中缅树鼩微卫星分子标记的筛选及特征分析
|
摘要
树鼩作为新兴的医学实验动物之一,因其在进化地位上与灵长类动物较为接近,体型小,繁殖快,易驯养和饲育,以及存在诸多自发性疾病等特性,可作为某些人类重大疾病研究的动物模型,在医学生物学上已呈现出广泛的应用前景。目前,树鼩已被应用于病毒、神经、免疫、肿瘤和神经性疾病的研究,但表现出对其应用研究多于基础研究的状态,遗传背景研究就更为少见,不利于实现树鼩实验动物标准化。微卫星是近十几年来发展起来的一种新的分子标记,它是指以少数几个核苷酸(1-6个)为单位多次重复的简单序列。由于微卫星遗传标记具有数量大、分布广且均匀、多态信息含量高、检测快速以及适合自动化分析等优点,已被广泛用于动植物基因图谱的构建、标记辅助选择、亲缘关系鉴定、遗传多样性评估及系统进化树的构建等。然而,当前树鼩的微卫星分子标记还很少,因此,筛选出可以应用的微卫星位点是一项非常基础和重要的工作。本研究通过构建树鼩小片段插入文库,利用地高辛标记(AC)15序列作为探针,筛选树鼩微卫星位点,丰富了中缅树鼩的特异性遗传标记,为进一步筛选大量的树鼩微卫星遗传标记奠定了基础,也为开展树鼩繁育工作与实现实验动物标准化提供一定的理论基础和遗传检测方法。
提取树鼩总DNA,限制性内切酶Sau 3AⅠ酶切,回收300-1000 bp之间的片段,连接质粒,转化入Top10感受态细胞中,蓝白斑筛选后培养扩增,建立中缅树鼩基因小片段插入文库。利用5’端地高辛标记的(AC)15探针进行菌落原位杂交,从约1500个菌落中选出36个阳性克隆。对这些克隆进行测序,发现其中15个含有重复序列,其中1个为重复克隆,1个因两端序列太短而不能设计引物。用Primer3软件设计13对引物。对树鼩种群进行PCR验证引物有效性,13对引物均有条带。退火温度分布在44℃到52℃之间。阳性克隆率为2.4%,微卫星克隆率为1%。本研究所使用的地高辛标记探针筛选树鼩微卫星分子标记所得的微卫星克隆率,可达到传统放射性同位素标记探针同等的效果,并可避免放射性危害。树鼩微卫星分子标记的筛选将为下一步进行基因组结构的分析、树鼩遗传连锁图谱的构建、分子进化和标记辅助选择等提供大量的微卫星标记。
为了进一步研究中缅树鼩微卫星位点多态性特征,结合微卫星DNA标记具有高度的多态性、共显性遗传、易于PCR稳定扩增等特点,用新筛选的13个微卫星座位对32只中缅树鼩进行PCR扩增及12%非变性聚丙烯酰胺凝胶电泳检测微卫星DNA的扩增产物,并计算其等位基因数、等位基因频率、多态性信息含量(PIC)、基因杂合度等指标。13个微卫星基因座共检测到77个等位基因,平均等位基因数为5.9231。TBC13座位的杂期望合度值(H)最高,为0.8646,最低为TBC11的0.5060,平均期望杂合度为0.7894。各微卫星座位的多态信息含量值(PIC)在0.3740-0.8324之间,平均值为0.7396,表明该树鼩种群具有较高的遗传多样性。结果表明,筛选的13个微卫星基因座具有较丰富的遗传多态性,可用于中缅树鼩的遗传多样性分析。
研究表明,该树鼩种群具有较丰富的遗传多态性；本研究建立树鼩小片段插入基因组文库,筛选出13个微卫星位点,并对这些位点进行了特征分析和多态性评估。多态性较高的树鼩种群有利于动物的繁殖育种,但要广泛应用于生物医学研究中还需要进一步推进实验动物标准化进程,以保证实验结果的一致性和可靠性。
As one of emerging animal models, Tree Shrews have been applied widely for biomedical research. These animals are phylogenetically close to primates in evolution. More characteristics were exhibited from the Tree Shrews, such as small size, easy to domestication, good reproduction ability, spontaneous or induced human-like diseases, especially as experimental animal model for HBV, HCV infection. Tree Shrews have been applied for lots of studies including virology, neurology, immunology, oncology and social biology and so on. However, people showed more interest to their applications research than the animal essential.Especially, there were rare studies about their genetic background, so that Tree Shrews were limited to perform standardization and application as experimental animal. Microsatellites have come into prominence over the last decade.They are simple tandemly repeated DNA sequence elements, the repeat unit including 1-6 nucleotides. Because microsatellite markers have many advantages, such as large quantities, widely and evenly distributed, high polymorphic information, rapid detection, fitted for automated analysis and so on. They have been applied widely for construction of gene mapping, marker-assisted selection, identification of genetic relationship, assessment of genetic diversity, construction of phylogenetic tree, et al. So, it is extremely basis and important to isolate those microsatellite markers in Tree Shrews due to its lack. A partial genomic library was constructed in Tree Shrews and microsatellite markers were isolated with (AC)15 probe labelled at the 5'end with Digoxin in this study. The specific genetic informations should be complement gradually, base on screening more microsatellite markers of Tree Shrews, and it could provide the theoretical basis and genetic detective methods for us to promote the standardization of laboratory Tree Shrews.
Extracted DNA from tree shrews(Tupaia Belangeri Chinensis), digested DNA by restriction enzymes Sau3A I, retrieved fragments between 300-1000 bp. The fragments connected into plasmid, then transformed into Top 10 competent cells.Made blue-white screening to construct a partial genomic library in Tree Shrews.36 positive clones were isolated from screening about 1500 clones of the genomic library by colony hybridization with (AC)15 probe labelled at the 5'end with Digoxin.Sequence these clones and 15 microsatellites were isolated which included one repeated clones and one short flank sequence.The other 13 primers were designed based on unique sequences flanking each motif with the software Primer3. PCR were performed with these primers, and all gave expected bands. Annealing temperature of these primers was between 44℃and 52℃. So the positive cloning efficiency is 2.4%, and the microsatellite cloning efficiency is 1%. The microsatellite cloning efficiency of isolating microsatellite markers in Tree Shrews with probe labelled by Digoxin is almost the same as probe labelled by radioisotope, and avoid the radioactive contamination.The new polymorphic microsatellite markers we have identified and characterized will contribute to the Tree Shrews genetic linkage mapping, molecular evolution and marker assistant selection.
