基于CGE技术的甘蔗SSR-PCR反应体系优化

Optimization and application of sugarcane SSR-PCR system based on CGE technology

【目的】优化基于毛细管凝胶电泳（CGE）技术的甘蔗SSR-PCR反应体系,为甘蔗遗传多样性和亲缘关系研究、分子辅助育种及遗传图谱构建提供技术支持。【方法】基于CGE技术,采用正交试验设计,选择dNTPs浓度、DNA聚合酶量、引物浓度和DNA模板量为考察因素进行优化,确定最佳SSR-PCR反应体系,并用于8个甘蔗种质资源材料的遗传多样性分析。【结果】最佳SSR-PCR反应体系（20μL）：dNTPs浓度0.15 mmol/L,DNA聚合酶1.5U,引物浓度0.50μmol/L,DNA模板量25 ng。利用该体系对8份来自不同国家地域的甘蔗种质材料进行遗传多样性分析,发现8对SSR引物共扩增出101个片段,其中多态性片段为93个,多态性比率为92.1%。聚类分析结果表明,参试材料间的遗传相似性系数为0.521-0.871,在相似性系数为0.614处,所有参试材料可分为两大类,3份国外种质材料PINDAR、CP72-1210、CP84-1198与ROC20、GT69-156聚为一类;而ROC26、GT02-237和GT97-69聚为另一类。【结论】基于CGE技术的SSR-PCR反应体系检测结果稳定,重复性好,适用于甘蔗的遗传分析及遗传作图。

[Objective]SSR-PCR reaction system of sugarcane based on capillary gel electrophoresis（CGE） technology was optimized to provide technical support for research on genetic diversity and genetic relationship, molecular-assisted breeding and genetic map construction of sugarcane. [Method]Four factors affecting SSR-PCR were optimized by orthogonal exprement based on CGE technology, including dNTPs concentration, primer concentration, Taq DNA polymerase amount and DNA template amount. Then the optimal SSR-PCR reaction system was confirmed and applied to genetic analysis of 8 sugarcane germplasm resources. [Result]The results showed that, the optimal reaction system（20 μL） was composed of 0.15 mmol/L dNTPs, 1.5 U Taq DNA polymerase, 0.50 μmol/L primers and 25 ng DNA templates. Using optimal reaction system and 8 pairs of SSR primers, a total of 101 fragments were amplified from 8 sugarcane germplasms of several coun-tries, 93 of which was polymorphic, with a polymorphism rate of 92.1%. The cluster analysis showed that, coefficient of genetic similarity among 8 germplasms ranged from 0.521 to 0.871. All tested materials could be clustered into two groups at similarity coefficient of 0.614, three foreign germplasms viz., PINDAR, CP72-1210, CP84-1198 and ROC20, GT69-156 were clustered into the same group, but ROC26, GT02-237 and GT97-69 were clustered into another group. [Con-clusion]The established SSR-CGE system is suitable for genetic diversity analysis and genetic mapping of sugarcane due to its stable detection result and good reproducibility.