Abstract: The orthogonal design was used to optimize ISSR amplification system of winterfat in five factors (Taq DNA polymerase, dNTP, primer, Mg²⁺ and DNA templet) at four levels respectively. The data were analyzed by software MINITAB. As a result, a satisfactory ISSR reaction system for winterfat with desirable repeatability and polymorphic bands was established. In a total volume of 20 μL ISSR-PCR system, it contained 1×buffer, 1.5 U Taq DNA polymerase, 0.2 mmol·L^-1 dNTP, 0.5 μmol·L^-1 primer, 2.5 mmol·L^-1 Mg²⁺ and 10 ng template DNA. This optimized ISSR reaction system would provide the basis for the analysis of diversity, map construction and genetic variation of important traits of Ceratoides with ISSR markers.

Key words: orthogonal design, winterfat, ISSR-PCR, reaction system