With the advantages of the microsatellite DNA maker, such as high polymorphism, codominant inheritance, and PCR amplification easily and steadily, we established the microsatellite genetic maker method to further analyze the genetic polymorphism of the new isolated microsatellite loci. PCR optimization was made in 32 Tree Shrews using 13 pairs of the primers.The PCR products were testd by 12% non-denaturing (neutral) polyacrylamide gel electrophoresis. Then the genetic index for 13 microsatellite loci, such as the number of alleles, allele frequency, polymorphism information content (PIC), heterozygosity were calculated.77 alleles were detected from the Tree Shrews population in 13 microsatellite loci. The average number of alleles was 5.9231.The expected heterozygosity of TBC13 was the highest 0.8646, the lowest for the TBC11 of 0.5060, the average of 0.7894. The polymorphism information content (PIC) of 13 microsatellite loci ranged from 0.3740 to 0.8324, with a mean of 0.7396. It showed that the Tree Shrews population had higher genetic diversity. The results indicated that the selected 13 microsatellite loci with high genetic polymorphism could be used to analyze the genetic diversity of the Tree Shrews population.
These results showed that the Tree Shrews population had higher genetic polymorphism. Here, a partial genomic library was constructed in Tree Shrews,13 microsatellites were isolated, and analyzed the feature and genetic polymorphism of the new isolated microsatellite loci. The high polymorphism of Tree Shrews is propitious to animal breeding, but using these animals more extensively in the biomedical research, we also need to promote the standardization of laboratory animals to ensure the consistency and reliability of experimental results.
提取树鼩总DNA,限制性内切酶Sau 3AⅠ酶切,回收300-1000 bp之间的片段,连接质粒,转化入Top10感受态细胞中,蓝白斑筛选后培养扩增,建立中缅树鼩基因小片段插入文库。利用5’端地高辛标记的(AC)15探针进行菌落原位杂交,从约1500个菌落中选出36个阳性克隆。对这些克隆进行测序,发现其中15个含有重复序列,其中1个为重复克隆,1个因两端序列太短而不能设计引物。用Primer3软件设计13对引物。对树鼩种群进行PCR验证引物有效性,13对引物均有条带。退火温度分布在44℃到52℃之间。阳性克隆率为2.4%,微卫星克隆率为1%。本研究所使用的地高辛标记探针筛选树鼩微卫星分子标记所得的微卫星克隆率,可达到传统放射性同位素标记探针同等的效果,并可避免放射性危害。树鼩微卫星分子标记的筛选将为下一步进行基因组结构的分析、树鼩遗传连锁图谱的构建、分子进化和标记辅助选择等提供大量的微卫星标记。
为了进一步研究中缅树鼩微卫星位点多态性特征,结合微卫星DNA标记具有高度的多态性、共显性遗传、易于PCR稳定扩增等特点,用新筛选的13个微卫星座位对32只中缅树鼩进行PCR扩增及12%非变性聚丙烯酰胺凝胶电泳检测微卫星DNA的扩增产物,并计算其等位基因数、等位基因频率、多态性信息含量(PIC)、基因杂合度等指标。13个微卫星基因座共检测到77个等位基因,平均等位基因数为5.9231。TBC13座位的杂期望合度值(H)最高,为0.8646,最低为TBC11的0.5060,平均期望杂合度为0.7894。各微卫星座位的多态信息含量值(PIC)在0.3740-0.8324之间,平均值为0.7396,表明该树鼩种群具有较高的遗传多样性。结果表明,筛选的13个微卫星基因座具有较丰富的遗传多态性,可用于中缅树鼩的遗传多样性分析。
研究表明,该树鼩种群具有较丰富的遗传多态性；本研究建立树鼩小片段插入基因组文库,筛选出13个微卫星位点,并对这些位点进行了特征分析和多态性评估。多态性较高的树鼩种群有利于动物的繁殖育种,但要广泛应用于生物医学研究中还需要进一步推进实验动物标准化进程,以保证实验结果的一致性和可靠性。
As one of emerging animal models, Tree Shrews have been applied widely for biomedical research. These animals are phylogenetically close to primates in evolution. More characteristics were exhibited from the Tree Shrews, such as small size, easy to domestication, good reproduction ability, spontaneous or induced human-like diseases, especially as experimental animal model for HBV, HCV infection. Tree Shrews have been applied for lots of studies including virology, neurology, immunology, oncology and social biology and so on. However, people showed more interest to their applications research than the animal essential.Especially, there were rare studies about their genetic background, so that Tree Shrews were limited to perform standardization and application as experimental animal. Microsatellites have come into prominence over the last decade.They are simple tandemly repeated DNA sequence elements, the repeat unit including 1-6 nucleotides. Because microsatellite markers have many advantages, such as large quantities, widely and evenly distributed, high polymorphic information, rapid detection, fitted for automated analysis and so on. They have been applied widely for construction of gene mapping, marker-assisted selection, identification of genetic relationship, assessment of genetic diversity, construction of phylogenetic tree, et al. So, it is extremely basis and important to isolate those microsatellite markers in Tree Shrews due to its lack. A partial genomic library was constructed in Tree Shrews and microsatellite markers were isolated with (AC)15 probe labelled at the 5'end with Digoxin in this study. The specific genetic informations should be complement gradually, base on screening more microsatellite markers of Tree Shrews, and it could provide the theoretical basis and genetic detective methods for us to promote the standardization of laboratory Tree Shrews.
