Abstract: To construct and optimize the CDDP-PCR reaction system of Fortunella japonica and select the ideal primers for the CDDP-PCR analysis of Fortunella japonica, so as to provide reference basis for using CDDP marker technology to assist germplasm identification and molecular breeding, the DNA of Fortunella japonica genome was used as the template, and the orthogonal optimization test design scheme was adopted to design the 5 factor and 4 level test for dNTPs concentration, primer concentration, template DNA dosage, Mg²⁺ concentration and Taq DNA polymerase concentration. Range analysis method, variance analysis method and Duncan multiple comparison method were used to analyze the test results. The results showed that polymorphic bands could be amplified from 21 common primers of CDDP using 5 Fortunella japonica varieties. The optimal reaction system was as follows: DNA dosage 120 ng, Mg²⁺ concentration 1.5 mmol·L^-1, Taq DNA polymerase concentration 0.25 U, primer concentration 1.0 μmol·L^-1, dNTPs concentration 0.15 mmol·L^-1, and the remaining volume was supplemented by ddH₂O to 20 μL. The influences of various factors on the reaction system from the largest to the smallest were dNTPs>primer>template DNA>Mg²⁺>Taq DNA polymerase.

Key words: Fortunella japonica, CDDP, reaction system, optimization