Keywords: 
• Ilex kudingcha C. J. Tseng /
• RAPD marker /
• optimization /
• sex identification

Abstract: An orthogonal design was made to optimize RAPD-PCR amplification system of Ilex kudingcha in 5 factors （DNA template, Mg²⁺, primer, dNTPs, Taq polymerase） at 4 levels, respectively. Through comprehensive analysis, an optimized RAPD-PCR reaction system, was established: 10×buffer 2.5 μL, 20 ng DNA template, 2.5 mmol·L^-1 Mg²⁺, 0.3 μmol·L^-1 primers, 2.0 U Taq polymerase, and 200 μmol·L^-1 dNTPs in the 25 μL reaction system, and the optimized RAPD-PCR amplification program: predenaturing at 94 ℃ for 5 min, then denaturing at 94 ℃ for 30 s, elongation at 36 ℃ for 30 s and extension at 72 ℃ for 120 s, running for 40 cycles, and final extension at 72 ℃ for 10 min. The products were stored at 4 ℃. Furthermore, a total number of 91 random primers were screened in the RAPD-PCR. Polymorphic fragments were detected with 24 primers in female and male DNA samples pools. Two male-associated fragments （S164-900 and S191-800） were respectively generated with S164 primer and S191 primer. Repeated experiments indicated that these RAPD markers appeared stably in male individuals. So these RAPD markers can be applied to identify the sex of the young plantlets of Ilex kudingcha C. J. Tseng at the early stage.