爱玉ISSR反应体系的建立与优化

通过单因子试验和正交设计，对影响爱玉ISSR-PCR扩增效果的因素，如模板DNA用量、Taq酶用量、dNTPs浓度、引物浓度、延伸时间和循环次数等指标进行优化.实验确立了可用于爱玉ISSR-PCR分析最适宜的反应体系：20 μL反应体积中含1 ng模板DNA、1.0 U Taq酶、0.4 μmol/L引物和0.18 mmol/L dNTPs；PCR扩增程序为94 ℃预变性4 min；94 ℃变性45 s，53 ℃退火45 s，72 ℃延伸2.5 min，共35个循环；72 ℃延伸7min，4 ℃保存.应用该体系对14份爱玉种质进行扩增，证实了该体系的适用性和稳定性.

Establishment and Optimization of ISSR Reaction System for Ficus Pumila L. var. Awkeotsang

The factors affecting the ISSR-PCR amplification on Ficus pumila L. var. awkeotsang such as dosage of template DNA,Taq DNA polymerase,dNTPs,primers and extension time and cycle times were optimized through the combination of single factor and orthogonal design experiment.The results showed that the optimal reaction system for ISSR-PCR of Ficus pumila were as follows:In the 20 μL reaction volume,1ng template DNA,1.0U Taq DNA polymerase,0.4 μmol/L primers,0.18 mmol/L dNTPs.The optimal PCR amplification process was:4 minutes at 94 ℃ for predenaturation,then followed by 35 cycles,each with 45 seconds at 94 ℃ for denaturation,45 seconds at 53 ℃ for annealing,2 at 72 ℃ for extension,finally extension at 72 ℃ for 7 minutes and holding the samples at 4 ℃.The system was applied in the amplification of 14 varieties of indicated the suitability and stability of the system.