蒙古牛和日本和牛遗传多态性比较研究
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摘要
本文用12种识别6个碱基的限制性内切酶BamHⅠ、BglⅡ、EcoRⅠ、EcoRⅤ、HindⅢ、KpnⅠ、PstⅠ、PvuⅡ、SacⅠ、SalⅠ、ScaⅠ和XhoⅠ对13头蒙古牛进行了线粒体DNA的限制性片断长度多态性分析，研究其起源、分化和种群遗传结构，并与已知的日本和牛的mtDNA-RFLP结果(Watanabe等，1985)进行比较，以初步探讨其亲缘关系。在蒙古牛中BamHⅠ、BglⅡ、HindⅢ、PstⅠ和SacⅠ 5种酶的酶切类型存在多态性，共检测到31个酶切位点，17种限制性态型，归结为3种线粒体DNA单倍型。单倍型Ⅱ和Ⅲ(蒙古牛的基本单倍型)在蒙古牛2个地方群体中分布频率较高，分别为46.15％和46.15％，单倍型Ⅰ分布频率较低(7.69％)。蒙古牛的Ⅱ、Ⅲ两种单倍型属于普通牛型，Ⅰ单倍型属于瘤牛型。日本和牛相应的11种内切酶分析中只有1种酶(HindⅢ)的酶切类型存在多态，共出现34个酶切位点，13种限制性态型，归结为2种线粒体DNA单倍型(单倍型Ⅳ和Ⅴ)。在日本和牛中单倍型Ⅴ(日本和牛的基本单倍型)所占比例较大(71.43％)，单倍型Ⅳ只占28.57％。利用Nei氏公式计算了各基因单倍型间的遗传距离P、群体遗传多态度值以及各群体间的净遗传距离，结果显示日本和牛和蒙古牛2个基本单倍型之间的遗传距离分别为2.610％，2.703％，说明了它们之间的亲缘关系较远。按照哺乳动物mtDNA碱基突变率每百万年2％计算，单倍型Ⅱ和Ⅲ(遗传距离为0.478％)所代表的蒙
蒙古牛和日本和牛遗传多态性比较研究硕士学位论文
古牛群体之间的分化时间大约24万年，这比人类驯化牛10000的历史要久远
得多。I单倍型和11、m两种单倍型(遗传距离为1.467%，1.83870)所代表的
牛群体之间的分化时间大约73万年至92万年前。蒙古牛不同群体中三种单
倍型的分布频率有差异，以单倍型n为限制性态型的母系祖先对乌珠穆沁地
区蒙古牛群体的形成遗传贡献较大，而对锡林浩特市附近地区的蒙古牛而言，
单倍型m所代表的母系祖先的遗传贡献较大。按照哺乳动物mtDNA碱基突变
率每百万年2%计算，两个蒙古牛群体(净遗传距离为0.2831%)的分化时间大约
为14万年前。本研究中蒙古牛各基因单倍型间的平均遗传距离(P值)为
0.0126，mtDNA核昔酸歧异度(二值)为0.219%，处于被认为是较低的范围
内(0 .15%一0.47%)。
The restriction fragment length polymorphism( RFLP) of mitochondrial DNA(mtDNA) of 13 Mongolian cattle breeds were studied using 12 restriction endonucleases recognizing 6 basepair, BamH I ,Bgl II ,EcoR I ,EcoR V ,Hind ni,Kpn I ,Pst I ,Pvu II ,Sac I ,Sal I ,Sca I and Xho I ,and compared with Japanese black cattle (Watanabe et al,1985), for investigation of the phylogenetic relationship between them. In Mongolian cattle, 5 of these(BamH I ,Bgl II ,Hind III,Pst I and Sac I )were found to be polymorphisms. Total 31 restriction sites and 17 restriction morphs were detected , which could be sorted into 3 haplotypes. Haplotype II and III (main haplotypes of Mongolian cattle) are distributed in mongolian cattle breeds with relatively high frequency ,i.e. 46.15%and 46.15%, respectively. The frequency of haplotype I is lower(7.69%).Haplotype II and III are shown the type of yellow cattle(Bos taurus), and Haplotype I is shown in that of zebu cattle(Bos indicus). In Japanese black cattle, only one of these restriction
endonucleases, HindIII, was found to be polymorphisms. Total 34 restriction sites and 13 restriction morphs were detected, which could be sorted into 2 haplotypes (Haplotype IV and V ) . The frequency of haplotype V (main haplotype of Japanese black cattle)is higher(71.43%) and haplotypeIVis lower (28.57%) . The genetic distance between haplotypes and polymorphic degree( value) were also