Extracted DNA from tree shrews(Tupaia Belangeri Chinensis), digested DNA by restriction enzymes Sau3A I, retrieved fragments between 300-1000 bp. The fragments connected into plasmid, then transformed into Top 10 competent cells.Made blue-white screening to construct a partial genomic library in Tree Shrews.36 positive clones were isolated from screening about 1500 clones of the genomic library by colony hybridization with (AC)15 probe labelled at the 5'end with Digoxin.Sequence these clones and 15 microsatellites were isolated which included one repeated clones and one short flank sequence.The other 13 primers were designed based on unique sequences flanking each motif with the software Primer3. PCR were performed with these primers, and all gave expected bands. Annealing temperature of these primers was between 44℃and 52℃. So the positive cloning efficiency is 2.4%, and the microsatellite cloning efficiency is 1%. The microsatellite cloning efficiency of isolating microsatellite markers in Tree Shrews with probe labelled by Digoxin is almost the same as probe labelled by radioisotope, and avoid the radioactive contamination.The new polymorphic microsatellite markers we have identified and characterized will contribute to the Tree Shrews genetic linkage mapping, molecular evolution and marker assistant selection.
With the advantages of the microsatellite DNA maker, such as high polymorphism, codominant inheritance, and PCR amplification easily and steadily, we established the microsatellite genetic maker method to further analyze the genetic polymorphism of the new isolated microsatellite loci. PCR optimization was made in 32 Tree Shrews using 13 pairs of the primers.The PCR products were testd by 12% non-denaturing (neutral) polyacrylamide gel electrophoresis. Then the genetic index for 13 microsatellite loci, such as the number of alleles, allele frequency, polymorphism information content (PIC), heterozygosity were calculated.77 alleles were detected from the Tree Shrews population in 13 microsatellite loci. The average number of alleles was 5.9231.The expected heterozygosity of TBC13 was the highest 0.8646, the lowest for the TBC11 of 0.5060, the average of 0.7894. The polymorphism information content (PIC) of 13 microsatellite loci ranged from 0.3740 to 0.8324, with a mean of 0.7396. It showed that the Tree Shrews population had higher genetic diversity. The results indicated that the selected 13 microsatellite loci with high genetic polymorphism could be used to analyze the genetic diversity of the Tree Shrews population.
These results showed that the Tree Shrews population had higher genetic polymorphism. Here, a partial genomic library was constructed in Tree Shrews,13 microsatellites were isolated, and analyzed the feature and genetic polymorphism of the new isolated microsatellite loci. The high polymorphism of Tree Shrews is propitious to animal breeding, but using these animals more extensively in the biomedical research, we also need to promote the standardization of laboratory animals to ensure the consistency and reliability of experimental results.
引文
[1]Nie W, Fu B, O'Brien PC, et al. Flying lemurs--the'flying tree shrews'? Molecular cytogenetic evidence for a Scandentia-Dermoptera sister clade[J]. BMC Biol, 2008,6:18.
[2]刘世熠,张文远,王泽盛,等.树鼩24小时昼夜节律的探讨[J].动物学报,1982,28(24)：399-407.
[3]T. L. Yate. Insectivores, Elephant Shrews, Tree Shrews and Dermopterns. In:Orders and families of recent mammals of the world (Eds. Anderson, S. and J.K. Jones) [M]. Wiley-Interscience, New York,1984,117-144.
[4]徐新平,陈红波,贲昆龙.树鼩在医学生物学中的应用[J].中国实验动物学报,2005,13(3)：187-190.
[5]李媛,K. Baumgartner, D. MacMillan,等.树鼩实验种群的繁育[J].动物学杂志,2001,36(3)：32-36.
[6]Elliot O, Elliot M, Lisco H. Breast cancer in a tree shrew (Tupaia glis)[J]. Nature, 1966,211:1105.
[7]冼苏,黄松,苏建家,等.链脲佐菌素诱导树鼩糖尿病动物模型研究[J].广西医科大学学报,2000,17(6)：945-948.
[8]周化愚,罗其胜.树鼩的自发性鳞状细胞癌一例报告[J].动物学杂志,1994,29(6)：41-42.
[9]秦雪,伊瑶,毕胜利,等.树鼩应用于疾病动物模型的研究进展[J].中华实验和临床病毒学杂志,2006,20(2)：97-98.
[10]Martin R, Dixson A, Wickings E. Paternity in Primate:Genetic Tests and Theories[M], Karger,swiss:1992.
[11]Wang Z, Weber J L, Zhing G, et al.1994. Survey of plant short tandem DNA repeat[J]. The Applied Genetics,88:1-6.
[12]Miesfeld R, Krystal M. Arnheim N. A member of a new repeated sequence family that is conserved hroughout eukaryotic evolution is found between the human delta and beta globin genes[J]. Nucl Acids Res,1981,9:5931-5947.
[13]Hamada H, Pitrino MT, Kakunaga. A novel repeated element with Z2DNA2 forming potential is widely found in evolutionarily diverse eukaryotic genomes[J]. Proc Natl Acad Sci USA,1982,79:6462-6469.
[14]Tautz D, Renz M. Simple sequences are ubiquitious repetitive component s of eukaryote genomes[J], Nucl Acids Res,1984,12:4127-4138.
[15]Sarkar G, Paynton C, Sommer S S. Segments containing alternating purine and pyrimidine dinucleotides:patterns of polymorphism in humans and prevalence throughout phylogeny[J]. Nucl Acids Res,1991,19:631-636.
[16]Wang Z, Weber JL, Zhong G, Tanksley SD. Survey of plant short tandem DNA repeats[J]. Theor Appl Genet,1984,88:1-6.
[17]Ma ZQ, Roder M, Scorrells ME. Frequencies and sequence characteristics of di-, tri-, and tetranucleotide microsatellite in wheat[J]. Genome,1996,39:123-130.
[18]Weber JL. Informativeness of human (dC-dA)n.(dG-dT)n polymorphisms [J]. Genomics,1990,7:524-530.
[19]Saveliev A, Everett C, Sharpe T, et al. DNA triplet repeats mediate heterochromatin2protein212sensitive variegated gene silencing[J]. Nature,2003, 422(6934):909-913.
