越桔ISSR-PCR反应体系建立与优化
Establishment and Optimization of an ISSR-PCR Reaction System for Vaccinium Linn.

以改良CTAB法提取越桔(Vaccinium Linn.)品种薄雾DNA为材料，采用正交设计L₁₆(4⁵)表探讨了Tag酶；Mg²⁺；DNA；dntps及引物浓度5个主要因素对越桔ISSR-PCR扩增反应的影响，通过直观分析和方差分析结合，建立了越桔ISSR-PCR(20 μL)最佳反应体系，各组分的浓度为：Tag酶0.75 U，Mg²⁺1.875 mmol/L，DNA 0.8 mmol/L，dntp 0.1 mmol/L，引物0.2 μmol/L.在此基础上探讨了引物UBC835的最佳退火温度、循环次数和延伸时间，结果分别为54.2 ℃、35次、60 s.选用两个引物对21份越桔资源进行ISSR扩增，结果显示优化的体系稳定性较高.
Based on the genomic DNA extracted from Vaccinium Linn. cultivar Misty with a modified CTAB method, five factors influencing ISSR were optimized in an orthogonal-design experiment. Through intuitive analysis and variance analysis, an optimal ISSR-PCR reaction system was established for this fruit crop, namely 10×buffer 2 μL; 0.75 U Taq DNA polymerase; 1.875 mmol/L Mg²⁺; 80 ng template DNA; 0.1 mmol/L dNTPs; and 0.2 μmol/L primer. The optimal annealing temperature, number of cycles and extension time of primer UBC835 were investigated, and the results were 54.2 ℃, 35 cycles and 60s, respectively. Twenty-one Vaccinium accessions were used to test the stability of the optimized reaction system. The results indicated that the optimized reaction system was very stable