哈萨克羊、阿勒泰羊、巴什拜羊遗传多样性的AFLP分析
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摘要
新疆是我国绵羊品种最多、遗传品质最复杂的省区,在长期生产实践中育成了许多具有特色的地方品种,这些品种是世界动物资源开发和利用的宝贵财富。如哈萨克羊具有体大早熟、抗逆性强、适应性强等优良性状;阿勒泰羊是哈萨克羊的一个优良类群,具有体格大,羔羊生长发育快,增膘快,产肉脂量高的特点;巴什拜羊具有早熟、耐粗饲、适应性强,被毛呈棕红色,绒毛多,干死毛少等特点。这些地方品种是保护生物多样性的重要物种资源,是实现我国养羊业可持续发展的基础。本研究在国内首次运用AFLP标记从分子水平阐明了新疆哈萨克羊、阿勒泰羊、巴什拜羊品种资源的多态性,为地方品种的保护和合理利用提供科学依据。
本实验利用AFLP标记技术对新疆哈萨克羊、阿勒泰羊、巴什拜羊进行了遗传分析,统计了每对引物组合在各品种中检测到的总条带数和多态性条带数,计算了3个地方品种绵羊的遗传相似系数及遗传距离,并据此构建了UPGMA聚类关系图。研究结果如下:
1.比较了沸水浴法、Miller盐析法、CTAB法及改进的酚法对绵羊基因组DNA的提取质量,结果沸水浴法所提取DNA的纯度和浓度效果最差,改进的酚抽提法同Miller盐析法、CTAB法相比所提取的DNA产物纯度和浓度差异显著( P < 0.05),适宜用于AFLP分子标记技术对DNA模板的实验要求。
2.本研究共对64对MseI和EcoRI引物组合进行多次重复筛选,其中均扩增出条带多且多态性百分率高、带型丰富、条带清晰的引物组合11对(E01/M02、E01/M07、E02/M02、E04/M01、E05/M05、E05/M08、E06/M02、E06/M04、E06/M05、E07/M03、E07/M08)。在
3个地方绵羊品种中共检测到527条带,平均每个引物组合产生47.91条,变化范围在34～61条,其中多态性条带总数为46条,占扩增总带数的8.73%,每对引物平均扩增多态性带4.18条。表明我国新疆地方绵羊品种遗传多样性较为丰富。
3.筛选的11对应物中其中多态性条带较多的应物组合为:E01/M02(7条)、E07/M08(6条)、E06/M02(5条)、E06/M04(5条)、E07/M03(4条)、E02/M02(4条)、E05/M08(4条)。
4.根据11对AFLP引物组合的检测结果,计算了新疆3个地方绵羊品种的遗传相似系数,其中哈萨克绵羊与阿勒泰绵羊的遗传相似系数最大(0.699),哈萨克绵羊与巴什拜绵羊遗传相似系数最小(0.592),阿勒泰绵羊与巴什拜绵羊为(0.620)。表明我国地方绵羊品种之间有较高的遗传相似性,遗传变异频率不大。
5.利用NTSYSpc软件的UPGMA法,对我国新疆3个地方绵羊品种进行聚类分析,构建了UPGMA聚类图。
Xinjiang is the province of China ,where sheep breeds is the most largest and has complex genetic quality, it has bred a number of characteristics local varieties that is a valuable asset for development and utilization of the world's animal resources in the long-term production practice. Kazakhstan sheep has excellent traits such as large body , prematurity, resistance, and adaptability;Altay sheep is a good group of Kazakhstan sheep that have much feature for example large body and fast growth of lamb and fast fat and high-fat meat production; Bashibai sheep also has many features such as prematurity,resistant coarse feed,strong adaptability and brown-red hair and multi-pile and dead hair less dry and so on. These local varieties is the important species that is used to protect the biological diversity,and it is to achieve the basis of sustainable development of sheep-raising in China. In this study, for the first time use AFLP markers to clarify polymorphism of Xinjiang breeds resources among Kazak sheep, Altay sheep,and Bashibai sheep from the molecular level,it will provide a scientific basis for conservation and rational forthputting of the local species.
