应用微卫星DNA标记对延边圈养黑熊进行亲子鉴定和遗传多样性研究
|
摘要
黑熊,也称亚洲黑熊,别名狗熊、黑瞎子、月牙熊,是现代生存的陆生食肉目动物中体形最大者。黑熊原来没有被列入保护动物目录,在一些地区还被列为害兽,由于其经济价值和药用价值,经常有人进山猎杀,或捕捉后进行人工繁殖。随着野生种群的不断减少,我国现已将其列为国家二级保护动物,世界各地的学者也都逐渐开展了对熊的研究。随着养殖规模的壮大,为了进一步了解我国存在的黑熊亚种的遗传性状,更好的推动养熊业科学、规范管理,本研究选取19头成年黑熊,通过比较三个不同来源的模板DNA和其浓度对微卫星位点扩增效果的影响,找出适合于微卫星位点扩增的模板DNA浓度,探讨使用毛发作为样本材料进行遗传多样性研究的可行性。利用15对黑熊微卫星标记对延边地区繁殖的圈养黑熊115个个体进行了微卫星多态性测定和统计分析,并对延边地区养殖黑熊进行了亲子鉴定的研究,结果如下：
1.毛发根部DNA、血液DNA和组织DNA的PCR产物数量和质量并无明显差异。利用非损伤性采样法,收集100根带有毛囊的毛发可满足微卫星位点扩增模板DNA需求量,50ng/μL的模板DNA浓度为微卫星位点扩增反应的最佳模板浓度,本研究结果进一步证明毛发根部DNA可作为黑熊遗传多样性研究的试验材料。
2.本研究从19个微卫星位点中筛选出15个位点进行遗传多样性分析,在黑熊养殖群体中共检测到111个等位基因,每个微卫星位点的等位基因数量为4-11个,平均每个位点7.40个,基因频率分布在0-1之间,说明黑熊养殖群体的遗传多样性比较丰富。
3.15个微卫星位点的PIC值在0.4533-0.7422之间,平均值为0.6133,其中只有ABB1、ABB3、ABB7、UT36的PIC值处于0.25与0.5之间,属于中度多态性位点,其余均高于0.5,15个位点平均期望杂合度为0.6213,说明所选标记具有丰富的多态性,能够在分子水平上准确的反映黑熊物种的遗传关系。
4.利用15个微卫星标记进行了亲子鉴定研究,分别找出了14头仔熊的亲本父母,亲权鉴定的累积非父排除概率和亲权指数较高,累积非父排除概率达0.999985,亲权相对机会范围是99.9473%-99.9993%。
Asiatic black bear(Ursus thibetanus), which have several alternative names like "black bear" or "dog hunchback" or "black blind animal" or "crescent moon bear", is the most heavyweight land carnivores nowadays. In the past, black bear was not included within the national aegis animals. In the around of mountain, black bear has been considered as a harmfuL animal. It has always been killed or captured to be raised artificially for gaining its economic value and medicinal value. With the sharp decline of wild black bear's popuLation, our country has already listed it into the national Ⅱ aegis animals. Many scholars all over the world have started to launch more and more research works about black bear. Acording to the increasing scales from breeding industry of the black bear, it was necessary to further investigate the genetic diversity of black bear subspecies in our country for more scientific promotion of the bear breeding with standardized management. Therefore, in this study we take nineteen adult black bears as a representative animal, contrasting the results of the microsatellite amplification reaction influenced by different quality and quantity of template DNA extracted from three body samples including hair or blood or tissue, try to find out the best template DNA concentration for appropriate reaction of microsatellite amplification. This study also discussed the feasibility of making the genetic diversity research by using the hair of black bear as a testing material. Using microsatellite markers, this study screened one hundred and fifteen black bears specimens in the area of Yanbian to analyze the microsatellite diversity of them statistically. Father-son Parentage of black bear in Yanbian region has also been clearly identified. The results are in the follwing:
1. The PCR products using template DNA extracted from hair or blood or tissue had no obvious differences in its quality and quantity. The noninvasive sampling method collecting one hundred hairs with hair follicles was good for extracting enough template DNA for microsatellite amplification.50ng/μL was the best template DNA concentration for efficient microsatellite amplification reactions. The results of this study have further proved that the template DNA extracted from hair samples could be taken for black bear genetic diversity research as good testing materials.
2. In this study, fifteen loci screened among nineteen different microsatellite sites of black bear genomic DNA were taken to be done genetic diversity analysis. Total111loci and alleles were investigated in this study. It was calculated that each microsatellite locus has almost4-11allele types. Average number of each allele types investigated in this study was7.40, and the allele frequency was between0-1. These results showed there was relatively rich genetic diversities in black bear population farming at north-east area of china.
3. The PIC value of fifteen microsatellite loci invesigated in this study was between0.4533-0.7422and average number was0.6133. The PIC value of ABB3, ABB7, ABB12, UT36was between0.25to0.5, which were belonging to the moderate polymorphism loci and the others were over0.5. Average expectation heterozygosis of fifteen loci was0.6213. It can be explained that the selected markers have relatively rich polymorphism, which could reflect the genetic relationship of black bear accurately.
4. Fourteen little bears were confirmed its parents through paternity test using fifteen microsatellite loci mentioned above. Cumulative chance of exclusion (CCE) and paternity index between the parents and their off-springs were high. The CCE was0.999985and the RCP range was99.9473%-99.9993%respectively between the parents and their off-springs investigated in this study.
