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目的建立并优化夏枯草ISSR-PCR反应体系和扩增程序,为探讨夏枯草种质间遗传多样性奠定基础。方法采用单因子试验和正交设计方法,研究Mg2+、dNTP、引物、TaqDNA聚合酶、模板DNA、退火温度及循环次数对PCR扩增的影响。结果夏枯草ISSR-PCR的最佳反应体系为:在20μL的反应体系中含模板DNA30ng,Mg2+2.2mmol/L、dNTP175μmol/L、引物0.75μmol/L、TaqDNA聚合酶1.0U。在此基础上,从92条引物中筛选出18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。结论采用单因子试验和正交设计方法可以快速建立ISSR-PCR反应体系,经过24份夏枯草种质检验,证明该体系稳定可靠,可用于夏枯草遗传分析。
Objective To establish and optimize the ISSR-PCR reaction system and amplification program of Prunella vulgaris, and lay a foundation for exploring the genetic diversity among Prunella vulgaris germplasm. Methods The effects of Mg2 +, dNTP, primers, Taq DNA polymerase, template DNA, annealing temperature and the number of cycles on PCR amplification were studied by single factor test and orthogonal design. Results The optimal reaction system of Prunella vulgaris ISSR-PCR was as follows: 30ng of template DNA, 2.2mmol / L of Mg 2+, 175μmol / L of dNTP, 0.75μmol / L of primer and 1.0U of Taq DNA polymerase in 20μL reaction system. Based on this, 18 ISSR primers with stable amplification and abundant polymorphism were screened from 92 primers and the optimal annealing temperature was determined by gradient PCR. Conclusion The ISSR-PCR reaction system can be rapidly established by single factor test and orthogonal design. After 24 samples of Prunella vulgaris germplasm tested, the system was proved to be stable and reliable, and could be used for genetic analysis of Prunella vulgaris.