Establishment and optimization of ISSR-PCR reaction system in Asparagus cochinchinensis / 中草药

ABSTRACT

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.