黑麂（Muntiacus crinifrons）AFLP-SCAR种特异性探针研制
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摘要
黑麂(Muntiacus crinifrons)是我国特有的濒危鹿科动物,具有很高的学术研究价值。由于分布范围狭窄,加之传统狩猎习俗和经济价值等因素,该物种长期遭受非法捕猎的生存压力,野生种群数量已十分稀少,因而,黑麂被列为国家一级重点保护野生动物。尽管如此,野外盗猎现象仍时有发生,出售在市场上的黑麂被去皮后,执法人员几乎无法利用形态学特征等宏观手段进行鉴定。为了切实保护黑麂,为相关执法部门提供一种便捷、可靠、高效的工具来鉴定黑麂组织样品,本研究基于AFLP方法开发了黑麂种特异性SCAR探针。
首先,利用引物Ep7和Mp8获得了长度为707bp的黑麂AFLP特异性条带,对该特异性DNA片段进行纯化、克隆、测序。根据所测DNA片段序列信息,利用Primer Premier 5.0和Oligo 6.0软件设计了黑麂特异引物(P-F/P1-R),并获得了长度为298bp的SCAR。为了检测特异引物的可靠性,对引物进行了群体扩增检测。同时,利用哺乳动物cyt b通用引物L1和H1对所有待检样品DNA的质量进行了检测。结果显示,在全部9种43个个体样品中,25个黑麂样品全部扩增出2条目标条带,即长度298bp的黑麂特异条带和长度约为466bp的cyt b基因片段;而代表其它物种的18个个体均仅扩增出单一的cyt b基因片段。所有个体都能被哺乳动物cyt b通用引物扩增,说明待检样品DNA的质量全部符合PCR扩增要求,引物可靠性检测结果达到对目标动物进行法医学鉴定的目的。
综上所述:本研究已成功地将黑麂种特异性AFLP标记转化为SCAR标记,并得到了具有高度物种特异性的SCAR探针。利用本研究开发的SCAR特异探针,只要根据黑麂特异条带的有或无,就能快速、准确地鉴别出黑麂。因此,该探针可作为执法部门鉴定黑麂组织样品的有效工具,以加强对偷猎行为的执法力度。此外,利用该探针可以基于无损伤取样法(如粪便)对特定区域黑麂野生种群动态进行监测。
Black Muntjac (Muntiacus crinifrons), a rare and endangered deer endemic to China, holds a high value in academic research. The species is currently regarded as the state key protected wild animal due to its limited distribution range and small population in the wild. However, because of the economic value of the meat, antlers and skin, the species has been hunted as the main targeted animal in traditional hunting practices and is still suffered from poaching at the present time. Usually, in order to escape from punishment, the poached black muntjac was peeled and sold together with carcasses of other animals, such as Chinese muntjac or livestocks. To provide a convenient, reliable and effective tool for authentication of tissue specimen such as, meat, skin and other tissues of the species, in this study, a Sequence Characterized Amplified Region (SCAR) derived from the black muntjac species-specific Amplification Fragment Length Polymorphism (AFLP) marker has been developed.
Initially, a 707-bp species specific DNA fragment of black muntjac was detected by an AFLP primer pair (Ep7/Mp8). Then, the DNA fragment was purified, cloned and sequenced. Subsequently, based on the specific AFLP fragment sequence, a black muntjac-specific primer pair (P-F/P1-R) was designed by using Primer Premier 5.0 and Oligo 6.0, obtaining a 298-bp SCAR for the species. Finally, the reliability of the SCAR primers was verified across all the samples, PCR amplification with the cyt b universal primers as positive control. As expected, out of the all forty-three DNA samples representing nine different species, all the twenty-five black muntjac samples presented two bands (about 298bp for SCAR and 466bp for cyt b). But the others failed to produce the SCAR with merely showing one band, the 466-bp cyt b fragment. The results indicated that the black muntjac-specific AFLP marker has been converted into a SCAR marker successfully and the SCAR primers were highly species-specific. Just based on the presence and absence of the SCAR band, we could discriminate black muntjac from other species rapidly and accurately. Therefore, the SCAR primers developed in this study will provide an effective tool for forensic authentication of tissues from black muntjac and strengthen the detection of poaching. And also, the SCAR probe may be useful for population survey in the wild through noninvasive sampling method.
