[Objective] This study aimed to optimize the SCoT-PCR system for Citrus.Under the optimized SCoT-PCR amplification conditions, the genetic DNA of Youzishatangju and Wuzishatangju were amplified. [Method] Orthogonal design was adopted to optimize the five influencing factors on amplification effect of SCoT-PCR,including the DNA template, Mg2＋, primers, dNTPs and Taq DNA polymerse concentrations. With the optimized SCoT-PCR system, the genomic DNA of Youzishatangju and Wuzishatangju were amplified by 60 SCoT primers, respectively, and the specific bands for Youzishatangju or Wuzishatangju were selected for SCAR transformation. [Result] The optimized SCoT-PCR reaction system（20 μl） was as follows： Mg2＋1.5 mmol/L, d NTPs 0.35 mmol/L, primer 0.25 μmol/L, Taq enzyme 0.5 U, DNA template 30 ng. The optimum annealing temperature was 50.6 °C. With the optimized SCoT-PCR system, the genomic DNA of Youzishatangju and Wuzishatangju were amplified respectively by 60 SCoT primers, and a total of 42 pairs of primers were screened. Among the 42 pairs of primers, only one primer amplified different band pattern between Youzishatangju and Wuzishatangju. [Conclusion] It indicates that there is certain difference between Youzishatangju and Wuzishatangju at genomic DNA level.