1.毛发根部DNA、血液DNA和组织DNA的PCR产物数量和质量并无明显差异。利用非损伤性采样法,收集100根带有毛囊的毛发可满足微卫星位点扩增模板DNA需求量,50ng/μL的模板DNA浓度为微卫星位点扩增反应的最佳模板浓度,本研究结果进一步证明毛发根部DNA可作为黑熊遗传多样性研究的试验材料。
2.本研究从19个微卫星位点中筛选出15个位点进行遗传多样性分析,在黑熊养殖群体中共检测到111个等位基因,每个微卫星位点的等位基因数量为4-11个,平均每个位点7.40个,基因频率分布在0-1之间,说明黑熊养殖群体的遗传多样性比较丰富。
3.15个微卫星位点的PIC值在0.4533-0.7422之间,平均值为0.6133,其中只有ABB1、ABB3、ABB7、UT36的PIC值处于0.25与0.5之间,属于中度多态性位点,其余均高于0.5,15个位点平均期望杂合度为0.6213,说明所选标记具有丰富的多态性,能够在分子水平上准确的反映黑熊物种的遗传关系。
4.利用15个微卫星标记进行了亲子鉴定研究,分别找出了14头仔熊的亲本父母,亲权鉴定的累积非父排除概率和亲权指数较高,累积非父排除概率达0.999985,亲权相对机会范围是99.9473%-99.9993%。
Asiatic black bear(Ursus thibetanus), which have several alternative names like "black bear" or "dog hunchback" or "black blind animal" or "crescent moon bear", is the most heavyweight land carnivores nowadays. In the past, black bear was not included within the national aegis animals. In the around of mountain, black bear has been considered as a harmfuL animal. It has always been killed or captured to be raised artificially for gaining its economic value and medicinal value. With the sharp decline of wild black bear's popuLation, our country has already listed it into the national Ⅱ aegis animals. Many scholars all over the world have started to launch more and more research works about black bear. Acording to the increasing scales from breeding industry of the black bear, it was necessary to further investigate the genetic diversity of black bear subspecies in our country for more scientific promotion of the bear breeding with standardized management. Therefore, in this study we take nineteen adult black bears as a representative animal, contrasting the results of the microsatellite amplification reaction influenced by different quality and quantity of template DNA extracted from three body samples including hair or blood or tissue, try to find out the best template DNA concentration for appropriate reaction of microsatellite amplification. This study also discussed the feasibility of making the genetic diversity research by using the hair of black bear as a testing material. Using microsatellite markers, this study screened one hundred and fifteen black bears specimens in the area of Yanbian to analyze the microsatellite diversity of them statistically. Father-son Parentage of black bear in Yanbian region has also been clearly identified. The results are in the follwing:
1. The PCR products using template DNA extracted from hair or blood or tissue had no obvious differences in its quality and quantity. The noninvasive sampling method collecting one hundred hairs with hair follicles was good for extracting enough template DNA for microsatellite amplification.50ng/μL was the best template DNA concentration for efficient microsatellite amplification reactions. The results of this study have further proved that the template DNA extracted from hair samples could be taken for black bear genetic diversity research as good testing materials.
2. In this study, fifteen loci screened among nineteen different microsatellite sites of black bear genomic DNA were taken to be done genetic diversity analysis. Total111loci and alleles were investigated in this study. It was calculated that each microsatellite locus has almost4-11allele types. Average number of each allele types investigated in this study was7.40, and the allele frequency was between0-1. These results showed there was relatively rich genetic diversities in black bear population farming at north-east area of china.
3. The PIC value of fifteen microsatellite loci invesigated in this study was between0.4533-0.7422and average number was0.6133. The PIC value of ABB3, ABB7, ABB12, UT36was between0.25to0.5, which were belonging to the moderate polymorphism loci and the others were over0.5. Average expectation heterozygosis of fifteen loci was0.6213. It can be explained that the selected markers have relatively rich polymorphism, which could reflect the genetic relationship of black bear accurately.
4. Fourteen little bears were confirmed its parents through paternity test using fifteen microsatellite loci mentioned above. Cumulative chance of exclusion (CCE) and paternity index between the parents and their off-springs were high. The CCE was0.999985and the RCP range was99.9473%-99.9993%respectively between the parents and their off-springs investigated in this study.
引文
[1]Garshelis D L, Steinmetz R. Ursus thibetanus. In:IUCN 2009.IUCN List of Threatened Species. Version 2009,1.
[2]Smith AT,解焱.中国兽类野外手册[M].长沙：湖南教育出版社,2009：412-419.
[3]国家林业局主编.中国重点陆生野生动物资源调查[M].北京：中国林业出版社,2009：249-251.
[4]腾毅,杨海玲.我国养熊业及引流胆技术的历史与发展现状[J].中国中医药信息杂志,2010,(17)：4：8-9.
[5]吴修红,孙文军,腾毅,等.基于UPLC-Q-TOF联用技术的长期引流采胆熊及正常熊的代谢比较研究.江苏省博士研究生学术论坛文集,2006：587-593.
[6]姚贵芳,于春光,叶金堂.胆汁引流对黑熊健康状况影响的研究[J].黑龙江八一农垦大学学报,2010,22(4)：68-70.
[7]Morin P A, D S WoodruLf. Noninvasive genotyping for veterbrate conservation. In MolecuLar Genetic Approaches in Conservation[M]. Oxford University Press:New York.1996,298-313.
[8]Taberlet P, L P Waits, G Luikart. Nonivasive genetic sampling:look before you leap[J]. Trends Ecol. Evol.,1999,14:323-327.
[9]Gill P, A J Jeffreys, D J Werrent. Forensic application of DNA'fringerprints'[J]. Nature,1985, 318:577-579.
[10]Woodruff D S. Non-invasive genotyping of primates[J]. Primates,1993,34:333-346.
[11]Morin P A, J J Moore, R Chakraborty, et al. Kin selection,social structure,gene flow and evolution of chimpanzees[J]. Science,1994a,265:1193-1201.
[12]Morin P A, J J Moore, J Wallis, D S Woodruff. Paternity exclusion in a community of wild chimpanzees using hypervariable simple sequence repearts[J]. Mol Ecol.,1994b,3:469-479.
[13]Taberlet P, S Griffin, B Goossens, et al. Reliable genotyping of samples with very low DNA quantities using PCR[J]. Nucleic Acids Res.1996,24:3189-3194.
[14]Gerloff U,C Schlotterer, K Rassmann,et al. Amplification of hypervariable simple sequence repeats from excremental DNA of wild living Bonobos[J]. Mol. Ecol.,1995,4:515-518.
[15]Foran D R, K R Crooks, S C Minta. Species idenrifiction from scat:an unambiguous genetic method[J]. Wildlife Society BuLletin,1997,25:835-839.
[16]Kohn M H, E C York, D A Kamradt, et al. Estimating popuLation size by genotyping faeces. Proc. Royal. Soc(Series B),1999,266:657-663.
