摘要
本研究利用随机扩增多态性DNA技术对褐壳蛋鸡(HK)、白壳蛋鸡(BK)、粉壳蛋鸡(FK)、绿壳蛋鸡(LK)和海兰鸡种C系一代(HC-1)、C系二代(HC-2)、海兰鸡种A系一代(HA-1)、A系二代(HA-2)8个蛋鸡群体在DNA水平上进行了遗传多态性分析,寻找出不同蛋鸡群体的特异性分子标记,并探讨了群体间的遗传距离以及八个群体之间的亲缘关系。结果如下:
一、八个不同蛋鸡群体的特异性分子标记研究
(1)在优化PCR反应条件的基础上,建立了比较完善的RAPD技术体系。在50条随机引物中,30个引物未出现任何扩增产物,12个引物表现为单态,筛选出8个多态引物,每个多态引物的扩增条带数在2~10条之间,平均为5.3条。
(2)通过利用8个引物对八个不同的蛋鸡群体进行PCR扩增,发现在OPV_(15)位点上,共扩增出10条DNA片段,其中A条带可以作为区别HK与BK的分子标记,而E条带可作为HA-1与HA-2的特异性条带。在OPB_(08)位点上,共扩增出12条DNA片段,F条带可以作为HA-1与HA-2的特异性条带,在OPH_(05)位点上,共扩增出12条DNA条带,I条带可以作为区别BK与LK的条带,在OPH_(20)位点上,共扩增出11条DNA片段,C条带可以作为区别LK与FK、BK的特异性条带,但无法区别BK与FK,在OPH_(19)位点上,共扩增出14条DNA片段,C片段可以作为HC-1与HC-2的特异性条带。在OPH_(13)位点,共扩增出10条DNA条带,H片段可以作为HC-1与HC-2的特异性条带。
二、八个不同蛋鸡群体的遗传多样性分析
(1)HK群体内条带相似率最小(0.824);HA-2群体内的条带相似率最大(0.897)。说明HK群体中遗传纯度不高,具有较大的遗传变异范围;而HA-2群体则具有较大的群体一致度。
(2)HA-1群体中一条带的平均等位基因频率最大(0.7019),BK群体中条带的平均等位基因频率最小(0.4844)。群体内的杂合度可以反映群体内遗传多样性,表明群体内的遗传变异范围。HK群体的杂合度最大(0.5889),表明HK群体中遗传多样性高,具有较大的遗传变异范围;而BK群体中的杂合度最小(0.3766),表明这个群体的遗传多态性较低,群内一致性较高。
(3)在八个不同蛋鸡群体中,HK的遗传多样性指数最大(1.1598)。说明HK群体具有比其它群体较高的遗传多样性,可作为蛋鸡遗传改良的素材,而遗传多样性指数最小的是BK群体(0.6097),这可能与群体采用同质选配和近亲选配等育种手段有关。
(4)本研究采用SPSS软件进行统计分析,根据Nearest Neighbor下的7种聚类方
法进行聚类,其中欧氏距离、欧氏平方距离、夹角余弦、皮尔逊相关四种方法得出的
结果是基本一致的,较能反映实际来源情况。而车贝雪夫距离、街区距离、明考夫斯
基三种聚类方法与实际的群体来源分布有较大差异。各种聚类方法得出的结果有一定
的差异,这是由不同处理方法各自的特性决定的,也可能与遗传标记以及所研究的位
点等因素有关。
(5)从聚类结果可以看出:在8个不同的蛋鸡群体中,FK与LK关系最近,这与群
体的实际来源相符。HC一2与HA一2以及HC一1与HA一1亲缘关系最近,这与实际来源稍
有出入,但是HC与HA是海兰褐壳鸡的不同品系,具有较高的同源性。而BK、HK、FK、
LK与HC一1、HC一2、HA一1、队一2都表现出较远的遗传距离,说明引进品种和地方品种具
有较远的亲缘关系。
The genetic diversity of 8 layer populations were analyzed by using RAPD technic. Their relationships and genetic distance of intra-populations were studied and some characteristic molecule markers of 8 layer populations were found .The results showed that:
1. The study of the characteristic molecular marker on the 8 different layer populations
(1) On the basis of perfecting the PCR reaction conditions,more advanced RAPD technology was established. It showed that RAPD marker technic was suitable for the genetic analysis on layers. 30 primers of the 50 random primers amplified no any products, 12 primers of them amplified products to be monomorphic and 8 of 50 primers amplified products to be polymorphic. The numbers of amplified bands of each of 8 were between 2 and 10 with a mean 5.3.
