Abstract: This work is aimed to establish a stable ISSR-PCR system for Ranunculus nephelogenes var. nephelogenes. A L₁₆（4⁵）orthogonal design was used to screen 5 main parameters（Mg²⁺,dNTP,Taq DNA polymerase,primer,and template DNA）;then the process of ISSR-PCR reaction was optimized,and consequently the optimal reaction system and amplifying procedure for R. nephelogenes var. nephelogenes was established;further,the optimized reaction system and amplifying procedure was verified. Results showed that the optimal concentrations in 20 µL reaction mixture were template DNA 30 ng,Mg²⁺ 1.95 mmol/L,Taq DNA polymerase 0.04 U/µL,dNTP 0.150 mmol/L, and primer 0.5 µmol/L. The optimal reaction procedure was：pre-denaturalization for 5 mins at 94℃,denaturalization for 20 s at 94℃,renaturation for 1 min at 49.6-60.6℃,followed by 38 cycles of extension for 100 s at 72℃,and final extension for 6 min at 72℃. Under the above optimized reaction system and procedure,16 amplified primers were screened from 100 ISSR primers,and the optimal annealing temperature for each primer was determined. Verification in R. nephelogenes var. nephelogenes of different populations with the optimized ISSR system confirmed that bands amplified were clear and steady,thus the established ISSR-PCR could favor further studies on the genetic diversity of R. nephelogenes var. nephelogenes.

Key words: Ranunculus nephelogenes var. nephelogenes, ISSR-PCR, orthogonal design, optimization of system, primer selection