calculated according to the methods of Nei's. The results show that genetic distance between main haplotypes of Mongolian cattle and Japanese black cattle are 2.610% and 2.703% respectively. It implies that the relationship between Japanese black cattle and Mongolian cattle is more distant. According to that the rate of sequence divergence of mtDNA is estimated to be 2% per million years, Haplotype II and III of mongolian cattle' s mtDNA might be separate about 240 thousand years ago, which is earlier than the 10000-year history of cattle husbandry. Haplotype of yellow cattle (haplotype I )and zebu cattle(Haplotype II andIII) might be separate about 0.73-0.92 million years ago. Frequencies of haplotype I , II and III are different in each Mongolian cattle populations, maternal ancestors of haplotype II give more genetic contribution to Mongolian cattle breeds located in Wuzhumuqin region, but maternal ancestors of haplotype III give more genetic contribution to Mongolian cattle breeds located in Xilinhot reg
ion. They might be separate about 140 thousand years ago. The average genetic distances between haplotypes and the polymorphic degree of Mongolian cattle populations were 0.0126 and 0.219%, respectively. It is always considered quite low(0.15%-0.47%).
蒙古牛和日本和牛遗传多态性比较研究硕士学位论文
古牛群体之间的分化时间大约24万年，这比人类驯化牛10000的历史要久远
得多。I单倍型和11、m两种单倍型(遗传距离为1.467%，1.83870)所代表的
牛群体之间的分化时间大约73万年至92万年前。蒙古牛不同群体中三种单
倍型的分布频率有差异，以单倍型n为限制性态型的母系祖先对乌珠穆沁地
区蒙古牛群体的形成遗传贡献较大，而对锡林浩特市附近地区的蒙古牛而言，
单倍型m所代表的母系祖先的遗传贡献较大。按照哺乳动物mtDNA碱基突变
率每百万年2%计算，两个蒙古牛群体(净遗传距离为0.2831%)的分化时间大约
为14万年前。本研究中蒙古牛各基因单倍型间的平均遗传距离(P值)为
0.0126，mtDNA核昔酸歧异度(二值)为0.219%，处于被认为是较低的范围
内(0 .15%一0.47%)。
The restriction fragment length polymorphism( RFLP) of mitochondrial DNA(mtDNA) of 13 Mongolian cattle breeds were studied using 12 restriction endonucleases recognizing 6 basepair, BamH I ,Bgl II ,EcoR I ,EcoR V ,Hind ni,Kpn I ,Pst I ,Pvu II ,Sac I ,Sal I ,Sca I and Xho I ,and compared with Japanese black cattle (Watanabe et al,1985), for investigation of the phylogenetic relationship between them. In Mongolian cattle, 5 of these(BamH I ,Bgl II ,Hind III,Pst I and Sac I )were found to be polymorphisms. Total 31 restriction sites and 17 restriction morphs were detected , which could be sorted into 3 haplotypes. Haplotype II and III (main haplotypes of Mongolian cattle) are distributed in mongolian cattle breeds with relatively high frequency ,i.e. 46.15%and 46.15%, respectively. The frequency of haplotype I is lower(7.69%).Haplotype II and III are shown the type of yellow cattle(Bos taurus), and Haplotype I is shown in that of zebu cattle(Bos indicus). In Japanese black cattle, only one of these restriction
endonucleases, HindIII, was found to be polymorphisms. Total 34 restriction sites and 13 restriction morphs were detected, which could be sorted into 2 haplotypes (Haplotype IV and V ) . The frequency of haplotype V (main haplotype of Japanese black cattle)is higher(71.43%) and haplotypeIVis lower (28.57%) . The genetic distance between haplotypes and polymorphic degree( value) were also
