| 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25 μL体系中含有DNA 40 ng、dNTPs 1 mmol/L、引物10 μmol/L、DNA聚合酶1.0 U。在此基础上，通过单因素试验确定了湿地松、加勒比松SRAP反应关键因子的最适浓度，即25 μL反应体系中含有DNA 40 ng、dNTPs 1 mmol/L、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 |
| DOI： |
| 投稿时间：2011-03-01修订日期：2011-06-10 |
| 基金项目:国家林业公益性行业科研专项（201104014），广东省林业科技创新专项资金项目（2008KJCX005 01，2009KJCX007 01），广东省科技厅项目（2007A020200001 1），广东省林业局林业种苗与基地管理总站项目（2009），北京林业大学林木育种国家工程实验室开放课题项目(FOP2010 3)。 |
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| Optimization of SRAP Reaction System and Primers Screening \=of Pinus elliottii and P. caribaea |
| zhaofencheng |
| () |
| 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 25 μL, DNA 40 ng, dNTPs 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 singlefactor completely randomized design. Each 25 μL PCR reaction mixture consisted of 40 ng of genomic DNA, 1 mmol/L 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, moleular marker assisted breeding and linkage mapping for the P. elliottii and P.caribaea. |
| Key words: Pinus elliottii, P. caribaea, SRAP PCR, optimization of system, primers screening |
