.0003 0.0040 0.0006 **** 0.9921 0.9921 0.9960 0.9980 Longling 0.0105 0.0105 0.0050 0.0050 0.0020 0.0050 0.0006 0.0127 0.0079 **** 1.0000 0.9994 0.9980 Lincang 0.0105 0.0105 0.0050 0.0050 0.0020 0.0050 0.0006 0.0127 0.0079 0.0000 **** 0.9994 0.9980 Yuxi 0.0059 0.0059 0.0021 0.0021 0.0004 0.0021 0.0000 0.0076 0.0040 0.0006 0.0006 **** 0.9996 Lufeng 0.0034 0.0034 0.0007 0.0007 0.0000 0.0007 0.0004 0.0047 0.0020 0.0020 0.0020 0.0004 **** fruit bodies (55/56) and only one fruit body (1/56) be- Central and northwest Yunnan, and northwest Guizhou) longed to type C. In total, Yunnan T. matsutake showed a is probably the modern distribution centre of T. matsu- very low level of genetic diversity. It is well known that take[4]. Chapel and Garbelotto[17] reported that T. matsu- Jilin and Japanese T. matsutake exist mainly in symbio- take in Asia is derived from the migration of T. matsu- sis with Pine densiflora[15,16]. However, the major host take from western North America through the Bering plant is P. yunnanensis for Yunnan T. matsutake. Cur- Strait. Generally, the population from the origin centre rently, whether or not such differences in host plants of a species always shows a higher level of genetic di- [18] influence genetic differentiation among different T. ma- versity than populations from other regions . There- tsutake populations is an open question. Further study is fore, in comparison to Jilin and Japanese populations, a needed to address this issue. lower level of genetic diversity in Yunnan populations Some researchers have suggested that the southeast did not support the view that T. matsutake originated delta region of Hengduan Mountain in China (including from Yunnan. southeastern Qinghai-Tibetan plateau, western Sichuan, The authors are grateful to Dr. CHEN ShanYuan for helpful suggestions.

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