(2)The PCR amplified products on the 8 different layer populations by using the 8 polymorphic primers showed mat on OPV15 primer amplified 10 polymorphic bands, band A could be used as the molecular marker to differentiate HK and BK populations.on OPB08 primer amplified 12 polymorphic bands, band E and F could be seen as the characteristic bands of HA-1 and HA-2. on OPH05 primer amplified 12 polymorphic bands, band I could be used to differentiate BK and LK populations, on OPH20 primer amplified 11 polymorphic bands,band C could be as the characteristic band of differentiating LK and BK populations,but could not tell BK from FK populations, on OPH19 primer amplified 14 polymorphic bands ,while band C as the characteristic band of HC-1 and HC-2. on OPH13 amplified primer 10 polymorphic bands ,band H as the characteristic band of HC-1 and HC-2. 2. The analysis of genetic diversity in the 8 different layer populations
(1)The intra-population band similarity rate of HA-2 was largest (0.897),while HK was smallest (0.824),these indicated that HA-2 had lower intra-breed genetic pure level and HK had higher.
(2)Gene frequency of HA-1 population was largest(0.7019),BK was smallest(0.4844); The intra-population heterozygosity can stand for the intra-population genetic diversity and genetic variation grope.In this study ,the heterozygosity of HK population was the largest
(0.5889), showed that HK had higher genetic diversity and genetic variation grope,While the heterozygosity of BK was the smallest(0.3766),it showed that BK had lower genetic diversity and higher intra-population similarity.
(3)The index of genetic diversity of HK population was the largest(1.1598),among the 8 layer populations,and it showed that the population had higher genetic diversity than any other populations. So, HK could be seen as the material of the layers genetic improvementHowever, BK had the lowest genetic diversity(0.6097), perhaps, it was caused by applying the breeding methods of positive assortative mating and inbreeding mating.
(4) 7 polygenetic methods of Nearest Neighbor were analyzed by SPSS : Euclidean distance,Squared Euclidean Distance,Cosine of Vectors of Values,Pearson Correlation Between Vectors of Values,Chebychev Distance,City Block distance and Minkowski distance were used to analyze the genetic relationship between the 8 populations in this study.The results showed that Euclidean distance,Squared Euclidean Distance,Cosine of Vectors of Values and Pearson Correlation Between Vectors of Values were same , and they were in accordance with the facts and their geographical distribution.but Chebychev distance,City Block distanceand Minkowski distance were not in accordance with the facts and their geographical distribution.There were variations of each polygenetic methods, was determined by the character of different methods, and perhaps,it was caused by the genetic marker and the loci.
(5)The relationship between FK and LK was the nearest in the 8 layer populations from the analysis results and it was in accordance with the actual orgin. These between HC-2 and HA-2 ,and between HC-1 and HA-1 were the nearest.This was in accordance with the fact. However, HC and HA populations were the different line of HK population,so they ha
引文
1.白秀娟.肉鸡肥度性状DNA标记的研究[D].东北农业大学博士后研究工作报告,哈尔滨,2000
2.常洪.我国家畜遗传资源的优势与危机[M].《中国家畜遗传资源研究》,陕西人民教育出版社,1998
3.曾福玲,李宁等.鸡品种(系)的随机扩增多态性DNA指纹分析[C].第八次全国动物遗传育种学术研讨会论文集,北京,农业出版社,1995,41-464.