首先,利用引物Ep7和Mp8获得了长度为707bp的黑麂AFLP特异性条带,对该特异性DNA片段进行纯化、克隆、测序。根据所测DNA片段序列信息,利用Primer Premier 5.0和Oligo 6.0软件设计了黑麂特异引物(P-F/P1-R),并获得了长度为298bp的SCAR。为了检测特异引物的可靠性,对引物进行了群体扩增检测。同时,利用哺乳动物cyt b通用引物L1和H1对所有待检样品DNA的质量进行了检测。结果显示,在全部9种43个个体样品中,25个黑麂样品全部扩增出2条目标条带,即长度298bp的黑麂特异条带和长度约为466bp的cyt b基因片段;而代表其它物种的18个个体均仅扩增出单一的cyt b基因片段。所有个体都能被哺乳动物cyt b通用引物扩增,说明待检样品DNA的质量全部符合PCR扩增要求,引物可靠性检测结果达到对目标动物进行法医学鉴定的目的。
综上所述:本研究已成功地将黑麂种特异性AFLP标记转化为SCAR标记,并得到了具有高度物种特异性的SCAR探针。利用本研究开发的SCAR特异探针,只要根据黑麂特异条带的有或无,就能快速、准确地鉴别出黑麂。因此,该探针可作为执法部门鉴定黑麂组织样品的有效工具,以加强对偷猎行为的执法力度。此外,利用该探针可以基于无损伤取样法(如粪便)对特定区域黑麂野生种群动态进行监测。
Black Muntjac (Muntiacus crinifrons), a rare and endangered deer endemic to China, holds a high value in academic research. The species is currently regarded as the state key protected wild animal due to its limited distribution range and small population in the wild. However, because of the economic value of the meat, antlers and skin, the species has been hunted as the main targeted animal in traditional hunting practices and is still suffered from poaching at the present time. Usually, in order to escape from punishment, the poached black muntjac was peeled and sold together with carcasses of other animals, such as Chinese muntjac or livestocks. To provide a convenient, reliable and effective tool for authentication of tissue specimen such as, meat, skin and other tissues of the species, in this study, a Sequence Characterized Amplified Region (SCAR) derived from the black muntjac species-specific Amplification Fragment Length Polymorphism (AFLP) marker has been developed.
Initially, a 707-bp species specific DNA fragment of black muntjac was detected by an AFLP primer pair (Ep7/Mp8). Then, the DNA fragment was purified, cloned and sequenced. Subsequently, based on the specific AFLP fragment sequence, a black muntjac-specific primer pair (P-F/P1-R) was designed by using Primer Premier 5.0 and Oligo 6.0, obtaining a 298-bp SCAR for the species. Finally, the reliability of the SCAR primers was verified across all the samples, PCR amplification with the cyt b universal primers as positive control. As expected, out of the all forty-three DNA samples representing nine different species, all the twenty-five black muntjac samples presented two bands (about 298bp for SCAR and 466bp for cyt b). But the others failed to produce the SCAR with merely showing one band, the 466-bp cyt b fragment. The results indicated that the black muntjac-specific AFLP marker has been converted into a SCAR marker successfully and the SCAR primers were highly species-specific. Just based on the presence and absence of the SCAR band, we could discriminate black muntjac from other species rapidly and accurately. Therefore, the SCAR primers developed in this study will provide an effective tool for forensic authentication of tissues from black muntjac and strengthen the detection of poaching. And also, the SCAR probe may be useful for population survey in the wild through noninvasive sampling method.
引文
Barrett BA, Kidwell KK. AFLP-based genetic diversity assessment among wheat cultivars from the Pacific Northwest. Crop Sci., 1998, 38: 1261-1271.
Blackett RS, Keim P. Big game species identification by deoxyribonucleic acid (DNA) probes. Journal of Forensic Sciences, 1992, 37(2): 590-596.
Boumedine K, Rodolakis A. AFLP allows the identification of genomic markers of ruminant Chlamydia psittaci strains useful for typing and epidemiological studies. Research in Microbiology, 1998, 149(10): 735-744.
Brugmans B,hulst RM, Visser RGF, Lindhout P. A new and versatile method for the successful conversion of AFLP markers into simple single locus markers. Nucleic Acids Research, 2003, 31(10): e55.