[17]方盛国,陈冠群,冯文和,等.大熊猫DNA 指纹在野生种群数量调查中的应用[J].兽类学报,1996a,16：246-249.
[18]Hoss M, M Kohn, S Paabo. Excrement analysis by PCR[J]. Nature,1992,359:199.
[19]Ernest H B, M C Penedo, N P May,et al. MolecuLar tracking of mountain lions in the Yosemite Valley region in California:genetic analysis using microsatellites and facecal DNA[J]. Mol.Ecol., 2000,9:433-441.
[20]Parsons K M, J F Dallas, D E Claridge,et al. Amplifying dolphin mitochondrial DNA from faecal plumes[J]. Mol. Ecol.,1999,8:1753-1768.
[21]Savill M G, S R Murray, P Scholes, et al. Application of polymerase chain reaction(PCR) and TaqManTM PCR techniques to the detection and identification of phodococcus coprophilus in faecal samples[J]. Journal of Microbiological Methods,2001,47:355-368.
[22]Ding B, Y P Zhang, Q A Ryder. Extraction, PCR amplification, and sequencing of mitochondrial DNA from scent mark and feces in the giant panda[J]. Zoo Biology,1998,17:499-504.
[23]Reed J Z, D J Tollit,P M Thompson,et al. MolecuLar scatology:the use of molecuLar genetic analysis to assign species, sex and individual identity to seal faeces[J]. Mol. Ecol.,1997,6:225-234.
[24]Machiels B M, T Ruers, M Lindhout, et al. New protocol for DNA extraction of stool[J]. BioTechniques,2000,28:286-290.
[25]Lantz P G,M Matsson, T Wadstrom, et al. Removal of PCR inhibitors from human faecal samples through the use of an aqueous two-phase system for sample preparation prior to PCR[J]. Journal of Microbiological Methods,1997,28:159-167.
[26]Inoue M,O Takenaka. Japanese macaque microsatellite PCR primers for paternity testing[J]. Primates,1993,34:37-45.
[27]丁波,O A Ryder,张亚平等.大熊猫气味标记DNA的制备和序列分析[J].动物学研究,1998,19：344-349.
[28]Taberlet P, J Bouvet. A single plucked feather as a source of DNA for bird genetic studies[J]. The Auk,1991,108:959-960.
[29]Weissenbach J, Fyapay G, Dib C,et al. A second-generation linkage map of human genome[J]. Nature,1992,359:794-801.
[30]钱迎倩,马克平.遗传多样性及其检测方法[M].北京中国科技出版社,1994,123-140.
[31]季维智,宿兵.遗传多样性研究的原理与方法[M].杭州浙江科学技术出版社,1999,1-12.
[32]Grant V. The Evolutionary Process:A Critical Study of Evolutionary Theory[M]. New York: Columbia University Press,1991,137-142.
[33]Millar C I, Libby W J. Strategies for conserving clinal, ecotypic, and disjunct popuLation diversity in wildspread species. In:Falk D A and Holsinger K E eds. Genetics and conservation of rare plante[M]. New York:Oxford University Press,1991,149-170.
[34]Hamrick J L, Godt M J W. Allozyme diversity in plant species. In:Brown A H D, Cleggg M T, Kahler A L, Wier B S eds. Plant popuLation genetics, breeding, and genetic resource. Sunderland, Massachuestts:Sinauer Associates,1990,43-63.
[35]Merrell D J. (黄瑞复等泽)生态遗传学[M].北京：科学出版社,1991,89-95.
[36]邱芳,伏健民,金德敏等.遗传多样性的分子检测[J].生物多样性,1998,6(2)：143-150.
[37]Nei M, Roychoudhury A K. Genetic variation within and between the three major races of man, Caucasoids, Negroids, and Mongoids[J]. Am J Hum Genet.1974,26:421-443.
[38]Bostein D, White R L. Construction of a genetic linkage maps in man using restraction fragment length polymorphisms[J]. Amer. J.Hum. Genet.1980,32:314-331.
[39]Francis C Y, Yang R C, Tim B. POPGENE(1.32).2000, http://www.ualberta.ca/fyeh/download.-htm.
[40]Raymond M, Rousset F. Genepop(1.2):PopuLation genetics software for exact tests and ecumenicism[J]. Hered.1995,86(3):248-249.
[41]Raymond M, Rousset F. Genepop(3.3). Population genetics software for exact tests and ecumenicism.2001, URL:Wbiomed.curtin.edu.au/genepop
[42]Louis E J, Dempster E R. An exact test for Hardy-Weinberg and muLtiple alleles[J]. Biometrics, 1987,43:805-811.
[43]Guo SW, Thompson E A. Performing the exact test of Hardy-Weiberg proportions for muLtiple alleles[J]. Biometrics,1992,48:361-372.
[44]Botstein D, White R L, Skolnick M, Dacis R. Construction of a genetic linkage map in man using restriction fragment length polymorphisms[J]. Am J Hum Genet.1980,32:314-331.
[45]Dodgson J B, Cheng H H, Okimoto R. DNA marker technology:a revolution in animal genetics[J]. PouLt Sci.1997,76:1108-1114.
[46]张亚平等.熊超科的分子系统发生研究[J].遗传学报,1997,24(1)：15-22.
[47]Welsh J, Muclellard M. Fingerprinting genomes using PCR with arbitary primers[J]. Nucleic Acids Research.1990,18:7213-7218.
[48]Williams J G K, Ubelik K A R, Livok J,et al. DNA Polymorphisms amplified by arbitary primers are usefuL as genetic marker[J]. Nucleic Acid Research.1990,18:6531-6535.
[49]陆永久,张亚平.随机扩增多态DNA影响因素的研究[J].动物学研究.1997,18(2)：221-227.
[50]林峰,陈卫红等.马来熊脑源性营养因子基因编码区的克隆和序列分析[J].动物学报,1998,44(1)：86.
[51]Vos P, Hogers R, Bleeker M, et al. AFLP:a new technique for DNA fingerprinting[J]. Nucleic Acids Res.1995,23:4407-4414.
[52]Mueller U G, Wolfenbarger L L. AFLP genotyping and fingerprinting[J]. Trends Ecology Evolution.1999,14(10):389-394.