[20]Heale S M, Petes TD. The stabilization of repetitive tracts of DNA by variant repeats requires a functional DNA mismatch repair system[J]. Cell,1995, 83:539-545.
[21]Schlotterer C, Tauze D. Slippage synthesis of simple sequence DNA[J]. Nucleic Acids Res,1992,20:211-215.
[22]Hite JM, Echert KA, Chery KC. Factors affecting fidelity of DNA synthesis during PCR amplification of d(CA)n d(CT)n microsatellite repeats[J]. Nucleic Acids Res, 1996,24:2429-2434.
[23]Srikwan S, Hufford K, Eggert L, Woodruff SD. Variable microsatellite markers for genotyping Tree Shrews,Tupaia,and their potential use in genetic studies of fragmented population[J]. Science Asia,2002,28:93-97.
[24]Munshi-South J, Wilkinson GS. Isolation and characterization of polymorphic microsatellite loci in Bornean treeshrews(Tupaia spp.)[J]. Molecular Ecology Notes, 2006,6:698-699.
[25]Klukowska J, Szczerbal I, Wengi-Piasecka A, et al. Characterization and mapping of canine microsatellites isolated from BAC clones harbouring DNA sequences homologous to seven human genes[J]. Anim Genet,2004,35(5):404-407.
[26]Zhao F, Ambady S, Ponce de Leon FA, et al. Microsatellite markers from a microdissected swine chromosome 6 genomic library[J]. Anim Genet, 1999,30(4):251-255.
[27]闫守庆,李冰,祝万菊.貂微卫星DNA富集文库的构建与鉴定[J].东北林业大学学报,2008,36(6)：48-50.
[28]何保丽,沈培清,陈丽玲,等.树鼩微卫星DNA的多态性研究[J].中国实验动物学报,2009,17(2)：143-145.
[29]杜爽,仇雪梅.微卫星DNA检测方法的研究[J].生物技术通报,2008,3：174-177.
[30]Nei M. Estimation of average heterozygosity and genetic dis-tance from a small number of individuals[J]. Genetics,1978,89(3):583-590.
[31]Botstein D, White R L, Skolnick M, Davis R W. Construction of a genetic linkage map in man using restriction fragment length polymorphisms[J]. American Journal of Human Genetics,1980,32(3):314-331.
[32]杨泽宇,苗永旺,李大林,等.德宏水牛微卫星标记分析的群体遗传变异[J].动物学研究,2007,28(6)：659-663.
[33]Frankham R, Ballou JD, Briscoe DA.Introduction to Conservation Genetics[M]. Cambridge University Press,2002,29-62.
[34]Hines H C, Zikakis J P, Haenlein G F, Kiddy C G, Trowbridge C L. Linkage relationships among loci of polymorphisms in blood and milk of cattle[J]. Journal of Dairy Science,1981,64(1):71-76.
[35]李莉,孙振兴,杨树德,等.用微卫星标记分析皱纹盘鲍群体的遗传变异[J].遗传,2006,28(12)：1549-1554.
[36]Takezaki N, Nei M. Genetic distances and reconstruction of phylogenetic trees from microsatellite DNA[J]. Genetics,1996,144(1):389-399.
[37]黎家敏,孙晓梅,代杰解,等.应用RAPD标记技术对树鼩遗传多样性的分析[J].中国比较医学杂志,2010,20(2)：34-40.
[1]Hamada H, Petrino M G, Kakunaga T. A novel repeated element with Z-DNA forming potential is widely found in evolutionarily diverse eukaryotic genomes [J]. Proceedings of the National Academy of Sciences of the United States of America. 1982,79(21):6465-6469.
[2]Wang Z, Weber J L, Zhing G, et al.1994. Survey of plant short tandem DNA repeat [J]. The Applied Genetics,88:1-6.
[3]何蔚,沈富军,岳碧松.大熊猫多态性微卫星标记筛选[J].四川动物,2007,26(3)：531-533.
[4]匡刚桥,刘臻,鲁双庆等GenBank数据库中鳜鱼微卫星位点的筛选及特征分析[J].动物学报,2007,53(1)：184-189.
[5]徐玲玲,吴艳花,路静等.筛选适用于小型猪遗传检测的微卫星位点[J].中国比较医学杂志,2009,19(2)：11-16.
[6]许新,王江勇,区又君.杂色鲍EST微卫星的筛选和应用[A].2009年中国水产学会学术年会论文摘要集[C],2009.
[7]石耀华,洪葵,郭希明等.马氏珠母贝EST微卫星的筛选[J].水产学报,2008,(02).
[8]Eujayl I, Baum M, Wolters P, et al. Assessment of genotypic variation among cultivated durum wheat based on EST-SSRs and genomic SSRs[J]. Theoretical and Applied Genetics,2000,97:671-683.
[9]Rossetto M. Sourcing of SSR markers from related plant species[M]. Plant Genotyping:the DNA Fingerprinting of Plant,2001,211-224.
[10]Zucoloto RB, Priscilla Marqui Schimidt Villela,et al. Cross-species microsatellite amp lificati on in South American Caimans (Caimanspp. and Paleosuchus palpebrosus) [J]. Genetics and Molecular Biology,2006,29 (1):75-78.
[11]Kupper C, Horsburgh GJ, Deborah A,et al.Characterizati on of 36 polymorphic microsatellite loci in the Kentish plover (Charadrius alexandrinus) including two sex-linked loci and their amp lification in four other Charadrius species[J]. Molecular Ecology Notes,2007:35-39.
[12]蔡清秀,林柳,潘文婧等.勐养保护区业洲象微卫星位点筛选及种群遗传多样性分析[J].兽类学报,2008,28(2)：126-134]
[13]孙涛,王博石,刘志瑾等.近缘种扩增法对黑叶猴微卫星位点的筛选及特征分析[J].兽类学报,2010,30(3)：351-353.