Using of the AFLP markers, this experiment has accomplished genetic analysis to Kazakh sheep, Bashibai sheep Altay sheep in Xinjiang,has counted the polymorphic bands that each primer combinations detected in all varieties;has calculated genetic similarity coefficient genetic distance of three local varieties sheep ,and based on that, constructed the UPGMA cluster diagram. The results are as follows:
1. Comparison of the boiling water bath,Miller salting-out method,CTAB method and phenol extraction improved four kinds of sheep genomic DNA extraction method,which extracted in boiling water bath for purity and concentration of DNA was significantly lower than the other three kinds of methods. Phenol extraction improved with Miller salting-out method,CTAB extraction method of DNA compared to the purity and concentration of the product of a significant difference (P<0.05),suitable for AFLP molecular markers of DNA Experimental request template.
2. This study had gone through a number of repeatedly screening to a total of 64 MseI and EcoRI primer combinations, in which obtained 11 primer combinations with more bands and higher percentage of polymorphism, richer banding pattern,clearer strips (E01/M01,E01/M07, E02/M03, E04/M01, E05/M05, E05/M08, E06/M02, E06/M04, E06/M05, E07/M03, E07/M08). A total of 527 strips in the three local sheep varieties, on average,47.91 strips were amplified per primers combination, the scope of the changes is the 34 to 61,including 46 polymorphic markers,with a polymorphic frequency of 8.73%, on average 4.18 polymorphic markers polymorphic markers were amplified per primers combination.
3.The 11 selective primer combinations by which more polymorphic markers (showed in brackets)were obtained involved in E01/M02 ( 7 ) ,E07/M08(6),E06/M02 ( 5 ) ,E06/M04(5),E07/M03(4),E02/M02(4),E05/M08(4).
4. According to the calculation result of the 11 AFLP primer combination,it hascalculated genetic similarity coefficient genetic distance of three local sheep varieties, in which the biggest genetic similarity coefficient is Kazakhstan and Altay sheep(0.699), the smallest genetic similarity coefficient is Kazakhstan sheep and Bashibai sheep( 0.592), and the genetic similarity coefficient of Altay sheep and Bashibai sheep is 0.620.
5.Using UPGMA method of NTSYSpc software to carry on cluster analysist to the three local sheep of Xinjiang,and constructed the UPGMA cluster diagram.
本实验利用AFLP标记技术对新疆哈萨克羊、阿勒泰羊、巴什拜羊进行了遗传分析,统计了每对引物组合在各品种中检测到的总条带数和多态性条带数,计算了3个地方品种绵羊的遗传相似系数及遗传距离,并据此构建了UPGMA聚类关系图。研究结果如下:
1.比较了沸水浴法、Miller盐析法、CTAB法及改进的酚法对绵羊基因组DNA的提取质量,结果沸水浴法所提取DNA的纯度和浓度效果最差,改进的酚抽提法同Miller盐析法、CTAB法相比所提取的DNA产物纯度和浓度差异显著( P < 0.05),适宜用于AFLP分子标记技术对DNA模板的实验要求。
2.本研究共对64对MseI和EcoRI引物组合进行多次重复筛选,其中均扩增出条带多且多态性百分率高、带型丰富、条带清晰的引物组合11对(E01/M02、E01/M07、E02/M02、E04/M01、E05/M05、E05/M08、E06/M02、E06/M04、E06/M05、E07/M03、E07/M08)。在
3个地方绵羊品种中共检测到527条带,平均每个引物组合产生47.91条,变化范围在34～61条,其中多态性条带总数为46条,占扩增总带数的8.73%,每对引物平均扩增多态性带4.18条。表明我国新疆地方绵羊品种遗传多样性较为丰富。
3.筛选的11对应物中其中多态性条带较多的应物组合为:E01/M02(7条)、E07/M08(6条)、E06/M02(5条)、E06/M04(5条)、E07/M03(4条)、E02/M02(4条)、E05/M08(4条)。
4.根据11对AFLP引物组合的检测结果,计算了新疆3个地方绵羊品种的遗传相似系数,其中哈萨克绵羊与阿勒泰绵羊的遗传相似系数最大(0.699),哈萨克绵羊与巴什拜绵羊遗传相似系数最小(0.592),阿勒泰绵羊与巴什拜绵羊为(0.620)。表明我国地方绵羊品种之间有较高的遗传相似性,遗传变异频率不大。
5.利用NTSYSpc软件的UPGMA法,对我国新疆3个地方绵羊品种进行聚类分析,构建了UPGMA聚类图。