calculated according to the methods of Nei's. The results show that genetic distance between main haplotypes of Mongolian cattle and Japanese black cattle are 2.610% and 2.703% respectively. It implies that the relationship between Japanese black cattle and Mongolian cattle is more distant. According to that the rate of sequence divergence of mtDNA is estimated to be 2% per million years, Haplotype II and III of mongolian cattle' s mtDNA might be separate about 240 thousand years ago, which is earlier than the 10000-year history of cattle husbandry. Haplotype of yellow cattle (haplotype I )and zebu cattle(Haplotype II andIII) might be separate about 0.73-0.92 million years ago. Frequencies of haplotype I , II and III are different in each Mongolian cattle populations, maternal ancestors of haplotype II give more genetic contribution to Mongolian cattle breeds located in Wuzhumuqin region, but maternal ancestors of haplotype III give more genetic contribution to Mongolian cattle breeds located in Xilinhot reg
ion. They might be separate about 140 thousand years ago. The average genetic distances between haplotypes and the polymorphic degree of Mongolian cattle populations were 0.0126 and 0.219%, respectively. It is always considered quite low(0.15%-0.47%).
引文
[1] 田允波、葛长荣、杨亮宇，云南黄牛的起源与分类地位，黄牛杂志，1998，24(2)：55-60
[2] Anderson S, de Bruijn M H, Coulson A R et al. Complete sequence of bovine mitochondrial DNA. Conserved features of the mammalian genome. J M of Biol, 1982, 156:683-717
[3] Hauswirth WW, Laipis PJ. Mitochondrial DNA polymorphism in a maternal lineage of Holstein cows. Proc Natl Acad Sci U S A. 1982 Aug; 79(15):4686-4690.
[4] Watanabe T, Hayashi Y, Semba R et al. Bovine Mitochondrial DNA polymorphism in restriction endonuclease cleavage patterns and the location of the polymorphic sites. Biochemical Genetics, 1985, 23(11/12):947-957
[5] Watanabe T, Masangkay J, Wakana S et al. Mitochondrial DNA polymorphism in native Philippine cattle based on restriction endonuclease cleavage patterns. Biochemical genetics, 1989, 27:431-438
[6] Bhat P Pet al. Polymorphism of mitochondrial DNA(mtDNA)in cattle and buffaloes. Biochemical Genetics. 1990. 28 (7/8): 311-318
[7] Loftus R T, Machugh D E, Nger L Oet al. Mitochondrial genetic variation in European African and Indian cattle populations. Animal Genetics, 1994, 25:215-221
[8] Kikkawa Y, Amano T, Suzuki H. Analysis of Genetic diversity of domestic cattle in East and Southeast Asia in terms of variations in restriction sites and sequences of mitochondricalDNA. Biochemical Genetics, 1995, 33 (1/2): 123-127
[9] 兰宏、熊习昆、林世英等．云南黄牛和大额牛的mtDNA多态性研究．遗传学报，1993，20(5)：419-423
[10] 聂龙、陈永久、王文等．海南黄牛和徐闻黄牛线粒体DNA的多态性及其品种分化关系．动物学研究，1996，17(3)：269-274
[11] 何正权、张亚平、简承松等，贵州黄牛品种间mtDNA的限制性片段长度多态性研究，动物学研究，1999，20(1)：7-11
[12] 王文，施立明，一种改进的动物线粒体DNA提取方法．动物学研究，
1993, 14(2): 197-198.