[53]Jeffreys A J. Highly variable minisatellites and DNA fingerprints twenty-third colworth medal lecture[J]. Biochemical Society Transaction,1986,(9):309.
[54]陆惠玲,周斌,陈丽娴等.HRP标记α-珠蛋白-3’-HVR探针DNA指纹图法医应用的研究[J].法医学杂志,1996,12(3)：162-164.
[55]李光汉,杨智等.小熊猫圈养种群遗传多样性及其影响因子[J].生态学报,2002,20(2)：85-86.
[56]Lander E S. The new genomics:glabal views of biology[J]. Science,1996,274:536-539.
[57]姜运良.卫星小卫星和微卫星DNA-真核生物基因组的串状重复序列[J].生命的化学1998,18(3)：29-31.
[58]Ellegren H. Microsatellites:simple sequences with complex evolution [J].Nature Reviews Genetics,2004,5,435-445.
[59]Britten R J, Kohne, D E.Repeated sequences in DNA[J].Science,1968,161,529-540.
[60]Chambers G K, MacAvoy E S. Comparative Biochemistry and Physiology Part B,2000,126. 455-476.
[61]Kasai K, Nakamura Y, White R. Amplification of a variable number of tandem repeats (VNTR) locus (pMCT118) by the polymerase chain reaction (PCR) and its application to forensic science[J].Journal of Forensic Science,1990,35,1196-1200.
[62]Weber J L. Informativeness of human (dC-dT)n(dG-dC)n polymorphisms[J]. Genomics,1990,(7): 524-530.
[63]Tauz D. Hypervariability of simple sequences as a general source for polymorphic DNA markers[J]. Nucleic Acid Research.1989,17:6463-6471.
[64]吴登俊,马丁费尔斯特.家畜基因组遗传多态标记-微卫星标记研究进展(上)[J].国外畜牧科技,1999,26(1)：33-40.
[65]Edwards A L, Andrew C, Holly A H. DNA typing and genetic mapping with trimeric and terameric tandem repeats[J]. American Journal of Human Genetics.1991,49(4):746-750.
[66]王芳.应用微卫星标记进行圈养大熊猫遗传多样性的研究[D].中国农业大学,2007,6,19-20.
[67]韩春梅,张嘉保,高庆华.DNA多态在动物亲子鉴定中的应用[J].草食家畜,2004,122：11-13.
[68]Ron M, Blanc Y, Band M, et al. Misidentification rate in the Israeli dairy cattle population and its implications for genetic improvement[J]. Journal of Dairy Science.1996,79:676-681.
[69]杨庆恩.亲子鉴定DNA分型亲权指数的简化计算法[J].中国法医学杂志.1998,13(2)：90-92.
[70]Moran P, Garcia V E. MuLtiple parternity in Atlantic salmon:a way to maintain genetic variability in relicted populations[J]. Journal of Heredity.1998,89(6):551-553.
[71]郑秀芬.法医DNA分析[M].北京：中国人民公安大学出版社,2002：422-424.
[72]Luikart G. Biju Duval M P, Ertugrul O, et al. Power of 22 microsatellite markers in fluorescent multiplexes for parentage testing in goats (Capra hircus)[J]. Animal Genetics,1999,30:431-438.
[73]Ohno Y, Sebetn I. A simple method for calculating the probability of excluding paternity with any number of codominant alleles[J]. Forensic. Sic. Int.1982,19:93-98.
[74]Saitbekova N, Gailard C. Genetic diversity in swiss goat breeds based on microlatellite analysis[J]. Animal Genetics.1999,30:36-41.
[75]Jamieson A.The genetics of transferring in cattle[J]. Heredity.1965,20:419-441.
[76]Jamieson A. The effectiveness of using codominant polymorphic allelic series for (1) checking pedigrees and (2) distinguishing fuLlsib pair members[J]. Animal Genetics.1994,25(suppl.l): 37-44.
[77]张于光,李迪强,饶力群等.东北虎微卫星DNA遗传标记的筛选及在亲子鉴定中的应用[J].动物学报,2003,49(1)：118-123.
[78]朱滨,常剑波,谭细畅等.湖鲟微卫星DNA引物应用于中华鲟亲子关系分析的初步研究[J].水生生物学报,1999,23(6)：547-533.
[79]黄磊,王义权.使用SSR和mtVNTR分子标记识别扬子鳄个体[J].动物学报,2005,51(3)：501-506.
[80]Heyen D W, Beever J E, Day, et al. Exclusion probability of 22 bovine microsatellite marker in fluorescent multiplexes for semi-automated parentage testing[J].Animal Genetic,1997,28:21-27.
[81]张志和.华南虎保护遗传学研究[D].浙江大学,2006.
[82]Reiner W, Neil B,Sonja G,et al.A guide to methods and applications of DNA and protein separations[M].Electrophoresis in practice. N J:A Wiley Company,1997:101-106.
[83]Higuchi R,Von Beroldingen C H,Senasbauch C F,et al.DNA typing from single hairs[J]. Nature, 1988,332:543-546.
[84]Heath E,Rutty G N.Genomic DNA from shed canine hairs[EB/OL].[2007-12-02]. http:-www.epicentre.com.
[85]Gagneux P,Boesch C,Woodruff D S.Microsatellite scoring errors associated with noninvasive genotyping based on nuclear DNA amplified from shed hair[J]. Mol Ecol,1997,6:861-868.
[86]Pascali V L,Bisol G D,Dobosz M,et al.Chimpanzee DNA profiles on trial[J]. Nature,1994,367: 692-693.
[87]Goldberg T L,Ruvolo M. The geographic apportionment of mitochondrial genetic diversity in east African chimpanzees,Pan troglodytes schweinfurthii[J]. Mol Biol Evol,1997,14:976-984.
[88]Morin P A, WoodruLf D S. Noninvasive genotyping for vertebrate conservation. In MolecuLar Genetic Approaches in Conservation, eds. Smith, T. B. and R. K. Wayne, [M]. Oxford University Press:New York,1996,298-313.
[89]Gill P, Jeffreys A J, Werrett D J. Forensic application of DNA'fingerprints'[J]. Nature,1985,318: 577-579.