Choi YE, Ahn CH, Kim BB, Yoo ES. Development of species specific AFLP-derived SCAR marker for authentication of Panax japonicus C. A. MEYER. Biological & Pharmaceutical Bulletin, 2008, 31(1): 135-138.
Fang SG, Wan QH. A genetic fingerprinting test for identifying carcasses of protected deer species in China. Biological Conservation, 2002, 103(3): 371-373.
Fernandes CA, Ginja C, Pereira I, Tenreiro R, Bruford MW, Santos-Reis M. Species-specific mitochondrial DNA markers for identification of non-invasive samples from sympatric carnivores in the Iberian Peninsula. Conservation Genetics, 2008, 9(3): 681-690.
HsiehhM, ChianghL, Tsai LC, Lai SY,huang NE, Linacre A, Lee JCI. Cytochrome b gene for species identification of the conservation animals. Forensic Science International, 2001, 122(1): 7-18.
Karlsson AO,holmlund G. Identification of mammal species using species-specific DNA pyrosequencing. Forensic Science International, 2007, 173(1): 16-20.
Kethidi DR, Roden DB, Ladd TR, Krell PJ, Retnakaran A, Feng Q. Development of SCAR markers for the DNA-based detection of the asian long-horned beetle, Anoplophora glabripennis (Motschulsky). Archives of Insect Biochemistry and Physiology, 2003, 52: 193-204.
Levinson G, Gutman GA. Slipped-strand mispairing: a major mechanism for DNA sequence evolution. Molecular Biology and Evolution, 1987, 4: 203-221.
Linder CR, Moore LA, Jackson RB. A universal molecular method for identifying underground plant parts to species. Molecular Ecology, 2000, 9(10): 1549-1559.
Livia L, Francesca V, Antonella P, Fausto P, Bernardino R. A PCR-RFLP method on faecal samples to distinguish Martes martes, Martes foina, Mustela putorius and Vulpes vulpes. Conservation Genetics, 2007, 8(3): 757-759.
Lu HG, Sheng HL. Status of the black muntjac, Muntiacus crinifrons, in eastern China. Mammal Rev, 1984, 14(1): 29-36.
Matsunaga T, Chikuni K, Tanabe R, Muroya S, Nakaih, Shibata K, Yamada J, Shinmura Y. Determination of mitochondrial cytochrome b gene sequence for red deer (Cervus elaphus) and the differentiation of closely related deer meats. Meat Science, 1998, 49(4): 379-385.
Meksem K, Ruben E,hytem D. Conversion of AFLP bands intohigh-thoughput DNA markers. Genet Genomics, 2001, 265: 207-214.
Meng K, Yu J, Luo TL, WuhL, Wu XB. Generation of a sequence characterized amplified region probe for authentication of mainland serow (Capricornis sumatraensis). Molecular Ecology Resources, 2009, 9(3): 764-766.
Morin PA, Woodruff DS. Noninvasive genotyping for vertebrate conservation. Molecular genetic approaches in conservation, 1996, 298-313.
Noguera FJ, Capel J, Alvarez JI, Lozano R. Development and mapping of a codominant SCAR marker linked to the andromonoecious gene of melon. Theoretical and Applled Genetics, 2005, 110: 714-720.
Paran I, Michelmore RW. Development of reliable PCR-based markers linked to downy mildew resistance genes in lettuce. Theor Appl Genet, 1993, 85: 985-993.
Riddle AE, Pilgrim KL, Mills LS, McKelvey KS, Ruggiero LF. Identification of mustelids using mitochondrial DNA and non-invasive sampling. Conservation Genetics, 2003, 4(2): 241-243.
Sambrook J, Russell DW. Molecular Cloning: A Laboratory Manual (3rd edn), 2001, Cold Springharbor Laboratory Press, New York. ShenghL, LuhG. Current studies on the rare Chinese black muntjac. Journal of Naturalhistory, 1980, 14(6): 803-807.
Taberlet P, Waits LP, Luikart G. Noninvasive genetic sampling: look before you leap. TREE., 1999, 14(8): 323-327.
Tams SH, Melchinger AE, Bauer E. Genetic similarity among European winter triticale elite germplasms assessed with AFLP and comparisons with SSR and pedigree data.Plant Breeding, 2005, 124: 154-160.
