Abstract: This experiment was conducted to obtain the suitable ISSR-PCR reaction system of Columba domestica by orthogonal design. Columba domestica was chosen to study the affecting factors of ISSR-PCR amplification. Orthogonal design of five factors (Mg²⁺, dNTPs, primer, Taq DNA polymerase, template) at four levels was used to establish the suitable ISSR-PCR system by primer ISSR807 in Columba domestica, and the suitable anneal temperature was yielded from gradient PCR on temperature. The results showed that the optimized ISSR-PCR system(25 μL reaction volume) in Columba domestica was proved to be: 10×PCR Buffer 2.5 μL,2.6 mmol/L Mg²⁺,0.2 mmol/L dNTPs,0.4 μmol/L primer,0.5 U Taq DNA polymerase,DNA template 1.2 ng/μL;and suitable anneal temperature was 56.2 ℃. According to the optimum ISSR-PCR reaction system, the results lay a foundation for the analyses of genetic diversity, germplasm resources and genetic relationship of Columba domestica.

Key words: Columba domestica; orthogonal design; optimization; ISSR-PCR system 