[14]洪艳云,李迪强,易图永等.筛选普氏原羚粪便DNA中微卫星引物并应用于个体识别[J].农业生物技术科学,2008,24(9)：54-58.
[15]谭元卿,李薇,杜小燕等.种间转移扩增法筛选长爪沙鼠微卫星位点[J].中国实验动物学报,2011,19(1)：1-5.
[16]Primmer CR, Painter JN, Koskinen MT, et al. Factors affecting avian cross-species microsatellite amplification [J].Javian biology,2005,36:348-360.
[17]Srikwan S, Hufford K, Eggert L, et al. Variable microsatellite markers for genotyping Tree Shrews,Tupaia,and their potential use in genetic studies of fragmented population[J]. Science Asia,2002,28:93-97.
[18]Munshi-South J, Wilkinson GS. Isolation and characterization of polymorphic microsatellite loci in Bornean treeshrews (Tupaia spp.) [J]. Mol Ecol Notes,2006, 6:698-699.
[19]魏东旺,楼允东,孙效文等.鲤鱼微卫星分子标记的筛选[J].动物学研究,2001,22(3)：238-241.
[20]于颖,杨孔,王永等.藏鸡微卫星文库的构建与微卫星标记筛选[J].四川动物,2009,28(3)：358-362.
[21]袁慧,张修月,宋昭彬等.岩原鲤微卫星富集文库的构建及微卫星分子标记的筛选[J].四川动物,2008,27(2)：210-215.
[22]朱亮,蔡月琴,屠珏等.磁珠富集法筛选实验豚鼠微卫星分子标记[J].中国比较医学杂志,2010,20(6)：29-34.
[23]WANG Hong-zhe,YIN Qian-qian,FENG Zhi-gang,et al.Construction Fractional Genomic Libraries and Screening Microsatellites DNA of Esox reieherti Dybowski[J].动物学研究,2008,29(3):245-252.
[24]赵亮,高贵珍,张兴桃.利用磁珠富集法筛选大银鱼微卫星标记的初步研究[J].宿州学院学报,,2010,25(11)：24-27.
[25]李齐发,赵兴波,罗晓林等.牦牛基因组微卫星富集文库的构建与分析[J].遗传学报2004,31(5)：489-494.
[26]闫守庆,李冰,祝万菊.貉微卫星DNA富集文库的构建与鉴定[J].东北林业大学学报.2008,36(6)：48-50.
[27]李新国,田金菊,谷尚树等.日本扁柏核基因组微卫星的分离与鉴定[J].遗传学报,2004,31(4)：375-379.
[28]战爱斌,胡景杰,胡晓丽等.富集文库-菌落原位杂交法筛选栉孔扇贝的微卫星标记[J].水产学报,2008,32(3)：353-361.
[29]李石柱,王艺秀,马雅军等.湖北钉螺多态微卫星DNA位点筛选和特征初步分析[JJ..中国血吸虫病防治杂志,2010,22(2)：122-125.
[30]张丽,樊勇,马雅军.中华白蛉微卫星DNA序列的分离和多态位点筛选的初步研究[J]..中国寄生虫学与寄生虫病杂志,2009,27(6)：503-507.
[31]樊勇,马雅军.中华按蚊多态微卫星DNA位点的筛选和特征研究[J]..第四军医大学学报,2008,29(17)：1537-1539.
[32]马雅军,樊勇,吴静.雷氏按蚊多态微卫星DNA位点的筛选和特征[J]..寄生虫与医学昆虫学报,2008,15(3)：150-153.
[33]Paetgkau D. Microsatellites obtained using strand extension:an enrichment protocol [J]. Biotechniques,1999.26:690-697.
[31]Fisher P J, Gardner R C. Richardso T E. Single locus microsatellites isolated using 5'anchored PCR [J]. Nucleic Acids Research,1999,24:4369-4371.
[35]Uedo S，Yoshimaru H,Tomarun， et al. Development and characterization of microsatellite markers in Camellia japonica L[J].Mol Ecol,1999,8:335-346.
[36]Lunt D H, Hutchinson W F,Carvalho G R.An efficient method for PCR based isolation of microsatellite arrays(PIMA)[J].Mol Ecol.1999.8:891-894.
[37]Frydenber G J,Pertold I C,Dahlgaar D J,et al.Genetic variation in original and colonizing Drosophila buzzatii populations analysed by microsatelliteloci isolated with a new PCR screenin method[J].Mol Ecol,2002,11:181-190.
[38]Zeithlewica E, Rafalski A,Labuda D. Genome fingerprinting by simplesequence repeatO anchored polymersae chain reaction amplification [J]. Genomics, 1994,20:176-183.
[39]Arnold C, Hodgson I J. Vectorette PCR:a novel approach to genomicwalking PCR [J].Methods Appl,1991,1:39-42.
[40]Siebert P D, Chenchik A, Kellog D E, et al. An improved PCR method for walking in uncloned genomic DNA [J]. Nucl Acids Res,1995,23:1087-1088.
[41]Davila J A, Loarce Y, Ferrer E.Molecular characterization and genetic mapping of random amplified microsatellite polymorphism in barely [J]. Theor Appl Genet, 1999,98:265-273.
[42]Fisher P J,Gardner R C,RichardsonT E. Single locus micro satellite isolated using 5'anchored PCR[J].Nucl Acids Res,1996,24:4369-4371.
[43]Lench N J, Norris A, Baileya, et al. Vectorette PCR isolation of microsatellite repeat sequences using anchored dinucleotide repeat primers [J].Nucl Acids Res, 1996,24:2190-2191.
[44]Ujino T, Kawahara T, Tsumura Y, et al.Development and polymorphism of simple sequence repeat DNA markers for Shorea curtisii and other Diptero-carpaceae species [J].Heredity,1998,81:422-428.
[45]Vander N A,Steenkamp E T,WingfieldI B D,et al. Development of simple sequence repeat (SSR) markers in Eucalyptus from amplified inter-simple sequence repeats (ISSR) [J]. Plant Breed,2000,119:433-436.
