Genetic Diversity and Matrilineal Structure in Chinese Tree Shrews Inhabiting Kunming, China CHEN Shi-Yi 1, XU Ling 1,3, LÜ Long-Bao 2, YAO Yong-Gang 1,*

动物学研究 2011，Feb. 32(1): 17−23 CN 53-1040/Q ISSN 0254-5853 Zoological Research DOI：10.3724/SP.J.1141.2011.01017 Genetic diversity and matrilineal structure in Chinese tree shrews inhabiting Kunming, China CHEN Shi-Yi 1, XU Ling 1,3, LÜ Long-Bao 2, YAO Yong-Gang 1,* (1. Key Laboratory of Animal Models and Human Disease Mechanisms of the Chinese Academy of Sciences & Yunnan Province, Kunming Institute of Zoology, Kunming Yunnan 650223, China; 2. Experimental Animal Core Facility & Kunming Primate Research Center, Kunming Institute of Zoology, the Chinese Academy of Sciences, Kunming Yunnan 650223, China; 3. Graduate School of the Chinese Academy of Sciences, Beijing 100049, China) Abstract: Due to their special phylogenetic position in the Euarchontoglires and close affinity to primates, tree shrews have been proposed as an alternative experimental animal to primates in biomedical research. However, the population genetic structure of tree shrews has largely remained unknown and this has hindered the development of tree shrew breeding and selection. Here we sampled 80 Chinese tree shrews (Tupaia belangeri chinensis) in Kunming, China, and analyzed partial mtDNA control region sequence variation. Based on our samples and two published sequences from northern tree shrews (T. belangeri), we identified 29 substitutions in the mtDNA control region fragment (~ 604 bp) across 82 individuals and defined 13 haplotypes. Seventeen samples were selected for sequencing of the cytochrome b (Cyt b; 1134 bp) gene based on control region sequence variation and were analyzed in combination with 34 published sequences to solidify the phylogenetic pattern obtained from control region data. Overall, tree shrews from Kunming have high genetic diversity and present a remarkable long genetic distance to the two reported northern tree shrews outside China. Our results provide some caution when using tree shrews to establish animal models because of this apparent genetic difference. In addition, the high genetic diversity of Chinese tree shrews inhabiting Kunming suggests that systematic genetic investigations should be conducted before establishing an inbred strain for medical and biological research. Key words: Chinese tree shrews; mtDNA; Control region; Cytochorme b; Genetic diversity 昆明城郊中国树鼩群体线粒体 DNA 遗传多样性陈仕毅 1, 许凌 1,3, 吕龙宝 2, 姚永刚 1,* (1. 中国科学院和云南省动物模型与人类疾病机理重点实验室, 中国科学院昆明动物研究所, 云南昆明 650223; 2. 中国科学院昆明动物研究所实验动物中心, 云南昆明 650223; 3. 中国科学院研究生院, 北京 100049) 摘要：由于树鼩是灵长类动物的近亲, 且具有体型小、繁殖周期短、饲养管理成本低等优点, 长期以来被认为有望替代灵长类动物用于人类疾病的动物模型研究。然而, 目前对树鼩的群体遗传结构还知之甚少, 这极大地限制了其在疾病动物模型研究的应用, 也是其品系资源创制的瓶颈。本研究通过分析 80 只采自于云南省昆明周边地区的野生树鼩 (Tupaia belangeri chinensis) 线粒体 DNA (mtDNA) 多态性, 结合国外报道的 2 个树鼩 (Tupaia belangeri) 序列比较后发现, 在 604 bp 的 mtDNA 控制区片段中共检测到 29 个核苷酸替代变异, 这些变异共界定了 13 种单倍型, 表现较高的群体遗传多样度。另外, 昆明地区的树鼩与国外报道的 2 个树鼩间存在较大的遗传分化, mtDNA 控制区单倍型之间的核苷酸替换数大于 18 个, 远高于昆明地区树鼩群体内部不同单倍型之间的差异。选择含有代表性的 mtDNA 控制区单倍型的 17 个昆明地区树鼩个体进一步测定了细胞色素 b 基因片段 (1134 bp), 结合前人报道的数据分析, 结果进一步支持 mtDNA 控制区数据反映的遗传格局及揭示的昆明地区树鼩与国外报道树鼩之间的明显差异。本研究结果提示, 昆明地区树鼩与国外树鼩之间存在较大遗传差异, 在将树鼩用于人类疾病动物模型研究中要注意这些遗传差别。昆明城郊的树鼩群体具有较高的遗传多样度, 在开展近交系建立等工作时须考虑选取群体内部具有代表性的 mtDNA 世系。 Received date: 2010-12-02; Accepted date: 2010-12-27 Foundation items: This study was supported by grants from Yunnan Province (2009CI119), the Chinese Academy of Sciences (KSCX2-EW-R-11 and KSCX2-EW-J-23) and the Key Laboratory of Animal Models and Human Disease Mechanisms of the Chinese Academy of Sciences & Yunnan Province * Corresponding author (通信作者), Email: [email protected] 收稿日期：2010-12-02；接受日期：2010-12-27 18 Zoological Research Vol. 32 关键词：中国树鼩; 线粒体 DNA; 高变区; 细胞色素 b; 遗传多样性中图分类号：Q959.832; Q951.3; Q347 文献标志码：A 文章编号：0254-5853-(2011)01-0017-07 Tree shrews (Tupaia belangeri) are small mammals south Yunnan, northwest Guangxi and southwest of a squirrel-like appearance formerly placed in the Guizhou. Recently, Jia et al (2009) studied the Primates order despite a lack of derived features morphometrics of the skull and mandible of T. b. characteristic of primate species (Sargis, 2004). It is chinensis collected in Luquan, Jianchuan, Lijiang, and currently placed in the family of Tupaiidae, Scandentia, Yunlong Counties of Yunnan and morphological which together with Dermoptera and Primates, form the differences between localities. In combination these group Euarchonta (Murphy et al, 2001; Helgen, 2005). studies suggest that Chinese tree shrews show regional Although the exact phylogenetic position of tree shrews differentiation. remains resolved, this animal is still regarded as a close Despite growing attention and the strong potential relative to primates (Nie et al, 2008; Peng et al, 1991; of tree shrews as an animal model for human disease, Seiffert et al, 2003). Given that tree