[13] Nei M, Li W H, 1979. Mathematical model for studying genetic variation in terms of restriction endonucleases. Proc. Natl. Acad. Sci. USA, 76:5269-5273
[14] 于汝梁，黄牛起源和分类的研究现状及有待解决的问题，生物学通报，2000，359(9)：38-40
[15] 陈幼春等，中国黄牛品种多样性及其保护，生物多样性 2001，9(3)：275-283
[16] 于汝梁等．黄牛Y染色体多态性及品种起源演变的研究．中国农业科学，1993，26(5)：61-67．
[17] 文际坤，赵开典，朱芳贤，俞英，刘爱华，林世英，张亚平，1998．云南五个黄牛品种的染色体比较研究．养牛科学研究进展，172-175
[18] Brown W M, 1983. Evolution of Animal Mitochondrial DNA. In: Nei M R K Koehml (eds.). Evolution of Genes and Proteins. New York: Sinauer Assotiation Inc. Press. 62-88.
[19] Kikkawa Y, Takada T, Sutopo, Nomura K, Phylogenies using mtDNA and SRY provide evidence for male-mediated introgression in Asian domestic cattle. Animal Genetics. 2003, 34(2):96-101
[20] 潘军，日本和牛品种形成、特征与评价，黑龙江动物繁殖 1994，2(1)：4-6
[21] 王作洲，戈新，日本和牛，山东畜牧兽医，2001，1：39-40
[22] Namikawa T, Genetical aspect of domestication and phylogeny in the cattle. Jap. J. Zoothech. Sci. 1980, 49: 817-827.
[23] Ogawa, Y., M. Daigo and H. Amasaki, 1989 Craniometrical estimation of the native Japanese Mishima cattle. Anat. Anz. Jena. 168:197-203.
[24] Felius, M, 1995, Cattle Breeds--An Encyclopedia. Misset, Doetinchem, The Netherlands.
[25] H. Mannen, S. Tsuji, R. T. Loftus, D. G. Bradley(1998b) Mitochondrial DNA Variation and Evolution of Japanese Black Cattle (Bos taurus), Genetics 150:1169-1175
[26] Lan H, Shi L M. The origin and genetic differentiation of native breeds of pigs in southwest China:An approach from mitochondrial DNA polymorphism [J]. Biochemical genetics 1993, 31:51-60.
[27] 兰宏，王文，施立明西南地区家猪和野猪mtDNA遗传多样性研究，遗传学报，1995，22(1)：28-33
[28] 王文，刘爱华，林世英，麻栗坡县家马mtDNA多态性研究，云南畜牧兽医1995，(增
刊)：39-42
[29] 门正明，韩建林，权洁霞等．阿拉善双峰驼线粒体DNA限制性酶切类型的研究．兰州大学学报，1995，31(遗传学专辑)：1-7．
[2] Anderson S, de Bruijn M H, Coulson A R et al. Complete sequence of bovine mitochondrial DNA. Conserved features of the mammalian genome. J M of Biol, 1982, 156:683-717
[3] Hauswirth WW, Laipis PJ. Mitochondrial DNA polymorphism in a maternal lineage of Holstein cows. Proc Natl Acad Sci U S A. 1982 Aug; 79(15):4686-4690.
