利用正交设计方法对柳杉SSR反应体系中的Mg2+、DNA模板、 聚合酶、dNTP、引物等5个反应因子进行了优化, 同时对反应程序中的退火温度进行筛选。结果表明:(1)柳杉10 μL的SSR反应体系的最优条件为Mg2+浓度2.0 mmol/L、4种dNTP浓度均为0.15 mmol/L、正反引物均为0.3 μmol/L、Taq聚合酶为0.5 μmol/min、DNA模板为20 ng;(2)从柳杉属植物的106对SSR 引物中筛选出了多态性高, 稳定性好的26 对引物;(3)优化了筛选出的26对引物的退火温度。
Abstract
A orthogonal design was used to optimize SSR amplification for Cryptomeria fortunei in five factors:Mg2+,DNA template,Taq DNA polymerase,dNTP, primer. The results were shown as follows:(1)A suitable 10 μL SSR reaction system contained Mg2+ 2.0 mmol/L, each of four dNTPs 0.15 mmol/L, primers 0.3 μmol/L, Taq polymerase 0.5 μmol/min, DNA template 20 ng.(2)Twenty-six SSR primers were screened from 106 primers existing in Cryptomeria,which had high polymorphism and stability.(3)The best annealing temperature of twenty-six SSR primers were optimized. The results provided references for genetic diversity research of C. fortunei using SSR marker.
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基金
收稿日期:2013-01-22 修回日期:2013-04-07
基金项目:江苏省科技支撑计划(BE2010312); 江苏省高校自然科学研究重大合同项目(11KJA220003)
第一作者:徐进,教授。E-mail: xjinhsh@njfu.edu.cn。
引文格式:徐进,刘子梁. 柳杉SSR引物的筛选及反应体系的优化[J]. 南京林业大学学报:自然科学版,2013,37(6):17-21.
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