西藏野生披碱草遗传多样性的SSR与AFLP标记比较研究
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摘要
本研究利用SSR和AFLP两种分子标记对搜集的9份西藏披碱草属野生牧草及8份对照材料进行遗传多样性研究。旨在从分子水平上评价西藏地区野生披碱草属种质资源的遗传多样性,以期为西藏地区披碱草属植物种质资源的保护和利用提供科学依据。同时比较两种分子标记技术在评价披碱草属牧草种质资源遗传多样性中的有效性。主要结果如下:
1.在前人研究的基础上,通过对披碱草属的SSR反应体系和反应程序进行优化,成功建立了适于披碱草属SSR分析的最佳反应体系和反应程序,也证明了近缘属微卫星引物的通用性。
2.通过参考前人在研究其他植物上的AFLP反应体系和反应程序,首次建立了披碱草属植物的AFLP银染体系。
3.SSR标记:筛选32对引物,其中14对引物有扩增条带,具有多态性且扩增效果较好的引物有8对。用筛选出的8对引物对供试的17份材料进行微卫星分析,共检测出69个位点,其中59个位点有多态性,平均每对引物所产生的多态性位点数为7.4个位点;西藏9个居群间遗传相似系数为0.273～0.900,变异幅度较大,说明西藏地区野生披碱草属植物具有较丰富的遗传多样性。UPGMA聚类分析表明,在相似系数阈值为0.59时,17份材料可大致分为5类,基本上反映了材料间的亲缘关系,表明SSR标记能够用于披碱草属植物的遗传多样性研究。
4.AFLP标记:筛选16对引物组合,其中扩增效果较好的引物组合有3对,共扩增出232个位点,每对引物组合可扩增出70～83个位点。其中206个位点有多态性,平均每个引物产生68.67个多态性位点。17个居群的遗传相似性指数在0.284～0.955之间,UPGMA聚类分析表明,当相似系数为0.59时,17个居群可以明显地划分为4类。
5.SSR标记和AFLP标记比较:SSR标记产生的位点多态性百分率为85.5%,AFLP标记产生的位点多态性百分率为88.79%。SSR标记平均每对引物产生8.6个等位位点,而AFLP标记平均每对引物组合产生68.67个等位位点。与SSR标记相比较,AFLP标记能更有效地评价群体的遗传多样性。根据这两种分子标记实验结果所得到的聚类图,AFLP标记聚类图更加真实反映了17份供试材料的亲缘关系。
Genetic diversity of 9 Tibet populations with 8 control groups was studied by microsatellite method (SSR)and amplified fragment length polymorphism(AFLP) in order to evaluate the genetic diversity, quantity and distribution of germplasm resources of the Tibet wild Elymus L. on molecular level and provide a scientific basis for the protection and utilization while comparing the effectiveness of the two molecular marker technique in the evaluation of the genetic diversity of Elymus L.. The main results were as follows:
1.Based on the previous research, through optimizing the SSR reaction system and reaction procedure of Elymus L. the optimization reaction system and reaction procedure system for SSR analysis was successfully established and it also proved that the general availability of relative genus microsatellite primers.
2.Based on the previous research on AFLP reaction system and reaction procedure, the AFLP silver-staining analysis system suitable for genomic DNA of Elymus L.was established for the first time.
3. SSR marker: 32 primer pairs were used for polymorphism selection, 14 primer pairs obtained amplified products, 8 primer pairs produced specifically polymorphism products; using those 8 primer pairs to analyse the 17 materials by microsatellite method ,a total of 69 sites were observed,among them 59 sites showed polymorphism sites. The average number of polymorphism site produced by each primer was 7.4. Genetic identity ranged from 0.273 to 0.900 among the 9 populations with a high variation range, which showed that the Tibet germplasm resource of wild Elymus.L had a high genetic diversity. The result of cluster analysis with UPGMA showed that when genetic identity is 0.59, the17 populations can be dividing into 5 groups, which basically showed the genetic relationship amang 9 populations and also proved that SSR marker could be used for the analysis of genetic diversity of wild Elymus L.
4. AFLP marker: 16 primer combinations were used for polymorphism selection and 3 primer combinations produced specifically polymorphism products. A total of 232 sites were observed, among them 206 sites are polymorphism sites, the average number of polymorphism site produced by each primer combination was 68.67. Genetic identity ranged from 0.284 to 0.955 among the 17 populations. The cluster analysis with UPGMA showed that when genetic identity is 0.59, the 17 populations could be divided into 4 groups.
