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目的建立并优化桔梗ISSR-PCR反应体系和扩增程序。方法采用正交试验和单因子试验,对ISSR反应体系主要因子(dNTPs、Taq酶、引物、Mg2+、模板)进行筛选。之后通过单因子试验,进行引物退火温度、循环次数的筛选。结果桔梗ISSR-PCR的最佳反应体系(20μl)为:dNTPs 0.25mmol/L、Taq酶1.0U、引物0.30μmol/L、Mg2+1.50mmol/L、模板DNA10ng。利用此反应体系从54条引物中筛选出12条扩增稳定、多态性丰富的ISSR引物,确定了引物UBC853[序列为(TC)8RC]的最佳退火温度为55℃和循环次数为45次。结论建立的桔梗ISSR-PCR反应体系,经过白、紫花桔梗共20份样品检验,证明该体系具有稳定性和可靠性,可用于桔梗遗传多样性研究。
Objective To establish and optimize the ISSR-PCR reaction system and amplification program of Campanulaceae. Methods The main factors (dNTPs, Taq enzyme, primer, Mg2 +, template) of ISSR reaction system were screened by orthogonal test and single factor test. After a single factor test, primer annealing temperature, the number of cycles of screening. Results The optimal reaction system (20μl) for ISSR-PCR was 0.25mmol / L dNTPs, 1.0U Taq enzyme, 0.30μmol / L primer, Mg2 + 1.50mmol / L and 10ng template DNA. Using this reaction system, 12 ISSR primers with stable amplification and abundant polymorphism were screened from 54 primers, and the optimal annealing temperature of primer UBC853 [sequence (TC) 8RC] was 55 ℃ and the number of cycles was 45 Times. Conclusion The established ISSR-PCR reaction system of Campanulaceae was tested in 20 samples of white and purple bellflower, which showed that the system was stable and reliable and could be used for the genetic diversity of Campanulaceae.