湿地松、加勒比松SRAP反应体系的优化及引物筛选-广东林业科技.doc

湿地松、加勒比松SRAP反应体系的优化及引物筛选 李义良赵奋成吴惠姗 广东省林业科学研究院广州510520） * 摘 要 采用正交设计L9（34）对影响湿地松、加勒比松SRAP反应体系的DNA浓度、dNTPs浓度、引物用量、Taq DNA聚合酶浓度4个因素进行了优化试验，结果表明：扩增反应体系5优于其他体系，即25uL体系中含有DNA 40ngdNTP 1 mmol/L、引物10 μmol/LDNA聚合酶 U。SRAP反应关键因子的最适浓度，即25 uL反应体系中含有DNA 40 ng、dNTPs 1 mM、引物5 μM及Taq DNA聚合酶1.0 U。利用优化反应体系从216对引物组合中筛选出扩增条带清晰、稳定、多态性好的引物42对。优化的SRAP反应体系将为湿地松、加勒比松种质资源遗传多样性评价、分子标记辅助选择、遗传连锁图谱构建研究提供基础。 关键词 湿地松，加勒比松，SRAP-PCR，体系优化，引物筛选 Optimization of SRAP Reaction System and Primers Screening of Pinus elliottii and P. caribaea Li Yiliang* Zhao Fencheng Wu Huishan Zheng Yongle Guangdong Academy of Forestry, Guangzhou, 510520 Abstract The orthogonal design, L9（34） included 4 factors (DNA template, dNTPs, primers and Taq polymerase) and 3 levels was used to optimize SRAP reaction system for Pinus elliottii and P. caribaea. The results showed that: PCR reaction system 5 is superior to other systems, that is, the system contains 25uL, DNA 40ng, dNTP 1 mmol / L, primer 10 μmol / L, DNA polymerase 1.0 U. Based on the results of experiment from orthogonal design, the proper conditions of the key factors affecting the stability of SRAP system were determine by single-factor completely randomized design. Each 25μL PCR reaction mixture consisted of 40 ng of genomic DNA , 1 mM of dNTPs, 5 μM of primer and 1.0 unit of Taq polymerase. Total of 216 primer pairs were used in SRAP amplification and 42 combinations were selected because of their clear banding patterns, consistent amplifications and good polymorphism. The optimal SRAP reaction system was established, which would provide the basis for evaluation of genetic diversity, molecular marker assisted breeding and linkage mapping for the Pinus elliottii and P.caribaea. Key words Pinus elliottii, P. caribaea, SRAP-PCR, optimization of system, primers screening 湿地松（Pinus elliottii，PEE）原产美国东南部，加勒比松[包括3个变种，即古巴加勒比松（P. caribaea var. caribaea，PCC）、洪都拉斯加勒比松（P. caribaea var. hondurensis，PCH）、巴哈马加勒比松（P. caribaea var. bahmaen