Van der Wurff AWG, Chan YL, van Straalen NM, Schouten J. TE-AFLP: combining rapidity and robustness in DNA fingerprinting. Nucleic Acids Research, 2000, 28(24): e105.
Vos P,hogers R, Bleeker M, Reijans M, van de Lee T,hornes M, Frijters A, Pot J, Peleman J, Kuiper M, Zabeau M. AFLP: a new technique for DNA fingerprinting. Nucleic Acids Research, 1995, 23: 4407-4414.
Vuylsteke M, Peleman JD, van Eijk MJT. AFLP technology for DNA fingerprinting. Nature Protocols, 2007, 2(6): 1387-1398.
Wan QH, Fang SG. Application of species-specific polymerase chain reaction in the forensic identification of tiger species. Forensic Science International, 2003, 131(1): 75-78.
Wong KL, Wang J, But PPH, Shaw PC. Application of cytochrome b DNA sequences for the authentication of endangered snake species. Forensic Science International, 2004, 139(1): 49-55.
Woo PCY, Leung PKL, Leung KW, Yuen KY. Identification by 16S ribosomal RNA gene sequencing of an Enterobacteriaceae species from a bone marrow transplant recipient. Molecular Pathology, 2000, 53(4): 211-215.
Wu HL, Fang SG. Characterization of mtDNA d-loop of black muntjac.第九届哺乳动物学大会论文集, 2005,日本,札幌.
Wu HL, Wan QH, Fang SG. Microsatellite analysis of genetic variation and population subdivision for the black muntjac, Muntiacus crinifrons. Biochemical Genetics, 2007, 45: 775-788.
Wu HL, Wan QH, Fang SG. Population structure and gene flow among wild populations of the black muntjac (Muntiacus crinifrons) based on mitochondrial DNA control region sequences. Zoological Science, 2006, 23(4): 333-340.
Wu XB, Liuh, Jiang ZG. Identification primers for sika deer (Cervus nippon) from a sequence-characterised amplified region (SCAR). New Zealand Journal of Zoology, 2006, 33: 65-71.
Yau FCF, Wong KL, Wang J, But PPH, Shaw PC. Generation of a sequence characterized amplified region probe for authentication of crocodilian species. Journal of Experimental Zoology Part B: Molecular and Developmental Evolution, 2002, 294(4): 382-386.
Zhang JB, Cai ZP. Differentiation of the rainbow trout (Oncorhynchus mykiss) from
Atlantic salmon (Salmon salar) by the AFLP-derived SCAR. European Food Research and Technology, 2006, 223(3): 413-417.
鲍毅新,程宏毅,周襄武,陈良,胡知渊,葛宝明.黑麂(Muntiacus crinifrons)3个种群的遗传多样性.生态学报, 2008, 28(8): 4030-4036.
鲍毅新,郑祥,葛宝明.浙江黑麂栖息地评价及保护对策.生态学报, 2006, 26(8): 2425-2431.
程宏毅,鲍毅新,陈良,周襄武,胡知渊,葛宝明.黑麂皖-浙分布中心种群的遗传多样性.动物学报, 2008, 54(1): 96-103.
褚栋,张友军,丛斌,徐宝云,吴青君. SCAR分子标记鉴别烟粉虱和温室白粉虱.植物保护, 2004, 30(6): 27-30.
单祥年,施燕峰,张海军,徐春宏,李健,郑爱玲.小麂、赤麂、黑麂mtDNA序列变异性及反映的进化关系.动物学杂志, 2004, 39(4): 35-39.
丁红梅,丁志山,李海波,陈铌铍.红曲菌株鉴别中SCAR分子标记的应用.中国中药杂志, 2008, 33(4): 359-362.
段幸生,刘爱华,林世英.黑麂的核型.动物学研究, 1981, 2(4): 383-385.
高文娜,陈万权,王中康. AFLP双酶切和连接方法探讨.植物保护, 2003, 29(6): 46-48.
贺培建,阮向东,方盛国. 12S rRNA在黑麂和黄麂物种鉴定中的应用.兽类学报, 2004, 24(4): 350-352.
江幸福,罗礼智.利用近等基因系构建黏虫黑化型AFLP-SCAR标记.植物保护, 2007, 33(5): 94-95.
