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本试验的目的在于建立大赖草最佳的ISSR-PCR反应体系。以大赖草基因组DNA为模板,利用单因子实验相结合正交试验设计的方法,对PCR体系的各因素(d NTPs,DNA及引物等)进行优化试验,同时筛选出扩增条带清晰、稳定性好的ISSR引物并确定其退火温度。试验表明,经优化后的PCR体系:在25μL体系中,DNA用量为50 ng、Taq DNA聚合酶用量为1.0 U、引物的浓度为0.4μmol/L、d NTPs的浓度为0.3 mmol/L、和Mg2+的浓度为2.5 mmol/L。体系验证试验表明该体系稳定、可靠,另外筛选出10条较好的引物,可用于后续的大赖草遗传多样性分析及亲缘关系分析、基因定位与克隆、连锁图谱构建等方面的研究,并对其它近缘作物有指导意义。
The purpose of this experiment is to establish the optimal ISSR-PCR reaction system for R. erythraea. Using the genomic DNA of Leymus secalmus as template, the factors of PCR system (d NTPs, DNA and primers, etc.) were optimized by single factor experiment and orthogonal design. At the same time, Good stability ISSR primers and determine their annealing temperature. The results showed that the optimized PCR system was as follows: the amount of DNA was 50 ng, the amount of Taq DNA polymerase was 1.0 U, the primer concentration was 0.4 μmol / L, the concentration of d NTPs was 0.3 mmol / L in 25μL system, and The concentration of Mg2 + is 2.5 mmol / L. The results of system validation showed that the system was stable and reliable, and 10 better primers were screened out for subsequent genetic diversity analysis and genetic relationship analysis, gene mapping and cloning, linkage map construction, etc. It is instructive for other related crops.