[90]徐艳春,马立新,白素英.应用PCR方法通过毛发进行哺乳动物性别鉴定[J].东北林业大学学报,2000,8(6)：72-77.
[91]余舰,刘从勇,邹继初.PCR技术鉴定毛发、陈旧血痕的性别[J].遵义医学院学报,1995,18(1)：21-23.
[92]伍新尧,罗超权,等.PCR技术分析DNA多态性作毛发的个体鉴别[J].广州医药,1994,4：7-8.
[93]李军林,舒青,蒙世杰,等.非损伤性取样在朱鹅种群遗传研究中的应用[J].遗传,2001,23(3)：217-219.
[94]Yoshii T, Tamura K, Ishiyama I. Presence of a PCR inhibitor in hairs[J]. Nippon Hoigaku zasshi, 1992,46(2):313-316.
[95]Reginaldo G B, Federizzi J, Deschamps J C, et al. Characterization of swine stress geneoby DNA testing using plucked hair as a source of DNA[J]. Genetics and MolecuLar Biology,2000, 23(4):815-817.
[96]杨胜林,Surintorn B不同数量猪毛中提取基因组DNA的比较试验[J].山地农业生物学报,2005,24(5)：457-459.
[97]杨胜林,夏先林,Surintorn Boonanuntan,等.猪不同基因组DNA模板来源和浓度与微卫星位点扩增效果的比较研究[J].安徽农业科学,2008,36(6)：2265-2267.
[98]Linda Vigilant.An evaluation of techniques for the extraction and amplification of DNA from naturally shed hairs[J]. Biol Chem,1999,380:1329-1331.
[99]David V A, Menotti R M. MolecuLar Genetic Analysis of PopuLation [M]. New York:Oxford University Press,1998:337-350.
[100]闰路娜,张德兴.种群微卫星DNA分析中样本量对各种遗传多样性度量指标的影响[J].动物学报,2004,50(2)：279-290.
[101]Nei M. Estimation of average heterozygosity and genetic distance from a small number of individual[J]. Genetics.1978,89:583-585.
[102]陈幼春.关于分子水平下遗传距离检测的模型和适宜样本数的讨论[J]Animal Biotechnology Bullettin.1996:130-132.
[103]Barker JSF. A global protocol for determining genetic distance among domestic livestock breeds. Proc.5th World Congr. Genet. Appl. Livest. Prod,1994,21:501-508.
[104]鲁双庆,刘臻,刘红玉等.鲫鱼4群体基因组DNA遗传多样性及亲缘关系的微卫星分析[J].中国水产科学,2005,12(4)：371-376.
[105]Debrauwere H, Gendrel C G, Lechat S, et al. Differences and similarities between various tandem repeat sequences:minisatellite and microsatellite[J]. Biochimie.1997,79 (9-10):577-586.
[106]况少青,王建民.应用多重PCR进行微卫星荧光标记-半自动基因组扫面[J].中华医学遗传学杂志.1998,15(2)：104-107.
[107]Moore S S, et al. The conservation of dinucleotide microsatellites among mammalian genomes allows the use of heterologous PCR primer pays in closely related species[J]. Genomics.1991,10: 654-670.
[108]Hua Wu, Shan Ning Zhang, Fu Wen Wei. Twelve novel polymorphic microsatellite loci developed from the Asiatic black bear (Ursus thibetanus)[J]. Conserv Genet.2009,4,10.
[109]Maudet C, Miller C, Bassano B, et al. Microsatellite DNA and recent statistical methods in wild conservation management:application in Alpine ibex Capra ibex(ibex)[J]. MolecuLar Ecology. 2002,11:421-436.
[110]Ball A O, Leonard S, Chapman R W. Characterization of (GT) microsatellites from native white shrimp Penaeus setlferus[J]. Mol Ecol.1998,7:1258-1253.
[111]Heame C M, Ghosh S, Todd J A. Microsatellites for linkage analysis of genetic traits[J]. Trend in Genetics.1992,8:288-294.
[112]Jurg O. Analysis of Human Genetic Linkage[M]. Revised edition. Baltimore:Johns Hopkins University Press,2001:112-115.
[113]赵兴春,季安全,胡兰,等.法医DNA鉴定试验室质量控制和质量保证[J].中国法医学杂志,2002.17(1)：46-47.
[114]管峰,杨利国,贾名成,等.用微卫星标记技术对动物进行亲子鉴定[J].黑龙江畜牧兽医,2003,4：43-44.
[115]胡杰,薛士杰,金静君,等.STR在亲子鉴定中的应用[J].福建医药杂志,2000,22(5)：104-105.
[116]张阮章,王沙燕,戴勇.用STR位点进行亲子鉴定一例报告[J].暨南大学学报,2002,4：74-75.
[117]吴继法.微卫早DNA在凉山半细毛羊亲子鉴定中的应用[D].四川农业大学硕士学位论文.2002：32-33.
[118]侯一平.第四讲法医DNA分析的进展[J].中国法医学杂志,2001,16(4)：23-25.
[2]Smith AT,解焱.中国兽类野外手册[M].长沙：湖南教育出版社,2009：412-419.
[3]国家林业局主编.中国重点陆生野生动物资源调查[M].北京：中国林业出版社,2009：249-251.
[4]腾毅,杨海玲.我国养熊业及引流胆技术的历史与发展现状[J].中国中医药信息杂志,2010,(17)：4：8-9.
[5]吴修红,孙文军,腾毅,等.基于UPLC-Q-TOF联用技术的长期引流采胆熊及正常熊的代谢比较研究.江苏省博士研究生学术论坛文集,2006：587-593.
[6]姚贵芳,于春光,叶金堂.胆汁引流对黑熊健康状况影响的研究[J].黑龙江八一农垦大学学报,2010,22(4)：68-70.
[7]Morin P A, D S WoodruLf. Noninvasive genotyping for veterbrate conservation. In MolecuLar Genetic Approaches in Conservation[M]. Oxford University Press:New York.1996,298-313.
[8]Taberlet P, L P Waits, G Luikart. Nonivasive genetic sampling:look before you leap[J]. Trends Ecol. Evol.,1999,14:323-327.
[9]Gill P, A J Jeffreys, D J Werrent. Forensic application of DNA'fringerprints'[J]. Nature,1985, 318:577-579.
