八个不同蛋鸡群体的RAPD标记研究
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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
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
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