SRAP-PCR System Optimization and Primer Selection for Juncao Studies

Objective SRAP-PCR system optimization and polymorphic SRAP primers identification for studying Juncao were conducted.

Method Genomic DNA extracted from Juncao was used as a template in a combined single-factor and orthogonal experimentation to optimize the primer, Mg²⁺, template DNA, dNTP, and Taq DNA polymerase for SRAP-PCR analysis. A set of polymorphic SRAP primers was selected using 4 varieties of Juncao as targets.

Result The optimized SRAP-PCR analysis required the concentrations of primer at 1.0 μmol·L^–1, Mg²⁺ at 2.50 mmol·L^–1, template DNA at 2.5 ng·μL^–1, dNTPs at 325 μmol·L^–1, and Taq DNA polymerase at 0.35 U. These parameters affected the SRAP amplification efficiency in the order of primer＞template DNA＞Taq DNA polymerase＞dNTPs＞Mg²⁺. From 35 pairs of SRAP primers, 18 with rich polymorphism and clear bands were selected. They had 3 to 10 polymorphic loci with an average polymorphism ratio of 77.36%.

Conclusion The optimized SRAP-PCR system was highly stable and reliable. It could be applied satisfactorily for analyzing genetic diversity, studying germplasm conservation, and broadening utilization of Juncao.