[10]Woodruff D S. Non-invasive genotyping of primates[J]. Primates,1993,34:333-346.
[11]Morin P A, J J Moore, R Chakraborty, et al. Kin selection,social structure,gene flow and evolution of chimpanzees[J]. Science,1994a,265:1193-1201.
[12]Morin P A, J J Moore, J Wallis, D S Woodruff. Paternity exclusion in a community of wild chimpanzees using hypervariable simple sequence repearts[J]. Mol Ecol.,1994b,3:469-479.
[13]Taberlet P, S Griffin, B Goossens, et al. Reliable genotyping of samples with very low DNA quantities using PCR[J]. Nucleic Acids Res.1996,24:3189-3194.
[14]Gerloff U,C Schlotterer, K Rassmann,et al. Amplification of hypervariable simple sequence repeats from excremental DNA of wild living Bonobos[J]. Mol. Ecol.,1995,4:515-518.
[15]Foran D R, K R Crooks, S C Minta. Species idenrifiction from scat:an unambiguous genetic method[J]. Wildlife Society BuLletin,1997,25:835-839.
[16]Kohn M H, E C York, D A Kamradt, et al. Estimating popuLation size by genotyping faeces. Proc. Royal. Soc(Series B),1999,266:657-663.
[17]方盛国,陈冠群,冯文和,等.大熊猫DNA 指纹在野生种群数量调查中的应用[J].兽类学报,1996a,16：246-249.
[18]Hoss M, M Kohn, S Paabo. Excrement analysis by PCR[J]. Nature,1992,359:199.
[19]Ernest H B, M C Penedo, N P May,et al. MolecuLar tracking of mountain lions in the Yosemite Valley region in California:genetic analysis using microsatellites and facecal DNA[J]. Mol.Ecol., 2000,9:433-441.
[20]Parsons K M, J F Dallas, D E Claridge,et al. Amplifying dolphin mitochondrial DNA from faecal plumes[J]. Mol. Ecol.,1999,8:1753-1768.
[21]Savill M G, S R Murray, P Scholes, et al. Application of polymerase chain reaction(PCR) and TaqManTM PCR techniques to the detection and identification of phodococcus coprophilus in faecal samples[J]. Journal of Microbiological Methods,2001,47:355-368.
[22]Ding B, Y P Zhang, Q A Ryder. Extraction, PCR amplification, and sequencing of mitochondrial DNA from scent mark and feces in the giant panda[J]. Zoo Biology,1998,17:499-504.
[23]Reed J Z, D J Tollit,P M Thompson,et al. MolecuLar scatology:the use of molecuLar genetic analysis to assign species, sex and individual identity to seal faeces[J]. Mol. Ecol.,1997,6:225-234.
[24]Machiels B M, T Ruers, M Lindhout, et al. New protocol for DNA extraction of stool[J]. BioTechniques,2000,28:286-290.
[25]Lantz P G,M Matsson, T Wadstrom, et al. Removal of PCR inhibitors from human faecal samples through the use of an aqueous two-phase system for sample preparation prior to PCR[J]. Journal of Microbiological Methods,1997,28:159-167.
[26]Inoue M,O Takenaka. Japanese macaque microsatellite PCR primers for paternity testing[J]. Primates,1993,34:37-45.
[27]丁波,O A Ryder,张亚平等.大熊猫气味标记DNA的制备和序列分析[J].动物学研究,1998,19：344-349.
[28]Taberlet P, J Bouvet. A single plucked feather as a source of DNA for bird genetic studies[J]. The Auk,1991,108:959-960.
[29]Weissenbach J, Fyapay G, Dib C,et al. A second-generation linkage map of human genome[J]. Nature,1992,359:794-801.
[30]钱迎倩,马克平.遗传多样性及其检测方法[M].北京中国科技出版社,1994,123-140.
[31]季维智,宿兵.遗传多样性研究的原理与方法[M].杭州浙江科学技术出版社,1999,1-12.
[32]Grant V. The Evolutionary Process:A Critical Study of Evolutionary Theory[M]. New York: Columbia University Press,1991,137-142.
[33]Millar C I, Libby W J. Strategies for conserving clinal, ecotypic, and disjunct popuLation diversity in wildspread species. In:Falk D A and Holsinger K E eds. Genetics and conservation of rare plante[M]. New York:Oxford University Press,1991,149-170.
[34]Hamrick J L, Godt M J W. Allozyme diversity in plant species. In:Brown A H D, Cleggg M T, Kahler A L, Wier B S eds. Plant popuLation genetics, breeding, and genetic resource. Sunderland, Massachuestts:Sinauer Associates,1990,43-63.
[35]Merrell D J. (黄瑞复等泽)生态遗传学[M].北京：科学出版社,1991,89-95.
[36]邱芳,伏健民,金德敏等.遗传多样性的分子检测[J].生物多样性,1998,6(2)：143-150.
[37]Nei M, Roychoudhury A K. Genetic variation within and between the three major races of man, Caucasoids, Negroids, and Mongoids[J]. Am J Hum Genet.1974,26:421-443.
[38]Bostein D, White R L. Construction of a genetic linkage maps in man using restraction fragment length polymorphisms[J]. Amer. J.Hum. Genet.1980,32:314-331.
[39]Francis C Y, Yang R C, Tim B. POPGENE(1.32).2000, http://www.ualberta.ca/fyeh/download.-htm.
[40]Raymond M, Rousset F. Genepop(1.2):PopuLation genetics software for exact tests and ecumenicism[J]. Hered.1995,86(3):248-249.
[41]Raymond M, Rousset F. Genepop(3.3). Population genetics software for exact tests and ecumenicism.2001, URL:Wbiomed.curtin.edu.au/genepop
[42]Louis E J, Dempster E R. An exact test for Hardy-Weinberg and muLtiple alleles[J]. Biometrics, 1987,43:805-811.
[43]Guo SW, Thompson E A. Performing the exact test of Hardy-Weiberg proportions for muLtiple alleles[J]. Biometrics,1992,48:361-372.
[44]Botstein D, White R L, Skolnick M, Dacis R. Construction of a genetic linkage map in man using restriction fragment length polymorphisms[J]. Am J Hum Genet.1980,32:314-331.
