目的 建立并优化天门冬ISSR-PCR反应体系和扩增程序,为探讨天门冬种质间遗传多样性提供依据。方法 采用单因子试验和正交设计法,研究Mg2+、dNTP、引物、Taq DNA聚合酶、模板DNA、延伸时间及循环次数对PCR扩增的影响。结果 天门冬ISSR-PCR的最佳反应体系为25μL的反应体系中含模板DNA 40 ng、Mg2+1.25 mmol/L、dNTP 320μmol/L、引物1.2μmol/L、Taq DNA聚合酶1.5 U。在此基础上,从50条引物中筛选出13条扩增稳定、多态性丰富的ISSR引物,并通过梯度PCR试验,确定引物最佳退火温度。结论 建立的天门冬ISSR-PCR反应体系,经过17份天门冬种质检验,证明该体系稳定可靠,可用于天门冬遗传分析。

Objective To establish and optimize ISSR-PCR reaction system for Asparagus cochinchinensis and lay foundation for its genetic diversity research. Methods The single-factor test and orthogonal design were applied for optimizing seven factors in the ISSR-PCR reaction system including Mg2 +, dNTP, primers, Taq DNA polymerase, the template DNA, extension time, and cycle times. Results The suitable PCR reaction system contained 1.25 mmol/L Mg2 +, 320 μmol/L dN TP, 1.2 μmol/L primer, 1.5 U Taq DNA polymerase, and 40 ng template DNA in total 25 μL reaction solution. On this basis, 13 primers were screened with stable amplification and rich polymorphism from 50 ISSR primers. The optimal annealing temperature for ISSR-PCR reaction was proposed by gradient PCR. Conclusion It is proved that the established and optimized ISSR reaction system would be stable and credible by the germplasm testing result of 17 A. cochinchinensis populations. This would provide the basis for the genetic analysis of A. cochinchinensis.

湖南省高校创新平台开放基金项目(09K106);湖南省科技计划重点项目(2009FJ2008)