Xinjiang is the province of China ,where sheep breeds is the most largest and has complex genetic quality, it has bred a number of characteristics local varieties that is a valuable asset for development and utilization of the world's animal resources in the long-term production practice. Kazakhstan sheep has excellent traits such as large body , prematurity, resistance, and adaptability;Altay sheep is a good group of Kazakhstan sheep that have much feature for example large body and fast growth of lamb and fast fat and high-fat meat production; Bashibai sheep also has many features such as prematurity,resistant coarse feed,strong adaptability and brown-red hair and multi-pile and dead hair less dry and so on. These local varieties is the important species that is used to protect the biological diversity,and it is to achieve the basis of sustainable development of sheep-raising in China. In this study, for the first time use AFLP markers to clarify polymorphism of Xinjiang breeds resources among Kazak sheep, Altay sheep,and Bashibai sheep from the molecular level,it will provide a scientific basis for conservation and rational forthputting of the local species.
Using of the AFLP markers, this experiment has accomplished genetic analysis to Kazakh sheep, Bashibai sheep Altay sheep in Xinjiang,has counted the polymorphic bands that each primer combinations detected in all varieties;has calculated genetic similarity coefficient genetic distance of three local varieties sheep ,and based on that, constructed the UPGMA cluster diagram. The results are as follows:
1. Comparison of the boiling water bath,Miller salting-out method,CTAB method and phenol extraction improved four kinds of sheep genomic DNA extraction method,which extracted in boiling water bath for purity and concentration of DNA was significantly lower than the other three kinds of methods. Phenol extraction improved with Miller salting-out method,CTAB extraction method of DNA compared to the purity and concentration of the product of a significant difference (P<0.05),suitable for AFLP molecular markers of DNA Experimental request template.
2. This study had gone through a number of repeatedly screening to a total of 64 MseI and EcoRI primer combinations, in which obtained 11 primer combinations with more bands and higher percentage of polymorphism, richer banding pattern,clearer strips (E01/M01,E01/M07, E02/M03, E04/M01, E05/M05, E05/M08, E06/M02, E06/M04, E06/M05, E07/M03, E07/M08). A total of 527 strips in the three local sheep varieties, on average,47.91 strips were amplified per primers combination, the scope of the changes is the 34 to 61,including 46 polymorphic markers,with a polymorphic frequency of 8.73%, on average 4.18 polymorphic markers polymorphic markers were amplified per primers combination.
3.The 11 selective primer combinations by which more polymorphic markers (showed in brackets)were obtained involved in E01/M02 ( 7 ) ,E07/M08(6),E06/M02 ( 5 ) ,E06/M04(5),E07/M03(4),E02/M02(4),E05/M08(4).
4. According to the calculation result of the 11 AFLP primer combination,it hascalculated genetic similarity coefficient genetic distance of three local sheep varieties, in which the biggest genetic similarity coefficient is Kazakhstan and Altay sheep(0.699), the smallest genetic similarity coefficient is Kazakhstan sheep and Bashibai sheep( 0.592), and the genetic similarity coefficient of Altay sheep and Bashibai sheep is 0.620.
5.Using UPGMA method of NTSYSpc software to carry on cluster analysist to the three local sheep of Xinjiang,and constructed the UPGMA cluster diagram.
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