关键词: 厚朴(Magnolia officinalis), 简单重复序列(SSR), 引物筛选, 正交设计

Abstract:

 To explore for optimal conditions for SSR-PCR( simple sequence repeat anchored polymerase chain reaction) for Magnolia officinalis,the five factors that would affect PCR (DNA templates,primers,Mg^^2＋,dNTPs and Taq polymerase) were optimized to four levels using an orthogonal experimental design based on screening of SSR primers. Out of the70 pairs of primers isolated from Magnolia officinalis and its related species,13 pairs were picked out with distinct amplified bands and rich polymorphism. In terms of impact on PCR system at various levels,the five factors displayed an order of primer>Taq enzymecatalytic activity concentration >Mg^2＋> dNTPs> template DNA. The optimal PCR system for SSR analysis was the 25 μL^-1 system with template DNA being 2 ng·μL^-1,primer being 0. 6 μmol·L^-1,Mg^2＋concentration being 2. 0 mmol·L^-1,dNTPs concentration being 0. 5 mmol·L^-1 and Taq polymerase being 0. 03 U·μL^-1,and the best amplification program consisted of 4 minutes of predenaturation at 94 ℃,30 seconds of denaturation at 94 ℃,30 seconds of annealing at 48. 0- 59. 0 ℃（ annealing temperature depending on primers） for 30 seconds,1 minute of extension at 72 ℃,and 10 minutes of extension at 72 ℃ after 35 cycles of the preceding processes. The above findings may provide some technical parameters for using the SSR-PCR technology in studying population genetics and molecular ecology of the endangered species,Magnolia officinalis.

Key words: Magnolia officinalis, SSR, primers screening, orthogonal design