[45]Dodgson J B, Cheng H H, Okimoto R. DNA marker technology:a revolution in animal genetics[J]. PouLt Sci.1997,76:1108-1114.
[46]张亚平等.熊超科的分子系统发生研究[J].遗传学报,1997,24(1)：15-22.
[47]Welsh J, Muclellard M. Fingerprinting genomes using PCR with arbitary primers[J]. Nucleic Acids Research.1990,18:7213-7218.
[48]Williams J G K, Ubelik K A R, Livok J,et al. DNA Polymorphisms amplified by arbitary primers are usefuL as genetic marker[J]. Nucleic Acid Research.1990,18:6531-6535.
[49]陆永久,张亚平.随机扩增多态DNA影响因素的研究[J].动物学研究.1997,18(2)：221-227.
[50]林峰,陈卫红等.马来熊脑源性营养因子基因编码区的克隆和序列分析[J].动物学报,1998,44(1)：86.
[51]Vos P, Hogers R, Bleeker M, et al. AFLP:a new technique for DNA fingerprinting[J]. Nucleic Acids Res.1995,23:4407-4414.
[52]Mueller U G, Wolfenbarger L L. AFLP genotyping and fingerprinting[J]. Trends Ecology Evolution.1999,14(10):389-394.
[53]Jeffreys A J. Highly variable minisatellites and DNA fingerprints twenty-third colworth medal lecture[J]. Biochemical Society Transaction,1986,(9):309.
[54]陆惠玲,周斌,陈丽娴等.HRP标记α-珠蛋白-3’-HVR探针DNA指纹图法医应用的研究[J].法医学杂志,1996,12(3)：162-164.
[55]李光汉,杨智等.小熊猫圈养种群遗传多样性及其影响因子[J].生态学报,2002,20(2)：85-86.
[56]Lander E S. The new genomics:glabal views of biology[J]. Science,1996,274:536-539.
[57]姜运良.卫星小卫星和微卫星DNA-真核生物基因组的串状重复序列[J].生命的化学1998,18(3)：29-31.
[58]Ellegren H. Microsatellites:simple sequences with complex evolution [J].Nature Reviews Genetics,2004,5,435-445.
[59]Britten R J, Kohne, D E.Repeated sequences in DNA[J].Science,1968,161,529-540.
[60]Chambers G K, MacAvoy E S. Comparative Biochemistry and Physiology Part B,2000,126. 455-476.
[61]Kasai K, Nakamura Y, White R. Amplification of a variable number of tandem repeats (VNTR) locus (pMCT118) by the polymerase chain reaction (PCR) and its application to forensic science[J].Journal of Forensic Science,1990,35,1196-1200.
[62]Weber J L. Informativeness of human (dC-dT)n(dG-dC)n polymorphisms[J]. Genomics,1990,(7): 524-530.
[63]Tauz D. Hypervariability of simple sequences as a general source for polymorphic DNA markers[J]. Nucleic Acid Research.1989,17:6463-6471.
[64]吴登俊,马丁费尔斯特.家畜基因组遗传多态标记-微卫星标记研究进展(上)[J].国外畜牧科技,1999,26(1)：33-40.
[65]Edwards A L, Andrew C, Holly A H. DNA typing and genetic mapping with trimeric and terameric tandem repeats[J]. American Journal of Human Genetics.1991,49(4):746-750.
[66]王芳.应用微卫星标记进行圈养大熊猫遗传多样性的研究[D].中国农业大学,2007,6,19-20.
[67]韩春梅,张嘉保,高庆华.DNA多态在动物亲子鉴定中的应用[J].草食家畜,2004,122：11-13.
[68]Ron M, Blanc Y, Band M, et al. Misidentification rate in the Israeli dairy cattle population and its implications for genetic improvement[J]. Journal of Dairy Science.1996,79:676-681.
[69]杨庆恩.亲子鉴定DNA分型亲权指数的简化计算法[J].中国法医学杂志.1998,13(2)：90-92.
[70]Moran P, Garcia V E. MuLtiple parternity in Atlantic salmon:a way to maintain genetic variability in relicted populations[J]. Journal of Heredity.1998,89(6):551-553.
[71]郑秀芬.法医DNA分析[M].北京：中国人民公安大学出版社,2002：422-424.
[72]Luikart G. Biju Duval M P, Ertugrul O, et al. Power of 22 microsatellite markers in fluorescent multiplexes for parentage testing in goats (Capra hircus)[J]. Animal Genetics,1999,30:431-438.
[73]Ohno Y, Sebetn I. A simple method for calculating the probability of excluding paternity with any number of codominant alleles[J]. Forensic. Sic. Int.1982,19:93-98.
[74]Saitbekova N, Gailard C. Genetic diversity in swiss goat breeds based on microlatellite analysis[J]. Animal Genetics.1999,30:36-41.
[75]Jamieson A.The genetics of transferring in cattle[J]. Heredity.1965,20:419-441.
[76]Jamieson A. The effectiveness of using codominant polymorphic allelic series for (1) checking pedigrees and (2) distinguishing fuLlsib pair members[J]. Animal Genetics.1994,25(suppl.l): 37-44.
[77]张于光,李迪强,饶力群等.东北虎微卫星DNA遗传标记的筛选及在亲子鉴定中的应用[J].动物学报,2003,49(1)：118-123.
[78]朱滨,常剑波,谭细畅等.湖鲟微卫星DNA引物应用于中华鲟亲子关系分析的初步研究[J].水生生物学报,1999,23(6)：547-533.
[79]黄磊,王义权.使用SSR和mtVNTR分子标记识别扬子鳄个体[J].动物学报,2005,51(3)：501-506.
[80]Heyen D W, Beever J E, Day, et al. Exclusion probability of 22 bovine microsatellite marker in fluorescent multiplexes for semi-automated parentage testing[J].Animal Genetic,1997,28:21-27.
[81]张志和.华南虎保护遗传学研究[D].浙江大学,2006.
[82]Reiner W, Neil B,Sonja G,et al.A guide to methods and applications of DNA and protein separations[M].Electrophoresis in practice. N J:A Wiley Company,1997:101-106.
