Abstract:

The effects of various factors on ISSR-PCR reaction, such as concentration of Mg²⁺, dNTPs and primers, DNA template and Taq DNA polymerase, were investigated to optimize the ISSR-PCR reaction system of Freesia refracta through orthogonal tests. The optimized ISSR-PCR system of F. refracta incloded 1×Taq DNA polymerase buffer (10 mmol·L^-1 KCl, 8 mmol·L^-1(NH₄)₂SO₄, 10 mmol·L^-1 Tris·HCl, pH 9.0, 0.05% NP-40), 2 mmol·L^-1 MgCl₂, 0.06 U·μL^-1 Taq DNA polymerase, 0.4 μmol·L^-1 primer, 4.0 ng·μL^-1 DNA template, 0.6 mmol·L^-1 dNTPs in 25 μL PCR reaction system. By temperature gradient PCR, the optimum annealing temperature was determined as 51.5℃. The optimized system laid the groundwork for ISSR molecular analysis of F. refracta.

Key words: ISSR-PCR, Freesia refracta Klatt, optimization, orthogonal design