Abstract: In order to obtainthe opti mal SSR-PCRsystem for zoysiagrass(Zoysiajaponica),an experi mental design of orthog-onal diagramL16(45) was employed to evaluate five factors (template DNA,Mg²⁺,dNTP,Taq DNA polymerase,and primer) at four different levels.Three randomly selected SSR primers (RM84,RM220,RM531) with resembled Tmwere used to vali-date the reaction system.The results indicate that orthogonal design could be used to optimize the SSR-PCR system;the optimal systemof terminal volume 20μl consisted of 2μl 10×buffer,1.0 U Taq DNA polymerase,0.2mM primers,100ng template DNA,0.2 mM dNTP,and 2.0mM Mg⁺.The optimal annealing temperature was determined to be 56-58℃by gradient PCR;the suitable thermal cycling conditions with initial melting at 94℃ for 4min,followed by 35 cycles at 94℃ for 30 s,56 -58℃ for 40s,72℃ for 60 s;then keep the reaction mixture at 4℃ after a final extension step of 72℃ for 10 min.The optimized system would be effective as a solid foundation for zoysiagrass SSR analysis.

Key words: Zoysia japonicaSteud., SSR markers, Reaction system, Orthogonal design, Opti mization

摘要： 为了快速获得结缕草(Zoysia japonica)SSR反应体系,采用5因素(模板DNA,Mg²⁺,dNTP,Taq酶和引物)4水平正交设计筛选合适的结缕草SSR体系,并通过随机挑选3对Tm相近的引物对该优化体系进行验证。结果表明:正交设计可应用于SSR-PCR反应体系的优化,20μl最佳PCR反应体系中包括2μl10×buffer、1.0 U Taq酶、0.2mM引物、100ng模板DNA、0.2mMdNTP、2.0 mM Mg⁺;对结缕草进行梯度退火实验,其最佳退火温度为56-58℃;可行扩增程序是:94℃预变性4min、进行35个循环的94℃变性30s、56-58℃退火40s,72℃延伸1min;72℃延伸10min,4℃保存;该最适反应体系的建立,为今后结缕草SSR分析奠定了坚实基础。

关键词: 结缕草, SSR标记, 反应体系, 正交设计, 优化