结缕草SSR反应体系的正交优化(英文)

Optimization of the SSR Reaction System for Zoysiagrass Using Orthogonal Design

为了快速获得结缕草（Zoysia japonica）SSR反应体系，采用5因素（模板DNA，Mg^2＋，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2μl 10×buffer、1．0U Taq酶、0．2mM 引物、100ng 模板 DNA、0．2mM dNTP、2．0mM 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。

In order to obtain the optimal SSR-PCR system for zoysiagrass （Zoysia japonica ）, an experimental design of orthogonal diagram L16 （4^5） was employed to evaluate five factors （template DNA, Mge＋ , dNTP, Taq DNA polymerase, and primer） at four different levels. Three randomly selected SSR primers （RM84, RM220, RM531） with resembled Tm were used to validate the reaction system. The results indicate that orthogonal design could be used to optimize the SSR-PCR system; the optimal system of terminal volume 20μl consisted of 2μl 10× buffer, 1.0U Taq DNA polymerase, 0. 2 mM primers, 100ng template DNA, 0.2 mM dNTP, and 2.0 mM 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 4 min, followed by 35 cycles at 94℃ for 30 s, 56-58℃ for 40 s, 72℃ for60 s; then keep the reaction mixture at 4~C after a final extension step of 72℃ for 10 min. The optimized system would be effective as a solid foundation for zoysiagrass SSR analysis.