论文部分内容阅读
采用正交设计试验,利用引物UBC834对长序榆ISSR-PCR扩增的主要影响因子Buffer(含Mg2+)浓度、模板DNA、引物浓度、Taq DNA聚合酶浓度、dNTPs浓度进行优化分析,并在此基础上对PCR反应过程中的退火温度、循环次数、延伸时间进行检测。结果表明,在25μL ISSR-PCR反应体系中各因素的最佳浓度为2.5μLBuffer(含Mg2+),50 ng模板DNA,2.5 U Taq DNA聚合酶,1.2μmol.L-1引物,0.2 mmol.L-1dNTPs,引物UBC834的最佳退火温度为57℃,最佳循环次数和延伸时间分别为35个循环和1 min。此外,还利用优化的反应体系成功筛选出11条ISSR引物,并用筛选的引物对长序榆的遗传多样性进行了分析,结果发现,在物种水平上,长序榆的遗传多样性较高,多态位点百分率(PPB)为82.35%,Shannon信息指数(I)为0.395 9,Nei’s基因多样性指数(H)为0.258 5。而种群水平上的遗传多样性则较低,多态位点百分率(PPB)、Shannon信息指数(I)、Nei’s基因多样性指数(H)分别为29.07%、0.173 3和0.119 1。
Orthogonal design was used to analyze the concentration of Buffer (Mg2 +), template DNA, primer, Taq DNA polymerase and dNTPs concentration of ISSR-PCR amplified by primer UBC834, Based on the reaction temperature during the PCR reaction, the number of cycles, the extension of time to detect. The results showed that the optimum concentration of each factor in 25 μL ISSR-PCR reaction system was 2.5 μL Buffer (containing Mg2 +), 50 ng template DNA, 2.5 U Taq DNA polymerase, 1.2 μmol.L-1 primer, 0.2 mmol.L- 1dNTPs. The optimal annealing temperature of UBC834 was 57 ℃. The optimal number of cycles and the extension time were 35 cycles and 1 min, respectively. In addition, 11 ISSR primers were successfully screened by using the optimized reaction system, and the genetic diversity of the elm was analyzed with the selected primers. The results showed that the genetic diversity of elm was higher at the species level, The percentage of polymorphic loci (PPB) was 82.35%, Shannon’s information index (I) was 0.395 9, Nei’s gene diversity index (H) was 0.258 5. While the genetic diversity at the population level was low. The percentage of polymorphic loci (PPB), Shannon’s information index (I) and Nei’s gene diversity index (H) were 29.07%, 0.173 3 and 0.119 1 respectively.