西洋杜鹃ISSR-PCR反应体系的建立及优化

以西洋杜鹃(Rhododendron hybridum)叶片提取的基因组DNA为材料,用引物UBC880(序列为GGA GAG GAG AGG AGA)研究了PCR反应体系的主要成分及退火温度对该种植物ISSR扩增结果的影响.确立了可用于西洋杜鹃ISSR-PCR 分析的最适宜的PCR反应体系:20μL PCR反应体积中...

Establishment and optimization of ISSR-PCR system of Rhododendron hybridum

Taking the genomic DNA of Rhododendron hybridum leaves as the experimental materials,the concentrations of PCR components and the annealing temperatures,which affect the inter-simple sequences(ISSR) amplification,were optimized with the primer UBC880(primer sequence: GGA GAG GAG AGG AGA).The suitable ISSR-PCR system for R.hybridum was as follows: the 20 μL PCR volume includes 10 ng DNA templates,0.25 mmol · L-1 dNTPs,2.0 mmol · L-1 Mg2+,1.0 U Taq DNA polymerase and 0.4 μmol · L-1 primers.The optimal PCR process was 5 minutes at 94 ℃ for predenaturation,the follows by 40 cycles,each with 45 seconds at 94 ℃ for denaturation,45 seconds at 54.4 ℃ for annealing,1.5 minutes at 72 ℃ for extension,finally 7 minutes at 72 ℃ for extension and holding the samples at 4 ℃.The ISSR-PCR of 11 cultivars of R.hybridum approves the suitability and stability of the system.