[83]Higuchi R,Von Beroldingen C H,Senasbauch C F,et al.DNA typing from single hairs[J]. Nature, 1988,332:543-546.
[84]Heath E,Rutty G N.Genomic DNA from shed canine hairs[EB/OL].[2007-12-02]. http:-www.epicentre.com.
[85]Gagneux P,Boesch C,Woodruff D S.Microsatellite scoring errors associated with noninvasive genotyping based on nuclear DNA amplified from shed hair[J]. Mol Ecol,1997,6:861-868.
[86]Pascali V L,Bisol G D,Dobosz M,et al.Chimpanzee DNA profiles on trial[J]. Nature,1994,367: 692-693.
[87]Goldberg T L,Ruvolo M. The geographic apportionment of mitochondrial genetic diversity in east African chimpanzees,Pan troglodytes schweinfurthii[J]. Mol Biol Evol,1997,14:976-984.
[88]Morin P A, WoodruLf D S. Noninvasive genotyping for vertebrate conservation. In MolecuLar Genetic Approaches in Conservation, eds. Smith, T. B. and R. K. Wayne, [M]. Oxford University Press:New York,1996,298-313.
[89]Gill P, Jeffreys A J, Werrett D J. Forensic application of DNA'fingerprints'[J]. Nature,1985,318: 577-579.
[90]徐艳春,马立新,白素英.应用PCR方法通过毛发进行哺乳动物性别鉴定[J].东北林业大学学报,2000,8(6)：72-77.
[91]余舰,刘从勇,邹继初.PCR技术鉴定毛发、陈旧血痕的性别[J].遵义医学院学报,1995,18(1)：21-23.
[92]伍新尧,罗超权,等.PCR技术分析DNA多态性作毛发的个体鉴别[J].广州医药,1994,4：7-8.
[93]李军林,舒青,蒙世杰,等.非损伤性取样在朱鹅种群遗传研究中的应用[J].遗传,2001,23(3)：217-219.
[94]Yoshii T, Tamura K, Ishiyama I. Presence of a PCR inhibitor in hairs[J]. Nippon Hoigaku zasshi, 1992,46(2):313-316.
[95]Reginaldo G B, Federizzi J, Deschamps J C, et al. Characterization of swine stress geneoby DNA testing using plucked hair as a source of DNA[J]. Genetics and MolecuLar Biology,2000, 23(4):815-817.
[96]杨胜林,Surintorn B不同数量猪毛中提取基因组DNA的比较试验[J].山地农业生物学报,2005,24(5)：457-459.
[97]杨胜林,夏先林,Surintorn Boonanuntan,等.猪不同基因组DNA模板来源和浓度与微卫星位点扩增效果的比较研究[J].安徽农业科学,2008,36(6)：2265-2267.
[98]Linda Vigilant.An evaluation of techniques for the extraction and amplification of DNA from naturally shed hairs[J]. Biol Chem,1999,380:1329-1331.
[99]David V A, Menotti R M. MolecuLar Genetic Analysis of PopuLation [M]. New York:Oxford University Press,1998:337-350.
[100]闰路娜,张德兴.种群微卫星DNA分析中样本量对各种遗传多样性度量指标的影响[J].动物学报,2004,50(2)：279-290.
[101]Nei M. Estimation of average heterozygosity and genetic distance from a small number of individual[J]. Genetics.1978,89:583-585.
[102]陈幼春.关于分子水平下遗传距离检测的模型和适宜样本数的讨论[J]Animal Biotechnology Bullettin.1996:130-132.
[103]Barker JSF. A global protocol for determining genetic distance among domestic livestock breeds. Proc.5th World Congr. Genet. Appl. Livest. Prod,1994,21:501-508.
[104]鲁双庆,刘臻,刘红玉等.鲫鱼4群体基因组DNA遗传多样性及亲缘关系的微卫星分析[J].中国水产科学,2005,12(4)：371-376.
[105]Debrauwere H, Gendrel C G, Lechat S, et al. Differences and similarities between various tandem repeat sequences:minisatellite and microsatellite[J]. Biochimie.1997,79 (9-10):577-586.
[106]况少青,王建民.应用多重PCR进行微卫星荧光标记-半自动基因组扫面[J].中华医学遗传学杂志.1998,15(2)：104-107.
[107]Moore S S, et al. The conservation of dinucleotide microsatellites among mammalian genomes allows the use of heterologous PCR primer pays in closely related species[J]. Genomics.1991,10: 654-670.
[108]Hua Wu, Shan Ning Zhang, Fu Wen Wei. Twelve novel polymorphic microsatellite loci developed from the Asiatic black bear (Ursus thibetanus)[J]. Conserv Genet.2009,4,10.
[109]Maudet C, Miller C, Bassano B, et al. Microsatellite DNA and recent statistical methods in wild conservation management:application in Alpine ibex Capra ibex(ibex)[J]. MolecuLar Ecology. 2002,11:421-436.
[110]Ball A O, Leonard S, Chapman R W. Characterization of (GT) microsatellites from native white shrimp Penaeus setlferus[J]. Mol Ecol.1998,7:1258-1253.
[111]Heame C M, Ghosh S, Todd J A. Microsatellites for linkage analysis of genetic traits[J]. Trend in Genetics.1992,8:288-294.
[112]Jurg O. Analysis of Human Genetic Linkage[M]. Revised edition. Baltimore:Johns Hopkins University Press,2001:112-115.
[113]赵兴春,季安全,胡兰,等.法医DNA鉴定试验室质量控制和质量保证[J].中国法医学杂志,2002.17(1)：46-47.
[114]管峰,杨利国,贾名成,等.用微卫星标记技术对动物进行亲子鉴定[J].黑龙江畜牧兽医,2003,4：43-44.
[115]胡杰,薛士杰,金静君,等.STR在亲子鉴定中的应用[J].福建医药杂志,2000,22(5)：104-105.
[116]张阮章,王沙燕,戴勇.用STR位点进行亲子鉴定一例报告[J].暨南大学学报,2002,4：74-75.
[117]吴继法.微卫早DNA在凉山半细毛羊亲子鉴定中的应用[D].四川农业大学硕士学位论文.2002：32-33.
[118]侯一平.第四讲法医DNA分析的进展[J].中国法医学杂志,2001,16(4)：23-25.