濒危植物香果树(Emmenopterys henryi)SRAP反应体系的建立与引物筛选

Establishment of Reaction System and Primer Screening for SRAP Marker on Endangered Plant Emmenopterys henryi

以珍稀濒危植物香果树(Emmenopterys henryi)基因组DNA为研究对象,对影响SRAP-PCR反应的因素如Mg^(2+)浓度、d NTPs浓度、Taq DNA聚合酶、引物浓度4因素进行优化。确定香果树SRAP反应体系为:20μL PCR反应体系,50 ng模板DNA,1×Buffer,2.5 mmol/L Mg^(2+),1 U Taq聚合酶,0.3 mmol/L d NTPs,正向和反向引物各0.3μmol/L;在香果树2份样品中进行引物初筛,利用已发表的SRAP引物,选取8条正向引物和8条反向引物,共计64对引物。从中筛选出10对重复性好,谱带清晰且稳定的引物。并将这些引物在香果树2个野生居群20份样品中进行遗传多样性分析,共得到72条扩增谱带,其中多态性谱带60条,多态性比率83.3%,平均每个引物组合产生6个多态性位点,供试材料间遗传变异相对丰富。

The concentration of Mg^（2＋）, template DNA, d NTPs, Taq DNA polymerase and primers which affected SRAP-PCR reaction were optimized, and selected using Emmenopterys henryi genomic DNA as the materials. The results showed that the optimum reaction system for SRAP-PCR of E. henryi were as follows： template DNA 50 ng,1×Buffer, Mg^（2＋）2.5 mmol/L, d NTPs 0.3 mmol/L, Taq DNA polymerase 1U, forward and reverse primers were all 0.3 μmol/L, and total volume of reaction system 20 μL. SRAP primer combinations which showed steadily,well-defined and scorable PCR amplification in 2 accessions（1 red bud and 1 green bud） were screened out from64 primer combinations（8 forward primers and 8 reverse primer）. 10 SRAP primer combinations had produced 72 amplified loci in 20 accessions of 2 populations, among which 60 were shown polymorphism level（average of 6bands per primer pairs）, with a polymorphic ratio of 83.3%.