[4] Watanabe T, Hayashi Y, Semba R et al. Bovine Mitochondrial DNA polymorphism in restriction endonuclease cleavage patterns and the location of the polymorphic sites. Biochemical Genetics, 1985, 23(11/12):947-957
[5] Watanabe T, Masangkay J, Wakana S et al. Mitochondrial DNA polymorphism in native Philippine cattle based on restriction endonuclease cleavage patterns. Biochemical genetics, 1989, 27:431-438
[6] Bhat P Pet al. Polymorphism of mitochondrial DNA(mtDNA)in cattle and buffaloes. Biochemical Genetics. 1990. 28 (7/8): 311-318
[7] Loftus R T, Machugh D E, Nger L Oet al. Mitochondrial genetic variation in European African and Indian cattle populations. Animal Genetics, 1994, 25:215-221
[8] Kikkawa Y, Amano T, Suzuki H. Analysis of Genetic diversity of domestic cattle in East and Southeast Asia in terms of variations in restriction sites and sequences of mitochondricalDNA. Biochemical Genetics, 1995, 33 (1/2): 123-127
[9] 兰宏、熊习昆、林世英等．云南黄牛和大额牛的mtDNA多态性研究．遗传学报，1993，20(5)：419-423
[10] 聂龙、陈永久、王文等．海南黄牛和徐闻黄牛线粒体DNA的多态性及其品种分化关系．动物学研究，1996，17(3)：269-274
[11] 何正权、张亚平、简承松等，贵州黄牛品种间mtDNA的限制性片段长度多态性研究，动物学研究，1999，20(1)：7-11
[12] 王文，施立明，一种改进的动物线粒体DNA提取方法．动物学研究，
1993, 14(2): 197-198.
[13] Nei M, Li W H, 1979. Mathematical model for studying genetic variation in terms of restriction endonucleases. Proc. Natl. Acad. Sci. USA, 76:5269-5273
[14] 于汝梁，黄牛起源和分类的研究现状及有待解决的问题，生物学通报，2000，359(9)：38-40
[15] 陈幼春等，中国黄牛品种多样性及其保护，生物多样性 2001，9(3)：275-283
[16] 于汝梁等．黄牛Y染色体多态性及品种起源演变的研究．中国农业科学，1993，26(5)：61-67．
[17] 文际坤，赵开典，朱芳贤，俞英，刘爱华，林世英，张亚平，1998．云南五个黄牛品种的染色体比较研究．养牛科学研究进展，172-175
[18] Brown W M, 1983. Evolution of Animal Mitochondrial DNA. In: Nei M R K Koehml (eds.). Evolution of Genes and Proteins. New York: Sinauer Assotiation Inc. Press. 62-88.
[19] Kikkawa Y, Takada T, Sutopo, Nomura K, Phylogenies using mtDNA and SRY provide evidence for male-mediated introgression in Asian domestic cattle. Animal Genetics. 2003, 34(2):96-101
[20] 潘军，日本和牛品种形成、特征与评价，黑龙江动物繁殖 1994，2(1)：4-6
[21] 王作洲，戈新，日本和牛，山东畜牧兽医，2001，1：39-40
[22] Namikawa T, Genetical aspect of domestication and phylogeny in the cattle. Jap. J. Zoothech. Sci. 1980, 49: 817-827.
[23] Ogawa, Y., M. Daigo and H. Amasaki, 1989 Craniometrical estimation of the native Japanese Mishima cattle. Anat. Anz. Jena. 168:197-203.
[24] Felius, M, 1995, Cattle Breeds--An Encyclopedia. Misset, Doetinchem, The Netherlands.
[25] H. Mannen, S. Tsuji, R. T. Loftus, D. G. Bradley(1998b) Mitochondrial DNA Variation and Evolution of Japanese Black Cattle (Bos taurus), Genetics 150:1169-1175
[26] Lan H, Shi L M. The origin and genetic differentiation of native breeds of pigs in southwest China:An approach from mitochondrial DNA polymorphism [J]. Biochemical genetics 1993, 31:51-60.
[27] 兰宏，王文，施立明西南地区家猪和野猪mtDNA遗传多样性研究，遗传学报，1995，22(1)：28-33
[28] 王文，刘爱华，林世英，麻栗坡县家马mtDNA多态性研究，云南畜牧兽医1995，(增
刊)：39-42
[29] 门正明，韩建林，权洁霞等．阿拉善双峰驼线粒体DNA限制性酶切类型的研究．兰州大学学报，1995，31(遗传学专辑)：1-7．