[46]Lian C,Zhou Z,Hogetsu T.A simple method for developing microsatellite markers using amplified fragments of inter 2simple sequence repeat (ISSR) [J].J Plant Res,2001,114:381-385.
[47]Burgess T,Wingfield M J,Wingfield B D. Simple sequence repeat (SSR) markers distinguish between morphotypes of Sphaeropsis sapinea[J].App Enviro Micro,2001,67:354-362.
[48]Hayden M J, Sharp P J. Sequence 2tagged microsatellite profiling (STMP):a rapid technique for developing SSR markers[J]. Nucl Acids Res,2001,29:43-53.
[49]Hayden M J, Sharp P J. Targeted development of informative microsatellite (SSR) markers [J].Nucleic Acids Res,2001,29(8):44-54.
[50]Hakki E E, Akkaya M S. Microsatellite isolation using amplified fragmentlength polymorphism markers:no cloning,no screening[J].Mol Ecol,2000(9):2152 2154.
[51]Tautz D, Renz M. Simple sequences are ubiquitious repetitive component s of eukaryote genomes [J]. Nucl Acids Res,1984,12:4127-4138.
[52]Maguire T L, et al. Characterisati on and analysis of microsatellite loci in a mangr ove s pecies, Avicenniamarina (Fork) vierh (Avicenniaceae) [J]. Theor Appl Genet, 2000,101:279～285
[53]杜爽,仇雪梅.微卫星DNA检测方法的研究[J].生物技术通报,2008,3：174-177.
[2]刘世熠,张文远,王泽盛,等.树鼩24小时昼夜节律的探讨[J].动物学报,1982,28(24)：399-407.
[3]T. L. Yate. Insectivores, Elephant Shrews, Tree Shrews and Dermopterns. In:Orders and families of recent mammals of the world (Eds. Anderson, S. and J.K. Jones) [M]. Wiley-Interscience, New York,1984,117-144.
[4]徐新平,陈红波,贲昆龙.树鼩在医学生物学中的应用[J].中国实验动物学报,2005,13(3)：187-190.
[5]李媛,K. Baumgartner, D. MacMillan,等.树鼩实验种群的繁育[J].动物学杂志,2001,36(3)：32-36.
[6]Elliot O, Elliot M, Lisco H. Breast cancer in a tree shrew (Tupaia glis)[J]. Nature, 1966,211:1105.
[7]冼苏,黄松,苏建家,等.链脲佐菌素诱导树鼩糖尿病动物模型研究[J].广西医科大学学报,2000,17(6)：945-948.
[8]周化愚,罗其胜.树鼩的自发性鳞状细胞癌一例报告[J].动物学杂志,1994,29(6)：41-42.
[9]秦雪,伊瑶,毕胜利,等.树鼩应用于疾病动物模型的研究进展[J].中华实验和临床病毒学杂志,2006,20(2)：97-98.
[10]Martin R, Dixson A, Wickings E. Paternity in Primate:Genetic Tests and Theories[M], Karger,swiss:1992.
[11]Wang Z, Weber J L, Zhing G, et al.1994. Survey of plant short tandem DNA repeat[J]. The Applied Genetics,88:1-6.
[12]Miesfeld R, Krystal M. Arnheim N. A member of a new repeated sequence family that is conserved hroughout eukaryotic evolution is found between the human delta and beta globin genes[J]. Nucl Acids Res,1981,9:5931-5947.
[13]Hamada H, Pitrino MT, Kakunaga. A novel repeated element with Z2DNA2 forming potential is widely found in evolutionarily diverse eukaryotic genomes[J]. Proc Natl Acad Sci USA,1982,79:6462-6469.
[14]Tautz D, Renz M. Simple sequences are ubiquitious repetitive component s of eukaryote genomes[J], Nucl Acids Res,1984,12:4127-4138.
[15]Sarkar G, Paynton C, Sommer S S. Segments containing alternating purine and pyrimidine dinucleotides:patterns of polymorphism in humans and prevalence throughout phylogeny[J]. Nucl Acids Res,1991,19:631-636.
[16]Wang Z, Weber JL, Zhong G, Tanksley SD. Survey of plant short tandem DNA repeats[J]. Theor Appl Genet,1984,88:1-6.
[17]Ma ZQ, Roder M, Scorrells ME. Frequencies and sequence characteristics of di-, tri-, and tetranucleotide microsatellite in wheat[J]. Genome,1996,39:123-130.
[18]Weber JL. Informativeness of human (dC-dA)n.(dG-dT)n polymorphisms [J]. Genomics,1990,7:524-530.
[19]Saveliev A, Everett C, Sharpe T, et al. DNA triplet repeats mediate heterochromatin2protein212sensitive variegated gene silencing[J]. Nature,2003, 422(6934):909-913.
[20]Heale S M, Petes TD. The stabilization of repetitive tracts of DNA by variant repeats requires a functional DNA mismatch repair system[J]. Cell,1995, 83:539-545.
[21]Schlotterer C, Tauze D. Slippage synthesis of simple sequence DNA[J]. Nucleic Acids Res,1992,20:211-215.
[22]Hite JM, Echert KA, Chery KC. Factors affecting fidelity of DNA synthesis during PCR amplification of d(CA)n d(CT)n microsatellite repeats[J]. Nucleic Acids Res, 1996,24:2429-2434.
[23]Srikwan S, Hufford K, Eggert L, Woodruff SD. Variable microsatellite markers for genotyping Tree Shrews,Tupaia,and their potential use in genetic studies of fragmented population[J]. Science Asia,2002,28:93-97.
[24]Munshi-South J, Wilkinson GS. Isolation and characterization of polymorphic microsatellite loci in Bornean treeshrews(Tupaia spp.)[J]. Molecular Ecology Notes, 2006,6:698-699.
[25]Klukowska J, Szczerbal I, Wengi-Piasecka A, et al. Characterization and mapping of canine microsatellites isolated from BAC clones harbouring DNA sequences homologous to seven human genes[J]. Anim Genet,2004,35(5):404-407.
