摘要
【目的】优化大旗瓣凤仙ISSR-PCR反应体系,为大旗瓣凤仙遗传多样性分析提供技术参考。【方法】利用正交试验设计对大旗瓣凤仙ISSR-PCR反应体系的5个因素（Mg2＋、dNTP、引物、Taq DNA聚合酶、模板DNA）4水平进行优化分析,并在此基础上对PCR反应过程中的退火温度进行筛选。【结果】建立了大旗瓣凤仙ISSR-PCR的优化反应体系,即在25.0μL反应体系中含1×PCR Buffer、Mg2＋1.5 mmol/L、dNTP 0.25 mmol/L、引物0.8μmol/L、Taq DNA聚合酶0.75~1.00 U、模板DNA 100 ng。通过该体系可以筛选出6条对大旗瓣凤仙种群扩增效果较好的引物。【结论】优化的ISSR-PCR反应体系具有较高的稳定性,可用于大旗瓣凤仙及凤仙属植物种群的ISSR遗传多样性分析。
【Objective】The present experiment was conducted to explore the best ISSR-PCR reaction for genetic diversity analysis of Impatiens macrovexilla Y. L. Chen. 【Method】Orthogonal design method was used to optimize the ISSR-PCR amplification system of Impatiens macrovexilla Y. L. Chen in five factors（concentration of Mg2＋, dNTP, primers, Taq DNA polymerase, and template DNA） at four levels. Then, based on the optimal ISSR-PCR amplification system, annealing temperature was screened by gradient PCR. 【Result】An optimal reaction system（25.0 μL） was established with containing 1× PCR Buffer,1.5 mmol/L Mg2＋, 0.25 mmol/L dNTP, 0.8 μmol/L primers, 0.75-1.00 U Taq DNA polymerase, and 100 ng temple DNA. Six ISSR primers with better effects were screened by this reaction system. 【Conclusion】This optimal ISSR-PCR reaction system showed a very stable result, which could be used for ISSR analysis of genetiec diversity in Impatiens macrovexilla Y. L. Chen.
出处
《南方农业学报》
CAS
CSCD
北大核心
2014年第2期184-188,共5页
Journal of Southern Agriculture
基金
财政部战略生物资源科技支撑运转专项项目(KSCX2-YW-Z-1009)
关键词
大旗瓣凤仙
ISSR-PCR
反应体系
优化
正交设计
Impatiens macrovexilla Y.L.Chen
ISSR-PCR
reaction system
optimization
orthogonal design