4.常洪.中国家禽遗传资源的保存[J].畜牧兽医杂志,1985(2):1-9,1985(3):29-30
5.陈国宏.鸡血浆碱性磷酸酶与生产性能关系的研究[J].江苏农学院学报,1990(4):37-39
6.程端仪,慎伟杰等.隐性白羽鹌鹑的发现及其应用研究[J].遗传,1992,14(4):23-25
7.陈奕新,左正宏.RAPD技术探讨几种家鸭遗传多样性及亲缘关系[J].厦门大学学报,2001,40(1):141-145
8.程光潮,刘树俊等.利用标记基因选配杂交亲本[C].第四次全国畜禽遗传标记研讨会论文集,1993,11
9.戴有理等.绿壳蛋鸡遗传结构的RAPD分析[J].中国家禽,2001,23(22):47-50
10.杜立新.我国畜禽遗传资源系统管理初探[J].畜牧兽医杂志,1991(4):39-42
11.樊斌.微卫星标记评估中国猪遗传多样性与标记辅助选择效率若干制约因素的研究[D].华东农业大学硕士学位论文,武汉,1999
12.高雪.中国驴种来源的遗传学研究[D].西北农林科技大学硕士论文,2001,6
13.龚允陈,王金玉,陈国宏.鸡的DNA指纹条带数目与屠宰性能的相关性研究,江苏农学院学报[J].1998,19(1):55-58
14.龚道清.肉鸡血浆VLDL浓度的选择效应及肥度性状的分子标记,东北农业大学博士学位论文[D].哈尔滨,1999
15.龚道清,张军,李辉.RAPD标记检测与肉鸡肥度性状关联的QTL[A].全国动植物数量遗传与育种学术研讨会论文集,扬州,2000:267-271
16.耿社民.四个黄牛群体外形特征的表型分化研究[A].第五次全国畜禽遗传标记研讨会论文集,1996,6
17.龚炎长.猪微卫星DNA的克隆、特性分析及应用研究[D].中国农业大学博士学位论文,北京,1997
18.黄海根.牛的高变异微卫星序列的克隆、结构分析及应用[A].第五次全国畜禽遗传标记研讨会论文集。1996,6
19.胡刚安等.鸡产肉性状基因位点的分子标记及效应分析[A].第六次全国畜禽遗传
标记研讨会论文集,常州,1998:133
20.黄海根,孟安明.齐顺章等,DNA指纹图带与鸡的蛋重性状的遗传相关分析[A].遗传,1998,20(3):13-15
21.郝家胜,周开亚.中国鹅5个品种遗传多样性随机扩增DNA分析[J].南京农业大学学报,2000,23(1):58-62
22.简承松,张永亮.中国部分地方鸡品种五个血浆同工酶位点的遗传多样性及聚类分析[C].第八次全国畜禽遗传标记研讨会论文集。2001.10,中国杨凌
23.刘荣宗,罗玉英.家畜遗传资源保护的新途径[J].畜牧兽医杂志,1998,17(2):16-19
24.吕雪梅.蛋鸡品系群体遗传结构及杂种优势的RAPD分析[A].华南农业大学硕士学位论文,1997
25.李亮,朱庆.四川地方乌骨鸡遗传多样性的微卫星分析[J].2002,四川畜牧兽医杂志,2001
26.李辉.肉鸡肥度性状的遗传标记及其应用研究[D].东北农业大学博士后研究工作报告,1998,哈尔滨
27.刘娣.鸡品种随机引物扩增多态DNA的研究[J].东北农业大学学报,1999,30(4):349-354
28.李辉.肉鸡肥度性状的遗传标记及其应用研究[D].东北农业大学博士后研究工作报告,1998,哈尔滨.