[26]Zhao F, Ambady S, Ponce de Leon FA, et al. Microsatellite markers from a microdissected swine chromosome 6 genomic library[J]. Anim Genet, 1999,30(4):251-255.
[27]闫守庆,李冰,祝万菊.貂微卫星DNA富集文库的构建与鉴定[J].东北林业大学学报,2008,36(6)：48-50.
[28]何保丽,沈培清,陈丽玲,等.树鼩微卫星DNA的多态性研究[J].中国实验动物学报,2009,17(2)：143-145.
[29]杜爽,仇雪梅.微卫星DNA检测方法的研究[J].生物技术通报,2008,3：174-177.
[30]Nei M. Estimation of average heterozygosity and genetic dis-tance from a small number of individuals[J]. Genetics,1978,89(3):583-590.
[31]Botstein D, White R L, Skolnick M, Davis R W. Construction of a genetic linkage map in man using restriction fragment length polymorphisms[J]. American Journal of Human Genetics,1980,32(3):314-331.
[32]杨泽宇,苗永旺,李大林,等.德宏水牛微卫星标记分析的群体遗传变异[J].动物学研究,2007,28(6)：659-663.
[33]Frankham R, Ballou JD, Briscoe DA.Introduction to Conservation Genetics[M]. Cambridge University Press,2002,29-62.
[34]Hines H C, Zikakis J P, Haenlein G F, Kiddy C G, Trowbridge C L. Linkage relationships among loci of polymorphisms in blood and milk of cattle[J]. Journal of Dairy Science,1981,64(1):71-76.
[35]李莉,孙振兴,杨树德,等.用微卫星标记分析皱纹盘鲍群体的遗传变异[J].遗传,2006,28(12)：1549-1554.
[36]Takezaki N, Nei M. Genetic distances and reconstruction of phylogenetic trees from microsatellite DNA[J]. Genetics,1996,144(1):389-399.
[37]黎家敏,孙晓梅,代杰解,等.应用RAPD标记技术对树鼩遗传多样性的分析[J].中国比较医学杂志,2010,20(2)：34-40.
[1]Hamada H, Petrino M G, Kakunaga T. A novel repeated element with Z-DNA forming potential is widely found in evolutionarily diverse eukaryotic genomes [J]. Proceedings of the National Academy of Sciences of the United States of America. 1982,79(21):6465-6469.
[2]Wang Z, Weber J L, Zhing G, et al.1994. Survey of plant short tandem DNA repeat [J]. The Applied Genetics,88:1-6.
[3]何蔚,沈富军,岳碧松.大熊猫多态性微卫星标记筛选[J].四川动物,2007,26(3)：531-533.
[4]匡刚桥,刘臻,鲁双庆等GenBank数据库中鳜鱼微卫星位点的筛选及特征分析[J].动物学报,2007,53(1)：184-189.
[5]徐玲玲,吴艳花,路静等.筛选适用于小型猪遗传检测的微卫星位点[J].中国比较医学杂志,2009,19(2)：11-16.
[6]许新,王江勇,区又君.杂色鲍EST微卫星的筛选和应用[A].2009年中国水产学会学术年会论文摘要集[C],2009.
[7]石耀华,洪葵,郭希明等.马氏珠母贝EST微卫星的筛选[J].水产学报,2008,(02).
[8]Eujayl I, Baum M, Wolters P, et al. Assessment of genotypic variation among cultivated durum wheat based on EST-SSRs and genomic SSRs[J]. Theoretical and Applied Genetics,2000,97:671-683.
[9]Rossetto M. Sourcing of SSR markers from related plant species[M]. Plant Genotyping:the DNA Fingerprinting of Plant,2001,211-224.
[10]Zucoloto RB, Priscilla Marqui Schimidt Villela,et al. Cross-species microsatellite amp lificati on in South American Caimans (Caimanspp. and Paleosuchus palpebrosus) [J]. Genetics and Molecular Biology,2006,29 (1):75-78.
[11]Kupper C, Horsburgh GJ, Deborah A,et al.Characterizati on of 36 polymorphic microsatellite loci in the Kentish plover (Charadrius alexandrinus) including two sex-linked loci and their amp lification in four other Charadrius species[J]. Molecular Ecology Notes,2007:35-39.
[12]蔡清秀,林柳,潘文婧等.勐养保护区业洲象微卫星位点筛选及种群遗传多样性分析[J].兽类学报,2008,28(2)：126-134]
[13]孙涛,王博石,刘志瑾等.近缘种扩增法对黑叶猴微卫星位点的筛选及特征分析[J].兽类学报,2010,30(3)：351-353.
[14]洪艳云,李迪强,易图永等.筛选普氏原羚粪便DNA中微卫星引物并应用于个体识别[J].农业生物技术科学,2008,24(9)：54-58.
[15]谭元卿,李薇,杜小燕等.种间转移扩增法筛选长爪沙鼠微卫星位点[J].中国实验动物学报,2011,19(1)：1-5.
[16]Primmer CR, Painter JN, Koskinen MT, et al. Factors affecting avian cross-species microsatellite amplification [J].Javian biology,2005,36:348-360.
[17]Srikwan S, Hufford K, Eggert L, et al. Variable microsatellite markers for genotyping Tree Shrews,Tupaia,and their potential use in genetic studies of fragmented population[J]. Science Asia,2002,28:93-97.
[18]Munshi-South J, Wilkinson GS. Isolation and characterization of polymorphic microsatellite loci in Bornean treeshrews (Tupaia spp.) [J]. Mol Ecol Notes,2006, 6:698-699.
[19]魏东旺,楼允东,孙效文等.鲤鱼微卫星分子标记的筛选[J].动物学研究,2001,22(3)：238-241.
[20]于颖,杨孔,王永等.藏鸡微卫星文库的构建与微卫星标记筛选[J].四川动物,2009,28(3)：358-362.
[21]袁慧,张修月,宋昭彬等.岩原鲤微卫星富集文库的构建及微卫星分子标记的筛选[J].四川动物,2008,27(2)：210-215.
