鹰嘴豆ISSR反应体系的正交优化

Orthogonal Optimization of ISSR Reaction System for Chickpea

鹰嘴豆(Cicer arietinum)ISSR反应体系的建立可以为鹰嘴豆种质资源遗传多样性分析、遗传图谱构建和基因定位提供理论依据和技术参考。本研究以CTAB法提取的鹰嘴豆DNA为模板,采用L16(4~5)正交设计直观分析法,对鹰嘴豆ISSR-PCR反应中的4个主要因素(Mg^(2+)浓度,引物浓度,TaqDNA聚合酶浓度,dNTPs浓度)在4个水平上进行优化,并在最优ISSR-PCR反应体系的基础上对引物UBC826的最适退火温度进行筛选。结果表明:Mg2+和引物浓度对PCR扩增结果有显著影响,dNTPs和Taq酶的浓度变化对PCR扩增结果无显著影响。鹰嘴豆ISSR-PCR优化反应体系(25μL)为:3mmol·L^(-1) Mg^(2+),0.2mmol·L^(-1) dNTP,0.24μmol·L^(-1)引物,2U Taq DNA聚合酶。UBC826最适退火温度为56℃。

The establishment of the chickpea ISSR reaction system would provide theoretical bases and technical references for the analysis of chickpea diversity,the construction of the gene map and the localization of important traits for chickpea genotypes using ISSR markers.The DNA template for chickpea（Cicer arietinum）was extracted using CTAB method.An ISSR-PCR amplification system for chickpea was optimized by visual analysis and an orthogonal design L16（4~5）involving 4levels of 4main factors（concentrations of Mg^（2＋）,Primer,Taq DNA polymerase,and dNTPs）.The annealing temperature of the primer UBC826 was optimized based on the optimal ISSR-PCR amplification system.The results indicated that the concentrations of Mg^（2＋） and primer affected the PCR amplification significantly,while the concentrations of dNTPs and Taq DNA polymerase had no effects on it.The suitable ISSR-PCR amplification system（25μL）for chickpea included 3mmol·L^（-1） Mg^（2＋）,0.2mmol·L^（-1） dNTP,0.24μmol·L^（-1） primer and 2U Taq DNA polymerase.The suitable annealing temperature for the primer UBC 826 was 56℃.