鞠秀芝,杜胜利,宗兆锋,张桂华,韩毅科,王鸣. AFLP技术及其常见问题与解决方案.天津农业科学, 2004, 10(4): 6-9.
兰宏,施立明.麂属(Muntiacus)动物线粒体DNA多态性及其遗传分化.中国科学B辑:化学, 1993, 23(5): 489-497.
李珊,赵桂仿. AFLP分子标记及其应用.西北植物学报, 2003, 23(5): 830-836.
李璇,王晓东. AFLP的研究.甘肃教育学院学报(自然科学版), 2004, 18(1): 58-61.
刘平武,李赟,蔡强,宋传宇,何庆彪,杨光圣. AFLP分子标记技术的改进.生物技术通报, 2006年增刊: 255-260.
欧善华,盛和林,陆厚基.黑麂和毛冠鹿的食性.上海师范大学学报(自然科学版), 1981, 1(1): 111-115.
蒲志刚,刘太国,张敏,陈万权.小麦叶锈菌生理小种MFR的分子鉴定研究.植物病理学报, 2004, 34(5): 449-454.
邱冬梅,孙继英,刘金,颜亨梅.扩增片断长度多态性(AFLP)荧光标记和银染技术的比较分析.生命科学研究, 2006, 10(3): 17-21.
盛和林,陆厚基.黑麂(M. crinifrons)的繁殖.兽类学报, 1981, 1: 14-18.
盛和林,徐宏发,陆厚基.黑麂种群的初步研究.华东师范大学学报(哺乳动物生态专辑), 1990, 54-59.
盛和林.中国特产动物——黑麂.动物学杂志, 1987, 22(2): 45-48.
盛和林等.中国鹿类动物, 1992,华东师范大学出版社,上海, pp: 149-159.
童芳芳,汤明亮,杨星,彭智,刘思阳.用RAPD和SCAR复合分子标记对黄颡鱼属进行种质鉴定.水生生物学报, 2005, 29(4): 465-468.
汪松等.中国濒危动物红皮书(兽类), 1998,科学出版社,北京, pp: 282-284.
王斌,翁曼丽. AFLP的原理及其应用.杂交水稻, 1996, 5: 27-30.
王立新,常利芳,黄岚,王晓维,赵昌平.小麦AFLP片段序列多态性分析和AFLP-SCAR标记的研究.麦类作物学报, 2007, 27(6): 943-951.
王岐山.安徽兽类志, 1990,安徽科学技术出版社,合肥.
王晓维,王立新,常利芳,魏建民,赵昌平.小麦AFLP-SCAR标记的开发及应用.麦类作物学报, 2008, 28(5): 738-744.
魏麟,黎晓英,黄英,史宪伟.遗传标记及其发展概述.动物科学与动物医学, 2004, 21(10): 42-45.
吴海龙.黑麂保护遗传学研究.浙江大学, 2006.
吴学谦,李海波,魏海龙,付立忠,吴庆其,彭华正,朱睦元. SCAR分子标记技术在香菇菌株鉴定上的应用研究.菌物学报, 2005, 24(2): 259-266.
叶汪薇,朱彤,胡超超,张晨岭,万霞,周立志,常青,张保卫.对几起疑似野生动物肉食品的分子遗传学分析.兽类学报, 2008, 28(4): 434-439.
张海军,李健,施燕峰,张晓梅,徐春宏,单祥年.黑麂线粒体基因组序列分析.中国生物化学与分子生物学报, 2004, 20(4): 513-518.
郑祥,鲍毅新,葛宝明,郑荣泉.黑麂栖息地利用的季节变化.兽类学报, 2006, 26(2): 201-205.
周用武. DNA技术鉴定动物物种研究评述.通化师范学院学报, 2008, 29(2): 48-51.
朱伟铨,王义权. AFLP分子标记技术及其在动物学研究中的应用.动物学杂志, 2003, 38(2): 101-107.
庄南生,郑成木.植物AFLP标记转化为特异PCR标记的研究进展.华南热带农业大学学报, 2004, 10(3): 11-14.
Blackett RS, Keim P. Big game species identification by deoxyribonucleic acid (DNA) probes. Journal of Forensic Sciences, 1992, 37(2): 590-596.
Boumedine K, Rodolakis A. AFLP allows the identification of genomic markers of ruminant Chlamydia psittaci strains useful for typing and epidemiological studies. Research in Microbiology, 1998, 149(10): 735-744.