5. Comparison of SSR marker with AFLP marker: The ratio of polymorphic sites obtained by SSR marker was 85.5%, more than 88.79% obtained by AFLP marker. The average site produced by each SSR primer was 8.6, but the average site produced by each AFLP primer combination was 68.67. Compared with SSR marker, AFLP marker could be used effectively to evaluate population’s genetic diversity. According to the results of cluster analysis with UPGMA by the two methods, the cluster graph by AFLP marker could be better to response the true genetic relationship amang 17 populations.
1.在前人研究的基础上,通过对披碱草属的SSR反应体系和反应程序进行优化,成功建立了适于披碱草属SSR分析的最佳反应体系和反应程序,也证明了近缘属微卫星引物的通用性。
2.通过参考前人在研究其他植物上的AFLP反应体系和反应程序,首次建立了披碱草属植物的AFLP银染体系。
3.SSR标记:筛选32对引物,其中14对引物有扩增条带,具有多态性且扩增效果较好的引物有8对。用筛选出的8对引物对供试的17份材料进行微卫星分析,共检测出69个位点,其中59个位点有多态性,平均每对引物所产生的多态性位点数为7.4个位点;西藏9个居群间遗传相似系数为0.273～0.900,变异幅度较大,说明西藏地区野生披碱草属植物具有较丰富的遗传多样性。UPGMA聚类分析表明,在相似系数阈值为0.59时,17份材料可大致分为5类,基本上反映了材料间的亲缘关系,表明SSR标记能够用于披碱草属植物的遗传多样性研究。
4.AFLP标记:筛选16对引物组合,其中扩增效果较好的引物组合有3对,共扩增出232个位点,每对引物组合可扩增出70～83个位点。其中206个位点有多态性,平均每个引物产生68.67个多态性位点。17个居群的遗传相似性指数在0.284～0.955之间,UPGMA聚类分析表明,当相似系数为0.59时,17个居群可以明显地划分为4类。
5.SSR标记和AFLP标记比较:SSR标记产生的位点多态性百分率为85.5%,AFLP标记产生的位点多态性百分率为88.79%。SSR标记平均每对引物产生8.6个等位位点,而AFLP标记平均每对引物组合产生68.67个等位位点。与SSR标记相比较,AFLP标记能更有效地评价群体的遗传多样性。根据这两种分子标记实验结果所得到的聚类图,AFLP标记聚类图更加真实反映了17份供试材料的亲缘关系。
Genetic diversity of 9 Tibet populations with 8 control groups was studied by microsatellite method (SSR)and amplified fragment length polymorphism(AFLP) in order to evaluate the genetic diversity, quantity and distribution of germplasm resources of the Tibet wild Elymus L. on molecular level and provide a scientific basis for the protection and utilization while comparing the effectiveness of the two molecular marker technique in the evaluation of the genetic diversity of Elymus L.. The main results were as follows:
1.Based on the previous research, through optimizing the SSR reaction system and reaction procedure of Elymus L. the optimization reaction system and reaction procedure system for SSR analysis was successfully established and it also proved that the general availability of relative genus microsatellite primers.
2.Based on the previous research on AFLP reaction system and reaction procedure, the AFLP silver-staining analysis system suitable for genomic DNA of Elymus L.was established for the first time.
3. SSR marker: 32 primer pairs were used for polymorphism selection, 14 primer pairs obtained amplified products, 8 primer pairs produced specifically polymorphism products; using those 8 primer pairs to analyse the 17 materials by microsatellite method ,a total of 69 sites were observed,among them 59 sites showed polymorphism sites. The average number of polymorphism site produced by each primer was 7.4. Genetic identity ranged from 0.273 to 0.900 among the 9 populations with a high variation range, which showed that the Tibet germplasm resource of wild Elymus.L had a high genetic diversity. The result of cluster analysis with UPGMA showed that when genetic identity is 0.59, the17 populations can be dividing into 5 groups, which basically showed the genetic relationship amang 9 populations and also proved that SSR marker could be used for the analysis of genetic diversity of wild Elymus L.
4. AFLP marker: 16 primer combinations were used for polymorphism selection and 3 primer combinations produced specifically polymorphism products. A total of 232 sites were observed, among them 206 sites are polymorphism sites, the average number of polymorphism site produced by each primer combination was 68.67. Genetic identity ranged from 0.284 to 0.955 among the 17 populations. The cluster analysis with UPGMA showed that when genetic identity is 0.59, the 17 populations could be divided into 4 groups.
5. Comparison of SSR marker with AFLP marker: The ratio of polymorphic sites obtained by SSR marker was 85.5%, more than 88.79% obtained by AFLP marker. The average site produced by each SSR primer was 8.6, but the average site produced by each AFLP primer combination was 68.67. Compared with SSR marker, AFLP marker could be used effectively to evaluate population’s genetic diversity. According to the results of cluster analysis with UPGMA by the two methods, the cluster graph by AFLP marker could be better to response the true genetic relationship amang 17 populations.
引文
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