Abstract: Orthogonal design was applied to optimize SCoT-PCR amplification system of Chrysanthemum in five factors such as Mg²⁺，dNTPs，primer，Taq DNA polymerase and template DNA. An optimal reaction system of species from Chrysanthemum was completed；25 μL PCR reaction mixtures contained Mg²⁺ 1.2 mmol · L^-1，dNTPs 0.15 mmol · L^-1，primers 0.8 μmol · L^-1，Taq polymerase 1 U，template DNA 50 ng. The most suitable annealing temperature of primers was 49.4 ℃. The improved method for separating PCR products was 8% polyacrylamide gel electrophoresis followed by silver staining. Three materials from Chrysanthemum with different ploidy levels（2x，4x，6x）were used to test the optimized reaction system. Ideal results with clear polymorphic bands showed that the stable and reliable reaction system was obtained. SCoT markers were applied to study the genetic diversity and relationship of 18 species of Chrysanthemum and its related genera. Using 12 selected SCoT primers 209 bands were generate，of which 189（90.43%）were polymorphic. The Jaccard genetic similarity coefficients among the species were calculated with NTSYS-pc2.10e software and the values were between 0.4516 and 0.7035 with an average of 0.58. These coef?cients were utilized to construct a dendrogram using the unweight pair-group method with arithmetic averages（UPGMA）. The results showed that 18 species of Chrysanthemum and its related genera could be grouped into two distinct clusters based on similarity of 0.530. The others except Crossostephium chinense and Artemisia sericea were in one cluster，which was further divided into six groups when the similarity coefficient value was 0.615. The results demonstrate that the SCoT marker system is useful for studying genetic diversity and relationships among species of Chrysanthemum and its related genera.

Key words: Chrysanthemum, sart codon targeted polymorphism（SCoT）, genetic diversity, system optimization