29.龙火生,史宪伟,张英汉等.11个鸡群体遗传多样性的RAPD标记分析[C].第八次全国畜禽遗传标记研讨会论文集.2002,10
30.刘荣宗,施启顺.不同杂交组合猪的RAPD标记研究[C].中国动物遗传育种研究,1997,28-32
31.李海涛、杨山.不同蛋鸡品系间蛋白质(酶)多态遗传差异的研究[C].第四次全国畜禽遗传标记研讨会论文集,1993,11
32.孟安明,齐顺章等.用DNA指纹图测定北京白鸡的遗传结构[A].中国畜牧兽医学会数量遗传研究会.中国畜禽遗传育种进展,第七次全国畜禽育种讨论会论文集长沙,湖南科学技术出版社,1993:113-115
33.孟安明.畜禽的DNA多态性及其应用[C].第四次全国畜禽遗传标记研讨会论文集,1993,11
34.彭中镇,刘榜,余梅.生物技术与猪的育种[C].第十次全国动物遗传育种学术讨论会论文集,1998
35.潘庆杰等.鹅的产蛋性能RAPD标记研究[C].第11次全国动物遗传育种学术会议论文集.2001
36.庞有志.蛋用鹌鹑伴性羽基因用于遗传学实验的设计与分析[J].实验动物科学与
管理,2002,4:15-18
37.秦鹏春等.家畜遗传资源的储备[J].黑龙江畜牧兽医,1984(11):27-31
38.任军,黄路生,高军等.江西省主要地方鸡品种的RAPD分析及其群体遗传关系的研究[J].遗传,2001,23(4):301-305
39.孙兆军等.固原红鸡分子遗传标记的研究[J].中国家禽,2001,23(22):10-11
40.沈伟.绒山羊不同生产水平群体遗传标记的研究[D],2001,6,西北农林科技大学硕士论文。
41.孙金梅.遗传标记的发展及其在家畜系统分类中的应用[J].黄牛杂志,1998,24(6),7-9
42.慎伟杰.隐性白羽鹌鹑伴性遗传的发现与研究[J].中国家禽,1998,3:20-21
43.王惠.RAPD技术及其在迪卡鸡遗传检测中的应用[C].中国动物遗传育种研究进展。北京,中国农业科技出版社,1997,75-77
44.吴衡.随机扩增多态性DNA指纹分析在鸡品系鉴定与育种中的应用[D].复旦大学硕士论文,1997
45.王昕.中国地方猪种微卫星DNA指纹的群体遗传学研究[D].陕西杨凌:西北农林科技大学硕士论文,2001
46.吴桢方.分子标记在猪育种中的应用[A].第十次全国动物遗传育种学术讨论会论文集,1998
47.王克华.地方鸡种随机扩增多态DNA(RAPD)及其与群体遗传结构相关研究[C].Animal Biotechnology Bulletin, Vol6 NO.1,1998:110-116
48.王金玉,龚允陈,陈国宏等.鸡的DNA指纹与屠宰性能的相关性研究[J].遗传学报,1999,26(4):324-328
49.王志跃,陈国宏.鸡育种研究进展[J].禽业导刊,1999,16(9):8-9
50.徐刚.宁夏固原鸡外部遗传特征及血液蛋白质多态性的研究[D].西北农业大学硕士论文,1993
51.吴敏生,王守才,戴景瑞.RAPD分子标记与玉米杂种产量优势预测的研究[J].遗传学报,1999,26(5):578-584
52.易建明.黄牛血液蛋白质多态性与牛种分类问题的研究[D].1986,西北农学院硕士论文
53.杨建军.RAPD技术在动物遗传育种中的应用[J].草食家畜(季刊),2002,3(1)1-3
54.严华祥等.DNA指纹技术在畜禽品种(系)鉴别中应用的初步研究[A].第七次全国畜禽遗传育种学术讨论会论文集,湖南,1993
55.晏兆莉,张成忠.家畜育种中的MAS的遗传标记和QTL[J].西南民族学院学报(自然科学版)1996,22(2):206-210
56.杨华林等.鸡血浆脂酶多态性的研究[J].中国家畜,1990,3:25-27
57.姚贵华.三种类型鸡的RAPD变异[A].Animal Biotechnology Bulletin,Vol6 NO.1,1998:117-121
58.张细权.三种遗传标记及其在分析畜禽遗传多样性上的应用[C].第六次全国畜禽遗传标记研讨会论文集,1998,10
59.周怀军.RAPD技术对鸡种群体遗传结构的分析[C].中国动物遗传育种研究进展,北京,中国农业科技出版社,1997,66-70
60.张吉德,赵宗盛,李大全等.微卫星标记及其在遗传育种中的应用[J].黄牛杂志,2001,27(3):1-5
61.张细权,杨关福等.用微卫星和RAPD分析珍稀家鸡品种的遗传变异[A].第九次全国动物遗传育种学术讨论会论文集,泰安,1997:61-65
62.张建勤等.固原鸡RAPD的标记研究[J].中国家禽,2001,23(22):12-13
63.张细权,吕雪梅等.用微卫星多态性和RAPD分析广东地方鸡种的群体遗传变异[J].遗传学报,1998,25(2):112-119
64.张继权.DNA标记的应用现状[C].第九次全国动物遗传育种学术讨论会论文集,泰安,1997
65.张细权,吕雪梅,杨玉华等.用微卫星多态性和RAPD分析广东地方鸡种的群体遗传变异[J].遗传学报,1998,25(2):112-119
66.张细权,李加棋,杨关福.动物遗传标记[M],北京:中国农业大学出版社,1997
67. Arendonk JAM et al. Use of multiple genetic marker in prediction of breeding values[J].Communicated material,1993
68. Arranz J J, Bayon Y, San Primitivo F. comparison of protein markers and microsatellites in differentiation of cattle populations[J]. Anim Genet, 1996,27:415-419
69. Andersson-Eklund. L and Rendel. J. Linkage between anaylase-1 locis and a major gene for milk[J]. Genetics,1993(24):101-103
70. Alis S, et al. DNA fingerprinting by oligo-nucleotide probes specific for simple repeats[J].Human Genetics, 1996, 74:239-243
71. Brown.W.M.Evolution of animal mitochondrial DNA[J]. Genetics 1983
72. Brow.W.M.Evolution of animal mitochondrial DNA,In Evolution of genes and proteins[J]. Nei,M.andKoehnl,P.Kced,T, Sundarland, 1983,62-68
