【摘要】 目的:建立白芷的ISSR最佳反应体系及PCR扩增参数,为今后利用ISSR标记技术进行白芷鉴定及种质遗传多样性分析提供一个标准化程序。方法:以白芷基因组DNA为模板,分析了ISSR反应体系中模板DNA、Mg2+、dNTPs、引物浓度,TaqDNA聚合酶用量及退火温度对ISSR-PCR扩增的影响。结果:建立了重复性好,分辨率高的ISSR反应体系,即在25μL反应体系中,模板DNA浓度为10~30 ng,2.5μL 10×PCR buffer(Mg2+free),MgC l22.0 mmol/L,dNTPs 0.2 mmol/L,Taq酶1.0U,引物0.6μmol/L;扩增程序为:94℃预变性5 m in;94℃变性30 s,引物在筛选后的最佳退火温度退火45 s,72℃延伸2m in,共计39个循环,循环结束后在72℃延伸5 m in。结论:建立了适用于白芷的ISSR反应体系,为今后利用ISSR标记技术开展白芷遗传变异分析和构建遗传图谱奠定了基础。更多还原

【Abstract】 This paper studied the effecting factors in Angelica dahurica ISSR analysis.These factors included DNA,Mg2+,dNTPs,TaqDNA polymerase and primer concentration,and annealing temperature.A reaction system and amplified procedure suitable for Angelica dahurica were established,that is,25 μL amplification reactions system containing10~30ng template DNA、2.5 μL10×PCR buffer(Mg2+ free)、MgCl2 2.0 mmol/L、dNTPs 0.2 mmol/L、TaqDNA polymerase 1.0 U、primer0.6 μmol/L.The optimal amplified procedure were as follows: after a pre-denaturing of 5min at 94℃,39 cycles were performed with denaturing of 30 s at 94℃,annealing of 45 s due to denaturing temperature of different primer,extension of 2 min at 72℃,a final extension step of 7min at 72℃ and hold at 4℃.This optimal system laid the standardization program of the identification of Angelica dahurica and the efficient use of its germplasm resource.更多还原