Brugmans B,hulst RM, Visser RGF, Lindhout P. A new and versatile method for the successful conversion of AFLP markers into simple single locus markers. Nucleic Acids Research, 2003, 31(10): e55.
Choi YE, Ahn CH, Kim BB, Yoo ES. Development of species specific AFLP-derived SCAR marker for authentication of Panax japonicus C. A. MEYER. Biological & Pharmaceutical Bulletin, 2008, 31(1): 135-138.
Fang SG, Wan QH. A genetic fingerprinting test for identifying carcasses of protected deer species in China. Biological Conservation, 2002, 103(3): 371-373.
Fernandes CA, Ginja C, Pereira I, Tenreiro R, Bruford MW, Santos-Reis M. Species-specific mitochondrial DNA markers for identification of non-invasive samples from sympatric carnivores in the Iberian Peninsula. Conservation Genetics, 2008, 9(3): 681-690.
HsiehhM, ChianghL, Tsai LC, Lai SY,huang NE, Linacre A, Lee JCI. Cytochrome b gene for species identification of the conservation animals. Forensic Science International, 2001, 122(1): 7-18.
Karlsson AO,holmlund G. Identification of mammal species using species-specific DNA pyrosequencing. Forensic Science International, 2007, 173(1): 16-20.
Kethidi DR, Roden DB, Ladd TR, Krell PJ, Retnakaran A, Feng Q. Development of SCAR markers for the DNA-based detection of the asian long-horned beetle, Anoplophora glabripennis (Motschulsky). Archives of Insect Biochemistry and Physiology, 2003, 52: 193-204.
Levinson G, Gutman GA. Slipped-strand mispairing: a major mechanism for DNA sequence evolution. Molecular Biology and Evolution, 1987, 4: 203-221.
Linder CR, Moore LA, Jackson RB. A universal molecular method for identifying underground plant parts to species. Molecular Ecology, 2000, 9(10): 1549-1559.
Livia L, Francesca V, Antonella P, Fausto P, Bernardino R. A PCR-RFLP method on faecal samples to distinguish Martes martes, Martes foina, Mustela putorius and Vulpes vulpes. Conservation Genetics, 2007, 8(3): 757-759.
Lu HG, Sheng HL. Status of the black muntjac, Muntiacus crinifrons, in eastern China. Mammal Rev, 1984, 14(1): 29-36.
Matsunaga T, Chikuni K, Tanabe R, Muroya S, Nakaih, Shibata K, Yamada J, Shinmura Y. Determination of mitochondrial cytochrome b gene sequence for red deer (Cervus elaphus) and the differentiation of closely related deer meats. Meat Science, 1998, 49(4): 379-385.
Meksem K, Ruben E,hytem D. Conversion of AFLP bands intohigh-thoughput DNA markers. Genet Genomics, 2001, 265: 207-214.
Meng K, Yu J, Luo TL, WuhL, Wu XB. Generation of a sequence characterized amplified region probe for authentication of mainland serow (Capricornis sumatraensis). Molecular Ecology Resources, 2009, 9(3): 764-766.
Morin PA, Woodruff DS. Noninvasive genotyping for vertebrate conservation. Molecular genetic approaches in conservation, 1996, 298-313.
Noguera FJ, Capel J, Alvarez JI, Lozano R. Development and mapping of a codominant SCAR marker linked to the andromonoecious gene of melon. Theoretical and Applled Genetics, 2005, 110: 714-720.
Paran I, Michelmore RW. Development of reliable PCR-based markers linked to downy mildew resistance genes in lettuce. Theor Appl Genet, 1993, 85: 985-993.
Riddle AE, Pilgrim KL, Mills LS, McKelvey KS, Ruggiero LF. Identification of mustelids using mitochondrial DNA and non-invasive sampling. Conservation Genetics, 2003, 4(2): 241-243.
Sambrook J, Russell DW. Molecular Cloning: A Laboratory Manual (3rd edn), 2001, Cold Springharbor Laboratory Press, New York. ShenghL, LuhG. Current studies on the rare Chinese black muntjac. Journal of Naturalhistory, 1980, 14(6): 803-807.
Taberlet P, Waits LP, Luikart G. Noninvasive genetic sampling: look before you leap. TREE., 1999, 14(8): 323-327.