73. Brow.W.M.Evolution of genes and proteins[J]. Mci.M, and R.K.Koehneds, sunderland,1983,62-88
74. Buchanan FC, Adams L J, Littlejohn R P et al. Determination of evolutionary relationships among sheep breeds using microsatellites[J]. Genomics, 1994,22:397-403
75. Baker C M A, Manwell C. Heterozygosity of the sheep:polymorphism of malic enzyme,
isocitrate dehydrogenase(NADP+),catalase and esterase[J]. Australian Journal of Biological Science, 1977,30:127-140
76. Botstein D,et al.Constrction of a genetic linkage map in man using restriction fragment length polymorphism[J]. Am J Hum Genet, 1980, 32: 314-331
77. Barker J.S,Preservation and management of animal genetic resource[C].第三 届世界遗传学应用于畜牧生产会议论文集(摘要),1986
78. Charcosset A,Lefort-Buaon M Gallais.Relationship between heterosis and heterozygosity at marker loci:a theoretical computation[J].Theor. Appl. Genet, 1991, 81:571-575
79. Caetano M A,Anolles A P. A versatile and universal tool for genome analysis [J]. Plant Molecular Biology ,1994,25:1011-1026
80. Detine M.R,Using molecular biology to improve the accuracy of linkage selection[J] .communication materials, 1993
81. Detine .M. R, MAS in livestock improvent[J].Animal genetics,1992,(23) :8-9
82. Dunning E A,Gal O,siegel PB,etal,Deoxyribonucleic acid fingerprint comparisons between selected populations of chicken[J].Poultry science ,1991,70:463-472
83. Epplen T T. A simple repeated GATA/GACA sequence in animal genome[J]. Heredity, 1988, 79: 409-416
84. Georges M and Massey J. M, Velogenetic on the synergistic use of MAS and germ line manipulation,The Viogenology, 1991,(35) : 151-159
85. Georges J M, A S Leqquarre, M Cadtelii, R Hannet and G. Vassart. DNA fingerprints in domestic animal using four different minisatellite probes [J]Cytogent cell Genet, 1988,47: 127-132
86. Georges J M, A S Leqquarre, M Cadtelii, R Hannet and G.Vassart. DNA fingerprints in domestic animal using four different minisatellite probes[J]. Cytogent cell Genet,1988,47: 127-132
87. Gavvora J.S,对高产量和马立克氏病抗性进行选择鸡主要组织相容性单倍型频率 的变化[J].J.Animal,Breed and Genet,1986,103(3) :218-226
88. Hechet W.Studies on mitochondrial DNA in farm animals.In genome analysis in domestic animal[J].Gelderman H and Ellendorff F ceel Weinheim, 1990,259-268
89. Haberfeld .A et al, Heterosis and DNA fingerprinting in chicken[J]. poultry science, 1996,(75) : 951-953
90. Haberfeld A, et al.Heterosis and DNA fingerprint in chickens[J].PoultryScience,1996, 75:951-953
91. Jeffreys,A.J,et al.Hyper-variable mini-satellite region in human DNA [J]. Nature,1985,
314:67-73
92. Jeffreys A J and D B Morton. DNA fingerprints of dogs and cats [J]. Animal Genetics, 1987, 18:1-5
93. Kuhnlein U, Hillel. J. DNA fmgerprint,a tool for determining genetic distance between strains of poultry [J],Theor Apps genetic, 1989,77:669-672
94. Koning G J and weller J I, Efficiency of direct selection on quantitative trait loci for a two traits breeding objective[C]. TAG, 1994(88) :669-677