[22]朱亮,蔡月琴,屠珏等.磁珠富集法筛选实验豚鼠微卫星分子标记[J].中国比较医学杂志,2010,20(6)：29-34.
[23]WANG Hong-zhe,YIN Qian-qian,FENG Zhi-gang,et al.Construction Fractional Genomic Libraries and Screening Microsatellites DNA of Esox reieherti Dybowski[J].动物学研究,2008,29(3):245-252.
[24]赵亮,高贵珍,张兴桃.利用磁珠富集法筛选大银鱼微卫星标记的初步研究[J].宿州学院学报,,2010,25(11)：24-27.
[25]李齐发,赵兴波,罗晓林等.牦牛基因组微卫星富集文库的构建与分析[J].遗传学报2004,31(5)：489-494.
[26]闫守庆,李冰,祝万菊.貉微卫星DNA富集文库的构建与鉴定[J].东北林业大学学报.2008,36(6)：48-50.
[27]李新国,田金菊,谷尚树等.日本扁柏核基因组微卫星的分离与鉴定[J].遗传学报,2004,31(4)：375-379.
[28]战爱斌,胡景杰,胡晓丽等.富集文库-菌落原位杂交法筛选栉孔扇贝的微卫星标记[J].水产学报,2008,32(3)：353-361.
[29]李石柱,王艺秀,马雅军等.湖北钉螺多态微卫星DNA位点筛选和特征初步分析[JJ..中国血吸虫病防治杂志,2010,22(2)：122-125.
[30]张丽,樊勇,马雅军.中华白蛉微卫星DNA序列的分离和多态位点筛选的初步研究[J]..中国寄生虫学与寄生虫病杂志,2009,27(6)：503-507.
[31]樊勇,马雅军.中华按蚊多态微卫星DNA位点的筛选和特征研究[J]..第四军医大学学报,2008,29(17)：1537-1539.
[32]马雅军,樊勇,吴静.雷氏按蚊多态微卫星DNA位点的筛选和特征[J]..寄生虫与医学昆虫学报,2008,15(3)：150-153.
[33]Paetgkau D. Microsatellites obtained using strand extension:an enrichment protocol [J]. Biotechniques,1999.26:690-697.
[31]Fisher P J, Gardner R C. Richardso T E. Single locus microsatellites isolated using 5'anchored PCR [J]. Nucleic Acids Research,1999,24:4369-4371.
[35]Uedo S，Yoshimaru H,Tomarun， et al. Development and characterization of microsatellite markers in Camellia japonica L[J].Mol Ecol,1999,8:335-346.
[36]Lunt D H, Hutchinson W F,Carvalho G R.An efficient method for PCR based isolation of microsatellite arrays(PIMA)[J].Mol Ecol.1999.8:891-894.
[37]Frydenber G J,Pertold I C,Dahlgaar D J,et al.Genetic variation in original and colonizing Drosophila buzzatii populations analysed by microsatelliteloci isolated with a new PCR screenin method[J].Mol Ecol,2002,11:181-190.
[38]Zeithlewica E, Rafalski A,Labuda D. Genome fingerprinting by simplesequence repeatO anchored polymersae chain reaction amplification [J]. Genomics, 1994,20:176-183.
[39]Arnold C, Hodgson I J. Vectorette PCR:a novel approach to genomicwalking PCR [J].Methods Appl,1991,1:39-42.
[40]Siebert P D, Chenchik A, Kellog D E, et al. An improved PCR method for walking in uncloned genomic DNA [J]. Nucl Acids Res,1995,23:1087-1088.
[41]Davila J A, Loarce Y, Ferrer E.Molecular characterization and genetic mapping of random amplified microsatellite polymorphism in barely [J]. Theor Appl Genet, 1999,98:265-273.
[42]Fisher P J,Gardner R C,RichardsonT E. Single locus micro satellite isolated using 5'anchored PCR[J].Nucl Acids Res,1996,24:4369-4371.
[43]Lench N J, Norris A, Baileya, et al. Vectorette PCR isolation of microsatellite repeat sequences using anchored dinucleotide repeat primers [J].Nucl Acids Res, 1996,24:2190-2191.
[44]Ujino T, Kawahara T, Tsumura Y, et al.Development and polymorphism of simple sequence repeat DNA markers for Shorea curtisii and other Diptero-carpaceae species [J].Heredity,1998,81:422-428.
[45]Vander N A,Steenkamp E T,WingfieldI B D,et al. Development of simple sequence repeat (SSR) markers in Eucalyptus from amplified inter-simple sequence repeats (ISSR) [J]. Plant Breed,2000,119:433-436.
[46]Lian C,Zhou Z,Hogetsu T.A simple method for developing microsatellite markers using amplified fragments of inter 2simple sequence repeat (ISSR) [J].J Plant Res,2001,114:381-385.
[47]Burgess T,Wingfield M J,Wingfield B D. Simple sequence repeat (SSR) markers distinguish between morphotypes of Sphaeropsis sapinea[J].App Enviro Micro,2001,67:354-362.
[48]Hayden M J, Sharp P J. Sequence 2tagged microsatellite profiling (STMP):a rapid technique for developing SSR markers[J]. Nucl Acids Res,2001,29:43-53.
[49]Hayden M J, Sharp P J. Targeted development of informative microsatellite (SSR) markers [J].Nucleic Acids Res,2001,29(8):44-54.
[50]Hakki E E, Akkaya M S. Microsatellite isolation using amplified fragmentlength polymorphism markers:no cloning,no screening[J].Mol Ecol,2000(9):2152 2154.
[51]Tautz D, Renz M. Simple sequences are ubiquitious repetitive component s of eukaryote genomes [J]. Nucl Acids Res,1984,12:4127-4138.
[52]Maguire T L, et al. Characterisati on and analysis of microsatellite loci in a mangr ove s pecies, Avicenniamarina (Fork) vierh (Avicenniaceae) [J]. Theor Appl Genet, 2000,101:279～285
[53]杜爽,仇雪梅.微卫星DNA检测方法的研究[J].生物技术通报,2008,3：174-177.