关键词: 绿竹, ISSR, 正交试验设计, 反应体系, 扩增程序, 引物筛选

Abstract: This work is to establish the optimal ISSR-PCR reaction system and amplifying procedure，and to screen high polymorphism primers for ISSR-PCR analysis of Dendrocalamus oldhami（Munro）. First，the genomic DNA extracted from D. oldhami（Munro）was used as template for ISSR-PCR amplification，and the orthogonal design（L₁₆（4⁵））was to investigate the optimal concentrations of dNTPs，Mg²⁺，Taq DNA polymerase，primers and DNA template，and the results were analyzed by range and variance analysis method. Then，the annealing temperature and the number of cycles were screened for establishing the optimal D. oldhami（Munro）ISSR-PCR reaction system and amplifying procedure. Moreover，the optimized system was utilized to screen the 100 ISSR primers. Ultimately，the optimal reaction system was determined as follows：in 20 μL reaction system containing 0.2 mmol/L dNTPs，2.0 mmol/L Mg²⁺，1.5U Taq DNA polymerase，0.4 μmol/L primer，60 ng DNA template，2.0 μL 10×Taq Buffer，and ddH₂O completed. The order of the effect by the factors was in Mg²⁺ > dNTPs > DNA template > Taq DNA polymerase > primer. The optimal amplifying procedure was as follows：pre-denaturing for 5 min at 94℃，38 cycles were performed with denaturing of 45 s at 94℃，annealing of 30 s due to denaturing temperature of different primer，extension of 90 s at 72℃，a final extension step of 10 min at 72℃ and stored at 4℃. Using this optimized PCR system，14 of 100 primers was selected for their clarity，high polymorphism and repetition.

Key words: Dendrocalamus oldhami（Munro）, ISSR, orthogonal design, reaction system, amplifying procedure, primer screening