Tams SH, Melchinger AE, Bauer E. Genetic similarity among European winter triticale elite germplasms assessed with AFLP and comparisons with SSR and pedigree data.Plant Breeding, 2005, 124: 154-160.
Van der Wurff AWG, Chan YL, van Straalen NM, Schouten J. TE-AFLP: combining rapidity and robustness in DNA fingerprinting. Nucleic Acids Research, 2000, 28(24): e105.
Vos P,hogers R, Bleeker M, Reijans M, van de Lee T,hornes M, Frijters A, Pot J, Peleman J, Kuiper M, Zabeau M. AFLP: a new technique for DNA fingerprinting. Nucleic Acids Research, 1995, 23: 4407-4414.
Vuylsteke M, Peleman JD, van Eijk MJT. AFLP technology for DNA fingerprinting. Nature Protocols, 2007, 2(6): 1387-1398.
Wan QH, Fang SG. Application of species-specific polymerase chain reaction in the forensic identification of tiger species. Forensic Science International, 2003, 131(1): 75-78.
Wong KL, Wang J, But PPH, Shaw PC. Application of cytochrome b DNA sequences for the authentication of endangered snake species. Forensic Science International, 2004, 139(1): 49-55.
Woo PCY, Leung PKL, Leung KW, Yuen KY. Identification by 16S ribosomal RNA gene sequencing of an Enterobacteriaceae species from a bone marrow transplant recipient. Molecular Pathology, 2000, 53(4): 211-215.
Wu HL, Fang SG. Characterization of mtDNA d-loop of black muntjac.第九届哺乳动物学大会论文集, 2005,日本,札幌.
Wu HL, Wan QH, Fang SG. Microsatellite analysis of genetic variation and population subdivision for the black muntjac, Muntiacus crinifrons. Biochemical Genetics, 2007, 45: 775-788.
Wu HL, Wan QH, Fang SG. Population structure and gene flow among wild populations of the black muntjac (Muntiacus crinifrons) based on mitochondrial DNA control region sequences. Zoological Science, 2006, 23(4): 333-340.
Wu XB, Liuh, Jiang ZG. Identification primers for sika deer (Cervus nippon) from a sequence-characterised amplified region (SCAR). New Zealand Journal of Zoology, 2006, 33: 65-71.
Yau FCF, Wong KL, Wang J, But PPH, Shaw PC. Generation of a sequence characterized amplified region probe for authentication of crocodilian species. Journal of Experimental Zoology Part B: Molecular and Developmental Evolution, 2002, 294(4): 382-386.
Zhang JB, Cai ZP. Differentiation of the rainbow trout (Oncorhynchus mykiss) from
Atlantic salmon (Salmon salar) by the AFLP-derived SCAR. European Food Research and Technology, 2006, 223(3): 413-417.
鲍毅新,程宏毅,周襄武,陈良,胡知渊,葛宝明.黑麂(Muntiacus crinifrons)3个种群的遗传多样性.生态学报, 2008, 28(8): 4030-4036.
鲍毅新,郑祥,葛宝明.浙江黑麂栖息地评价及保护对策.生态学报, 2006, 26(8): 2425-2431.
程宏毅,鲍毅新,陈良,周襄武,胡知渊,葛宝明.黑麂皖-浙分布中心种群的遗传多样性.动物学报, 2008, 54(1): 96-103.
褚栋,张友军,丛斌,徐宝云,吴青君. SCAR分子标记鉴别烟粉虱和温室白粉虱.植物保护, 2004, 30(6): 27-30.
单祥年,施燕峰,张海军,徐春宏,李健,郑爱玲.小麂、赤麂、黑麂mtDNA序列变异性及反映的进化关系.动物学杂志, 2004, 39(4): 35-39.
丁红梅,丁志山,李海波,陈铌铍.红曲菌株鉴别中SCAR分子标记的应用.中国中药杂志, 2008, 33(4): 359-362.
段幸生,刘爱华,林世英.黑麂的核型.动物学研究, 1981, 2(4): 383-385.
高文娜,陈万权,王中康. AFLP双酶切和连接方法探讨.植物保护, 2003, 29(6): 46-48.
