Objective  The optimized ISSR-PCR reaction system and appropriate polymorphic primers were established to facilitate the authentication, breeding, and genetic studies on Fritillaria hupehensis Hsiao et K.C. Hsia.

  Method  A U₁₂(4³) uniform design method combined with a single factor screening test was employed to optimize the dosages of 2×Taq Master Mix, template DNA, and primer applied for ISSR-PCR and followed by a gradient temperature experiment to determine the primer annealing temperature.

  Result  The optimal ISSR reaction system used 10.5 μL of 2×Taq Master Mix, 0.8 μL of template DNA (40.0 ng), 2.2 μL (2.5 μmol·L⁻¹) of primers, and 6.5 uL of ddH₂O for a 5 min sequencing at 94 ℃ and 40 cycles at 94 ℃ for 0.75 min, 54.3–60.0 ℃ for 0.75 min, and 72 ℃ for 1.5 min, followed by 10 min at 72 ℃ prior to storage at 4 ℃. Six stable polymorphic primers, UBC848, UBC850, UBC853, UBC857, UBC859, and UBC866 with the optimal annealing temperatures of 59.3, 58.2, 56.9, 54.3, 59.3, and 60.0 ℃, respectively, were selected. On 12 F. hupehensis germplasms from various locations, the optimized ISSR-PCR yielded stable and reliable results that showed abundant genetic diversity.

  Conclusion  The optimized ISSR-PCR reaction system could be satisfactorily applied for the resource authentication and genetic analysis on F. hupehensis.