shrews are easy to scant biological data about these species have been raise, cheap to maintain, have a high reproductive rate, collected, especially at the molecular level (Peng et al, small body size, and close affinity to primates, this 1991). This deficit has constrained the utilization of tree animal may be an alternative to the use of primates in shrews as an animal model in biomedical studies. biomedical research. For example, tree shrews have been Although the critical problem regarding laboratory used in creating animal models for hepatitis B virus and breeding and domestication of wild tree shrews was hepatitis C virus infection (Amako et al, 2010; Cao et al, solved more than two decades ago (Peng et al, 1991), an 2003; Köck et al, 2001; Ren & Nassal, 2001; Su, 1987; inbred strain of tree shrews has not been established Xu et al, 2007; Yan et al, 1996). Tree shrews were also because, among other factors, of a lack of genetic data. successfully used in the study of pathogenesis of human Here we aimed to analyze the genetic structure of myopia and psychosocial stress diseases (Cao et al, 2003; tree shrews collected from urban Kunming, Yunnan, Fuchs, 2005; Norton et al, 2006). In addition, tree shrews China to provide useful information to establish several have the highest brain to body ratio of all mammals, inbreeding lines from these captured animals. We used including humans (Peng et al, 1991; Previc, 2009), which mitochondrial DNA (mtDNA) as a genetic marker as it is raises the possibility of investigating brain function and useful in revealing the origin and demographic history creating animal models for human neuro-degenerative and overall genetic diversity of domestic and wild diseases. species (He et al, 2008). There are four genera within the family Tupaiidae, 1 Materials and Methods consisting of Tupaia, Anathana, Urogale, and Dendrogale (Helgen, 2005). In genus Tupaia, a total of 1.1 Sampling, DNA amplification and sequencing 15 species are recognized and broadly distributed across We collected ear or muscle tissue from 80 Chinese Southeast Asia including southern China, India, tree shrews (T. b. chinensis) inhabiting the suburb of Philippines, Java, Borneo, Sumatra and Bali (Olson et al, Kunming, Yunnan, China. Animals were raised at the 2005; Peng et al, 1991). Chinese tree shrews (Tupaia experimental animal core facility of the Kunming belangeri) are distributed across southwest China. Institute of Zoology and procedures were approved by According to geographical distribution and the Institutional Animal Care and Use Committee of the morphological characteristics, Wang (1987) proposed Kunming Institute of Zoology, the Chinese Academy of that Chinese tree shrews could be divided into six Sciences. subspecies: Tupaia belangeri chinensis distributed in Genomic DNA was extracted from tissue samples most of parts of Yunnan, Yun-Gui plateau, and using the standard phenol/chloroform method. A southwest Sichuan; T. b. gaoligongensis distributed in fragment with a size of 1 488 bp that covers the entire central and north Gaoligong Mountain; T. b. modesta on mtDNA control region of Chinese tree shrews was Hainan Island; T. b. tonquinia distributed in southwest amplified using primer pairs L15356: 5'-CTCAAGGAA Guangxi; T. b. yaoshanensis distributed in northwest GAAGAACAATA-3' / H50: 5'-TGTATGTTTATGGAG Guangxi; and T. b. yunalis distributed in central and TCTATG-3'. Primers were named relative to their No. 1 CHEN Shi-Yi et al: Genetic diversity and matrilineal structure in Chinese tree shrews inhabiting Kunming, China 19 positions in the complete mtDNA sequence of northern We analyzed a fragment of mtDNA control region tree shrew (GenBank accession number NC_002521). (~604 bp) in 80 Chinese tree shrews, corresponding to PCR amplification was performed in a volume of 50 µL 15416th–16017th region in mitochondrial genome of T. reaction mixture containing 100 ng of DNA, 10 mmol/L belangeri (GenBank accession number NC_002521). Tris-HCl (pH 8.3), 2.5 mmol/L MgCl2, 50 mmol/L KCl, Two reported individuals of northern tree shrews (Tupaia 10 pmol/L of each primer, and one unit of LA Taq belangeri) outside China were retrieved from GenBank polymerase (Takara, Dalian, China) following 35 cycles and were aligned with those of Chinese tree shrews (Tab. of 50 s at 94°C, 50 s at 55°C and 60 s at 72°C. The 1 and Fig. 1). Sequence variations were exported using complete mitochondrial Cyt b gene was amplified and Mega 4.0 (Tamura et al, 2007). The available Cyt b gene sequenced in 17 Chinese tree shrews representing sequences of tree shrews in GenBank, including 32 different haplotypes as identified by mtDNA control Chinese tree shrews and two northern tree shrews outside region data. We used primer pair L14058: 5'- China (Tab. 1), were also analyzed together

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