贺培建,阮向东,方盛国. 12S rRNA在黑麂和黄麂物种鉴定中的应用.兽类学报, 2004, 24(4): 350-352.
江幸福,罗礼智.利用近等基因系构建黏虫黑化型AFLP-SCAR标记.植物保护, 2007, 33(5): 94-95.
鞠秀芝,杜胜利,宗兆锋,张桂华,韩毅科,王鸣. AFLP技术及其常见问题与解决方案.天津农业科学, 2004, 10(4): 6-9.
兰宏,施立明.麂属(Muntiacus)动物线粒体DNA多态性及其遗传分化.中国科学B辑:化学, 1993, 23(5): 489-497.
李珊,赵桂仿. AFLP分子标记及其应用.西北植物学报, 2003, 23(5): 830-836.
李璇,王晓东. AFLP的研究.甘肃教育学院学报(自然科学版), 2004, 18(1): 58-61.
刘平武,李赟,蔡强,宋传宇,何庆彪,杨光圣. AFLP分子标记技术的改进.生物技术通报, 2006年增刊: 255-260.
欧善华,盛和林,陆厚基.黑麂和毛冠鹿的食性.上海师范大学学报(自然科学版), 1981, 1(1): 111-115.
蒲志刚,刘太国,张敏,陈万权.小麦叶锈菌生理小种MFR的分子鉴定研究.植物病理学报, 2004, 34(5): 449-454.
邱冬梅,孙继英,刘金,颜亨梅.扩增片断长度多态性(AFLP)荧光标记和银染技术的比较分析.生命科学研究, 2006, 10(3): 17-21.
盛和林,陆厚基.黑麂(M. crinifrons)的繁殖.兽类学报, 1981, 1: 14-18.
盛和林,徐宏发,陆厚基.黑麂种群的初步研究.华东师范大学学报(哺乳动物生态专辑), 1990, 54-59.
盛和林.中国特产动物——黑麂.动物学杂志, 1987, 22(2): 45-48.
盛和林等.中国鹿类动物, 1992,华东师范大学出版社,上海, pp: 149-159.
童芳芳,汤明亮,杨星,彭智,刘思阳.用RAPD和SCAR复合分子标记对黄颡鱼属进行种质鉴定.水生生物学报, 2005, 29(4): 465-468.
汪松等.中国濒危动物红皮书(兽类), 1998,科学出版社,北京, pp: 282-284.
王斌,翁曼丽. AFLP的原理及其应用.杂交水稻, 1996, 5: 27-30.
王立新,常利芳,黄岚,王晓维,赵昌平.小麦AFLP片段序列多态性分析和AFLP-SCAR标记的研究.麦类作物学报, 2007, 27(6): 943-951.
王岐山.安徽兽类志, 1990,安徽科学技术出版社,合肥.
王晓维,王立新,常利芳,魏建民,赵昌平.小麦AFLP-SCAR标记的开发及应用.麦类作物学报, 2008, 28(5): 738-744.
魏麟,黎晓英,黄英,史宪伟.遗传标记及其发展概述.动物科学与动物医学, 2004, 21(10): 42-45.
吴海龙.黑麂保护遗传学研究.浙江大学, 2006.
吴学谦,李海波,魏海龙,付立忠,吴庆其,彭华正,朱睦元. SCAR分子标记技术在香菇菌株鉴定上的应用研究.菌物学报, 2005, 24(2): 259-266.
叶汪薇,朱彤,胡超超,张晨岭,万霞,周立志,常青,张保卫.对几起疑似野生动物肉食品的分子遗传学分析.兽类学报, 2008, 28(4): 434-439.
张海军,李健,施燕峰,张晓梅,徐春宏,单祥年.黑麂线粒体基因组序列分析.中国生物化学与分子生物学报, 2004, 20(4): 513-518.
郑祥,鲍毅新,葛宝明,郑荣泉.黑麂栖息地利用的季节变化.兽类学报, 2006, 26(2): 201-205.
周用武. DNA技术鉴定动物物种研究评述.通化师范学院学报, 2008, 29(2): 48-51.
朱伟铨,王义权. AFLP分子标记技术及其在动物学研究中的应用.动物学杂志, 2003, 38(2): 101-107.
庄南生,郑成木.植物AFLP标记转化为特异PCR标记的研究进展.华南热带农业大学学报, 2004, 10(3): 11-14.