Establishment and Optimization of ISSR Reaction System for Eriobotryae Folium from Fujian

OBJECTIVE To establish and optimize the ISSR-PCR reaction system for Eriobotryae Folium; to screen the suitable primers and determine their optimal annealing temperatures. METHODS Genomic DNA was extracted by CTAB method from Eriobotrya leaves. The main influencing elements in different levels were tested by single factor experiment. The gradient PCR was used to determine the optimal annealing temperatures of each selected primer. RESULTS The optimized PCR reaction system: total response volume 25 μL, including 60 ng template DNA, DNA polymerases dosage for 0.9 U, primer eventual concentration of 0.4 mol·L^{-1, dNTP eventual concentration was 200 μmol·L-1, 10×Buffer(including Mg2+) for 2.5 μL, sterilization deionized water to 25 μL. The optimal amplified procedure was as follows: after a pre-denaturing of 7 min at 94 ℃, 35 cycles were performed with denaturing of 1 min at 94 ℃, annealing of 1 min according to denaturing temperature of different primers, extension of 1 min at 72 ℃, a final extension step of 10 min at 72 ℃. Forteen primers which showed clearer and more bands from 100 primers were selected and the optimal annealing temperatures were 48-58 ℃(changed among different primers). CONCLUSION The stable and reproducible optimal ISSR-PCR reaction system and suitable annealing temperatures of 14 selected primers from 100 are established for Eriobotryae Folium which had laid the good foundation for ISSR analysis on studies of germplasm resources identification and genetic diversity.}