95. Lament S.J ETAL,Genetics markers linked to quantitative traits in poultry [J]. Animal Genetics, 1996(27) : 1-8
96. L.L.B.Lanza,et al.Genetic of inbreed lines and prediction of maize single-cross performance using RAPD markers[J]. Theoretical and Applied genetics, 1997, 94:1023-1030
97. Plotsky Y. DNAfingerprint band applied to linkage analysis with QTL in chickens [J]. Animal Genetics,1993,24:105-110
98. R.Lande and R.Thompson,Efficienting of marker assisted selection in the improvement of quantitative trait[J] . Genetic,1990(124) :743-756
99. Soller, M and Genizi A, The efficiency of experimental design for the detection of linkage between a marker locus and a locus affection a quantitative trait in segregation population[J].Biometics,1978,(84) :47-55
100. Soller,M and B ackmamm JS, genetic polymorphism in variety identification and genetic improvement[C].TAG,1983,67:25
101. Stom P,The use of marker loci in selection for quantitative characters,Exploiting new technologies in animal breeding genetics developments[J]. Oxford university press, 1989,170-182
102. Shuman, R.M. et al Sym-posium: Molecular Approaches to poultry Breeding Gene Transfer by Avian Retroviruse.[J] Poultry sci.!986,65:1437-1444
103. Saghai M.A.et al.Correlation between molecular distance and hydrid performance in U.S,southern long grain rice[J].Crop Science,1997,37:145-150
104. Skinner D M,et al,The sequence of a hermit crab satellite DNA is (TAGG)N-(ATCC)N [J]. Biochemistry ,1984,13:3930-3937
105. Schafer M,et al.Three random loci in the human genome with base pair change polymorphism [J].Cytogenet,1982,32:314-315
106. Smith E,John C P,Nestor K E,Use of random amplified polymorphic DNA markers for the genetic analysis of relatedness and diversity in chickens and turkey [J]. Poultry Science, 1996,75:579-584
107. Welsh J,Meclelland M. Fingerprinting genomes using PCR with arbitrary prints [J]. Nuci.Acid Res,1990. 18:7213-7218
108. Williams J G,Kubelik AR,Livak JL,et al,DNA Polymorphisms amplified by arbitrary primers are useful as genetic makers[J].Nucl Acid Res,1990,18:6531-6535
109. Willams J G,Kubelik AR,Livak J L,et al. DNA Polymorphisms amplified by arbitrary primers are useful as genetic Markers[J].Nucl Acid Res,1990 18:6531-6535
110. Welsh J, Meclelland M.Fingerprinting genomes using PCR with arbitrary primers[J] Nucl.Acid Res,1990,18:7213-7218
111. X.C Liu,et al. Predictioning heterosis through biochemical and RAPD markers in animal species[J].AnimalBiotechnology Bulletin,1998,6(1) :127-133
112. Zhou H,Lament S J.Genetic characterization of biodiversity in highly inbred chicken lines by microsatellite markers[J]. Anim Genet,1999,30(4) :256-264
113. Zhang Q.F.et al.Molecular marker heterozygosity and hybrid performance in indica and japonica rice[J].Theoretical and Applied Genetics ,1996,93:1212-1224
114. Zabeu M,et al,Vos P European patent, 0535858,1993,3:31.