摘要
为了对狗牙根进行SSR-PCR分子标记分析,本研究对狗牙根的SSR-PCR反应体系进行了优化,利用L16(45)正交设计对狗牙根SSR-PCR反应体系中的Taq酶、Mg^(2+)、dNTPs和引物4个因素的多个水平进行了筛选,PCR结果用SAS进行方差分析,并利用筛选出的最优体系对退火温度进行了筛选。结果表明:(1)Taq酶、Mg^(2+)和引物3个因素对狗牙根SSR-PCR扩增结果有极显著影响,影响程度为Taq酶>Mg^(2+)>引物,而d NTPs对扩增结果影响不显著;(2)筛选出了20μL的狗牙根SSR-PCR最优反应体系为0.5 U Taq酶、2 mmol/L Mg^(2+)、0.4μmol/L引物、0.2 mmol/L dNTPs、30~60 ng模板DNA;(3)退火温度50~62℃对本试验所选引物的SSR-PCR扩增结果影响不大,本研究选用55℃作为退火温度。
The SSR-PCR system of Cynodon dactylon was optimized in this study, in order to analysis Cynodon dactylon SSR-PCR molecular marker. The L16（45） orthogonal design was applied to evaluate the 4 factors （Taq polymerase, Mg2＋, dNTPs and primer） at several concentration levels, and the PCR results did variance analysis by software SAS. After that, the annealing temperature was selected using the selected the optimal system. The results showed that： （1） The 3 factors （Toq polymerase, Mg〉 and primer） had a highly significant influence in SSR-PCR amplification on Cynodon dactylon, the order of effects was Taq polymerase〉Mg2＋〉primer, but dNTPs had no significant influence in the amplification; （2） The optimal SSR-PCR reaction system （20 μL） on Cynodon dactylon was selected contained 0.5 U Taq polymerase, 2 mmol/L Mg2＋, 0.4 μmol/L primer, 0.2 mmol/L dNTPs and 30-60 ng template DNA; （3） The annealing temperature （50-62 ℃） had little influence on amplification of this primer selected SSR-PCR, for convenience, we selected 55 ℃ as the proper annealing temperature.
出处
《基因组学与应用生物学》
CAS
CSCD
北大核心
2016年第1期198-205,共8页
Genomics and Applied Biology
基金
中央级公益性科研院所基本科研业务费专项资金(1630032014028)
现代农业产业技术体系建设专项资金(No.CARS-35)共同资助
