山丹百合cpDNA-PCR反应体系建立与优化

Construction and Optimization of cpDNA-PCR System for Lilium pumilum DC.

本试验以采自青海东南部地区的山丹百合为材料,结合单因素与L_(16)(4~5)正交设计对山丹百合cpDNA-PCR反应体系的Mg^(2+)、d NTPs、引物、Taq酶和模板DNA浓度进行优化,建立最优的山丹百合cpDNA-PCR反应体系,为研究山丹百合遗传多样性及其演变提供技术支持。研究表明:在25μL反应体系中,以Mg^(2+)2.0 mmol/L、d NTPs 0.2 mmol/L、引物0.2μmol/L、Taq酶0.25 U、模板DNA 50 ng最佳,退火温度优化后为53.1℃。建立的反应体系在6个山丹百合居群中经验证具有较高的稳定性和重复性,可用于山丹百合遗传多样性和分子谱系地理学方面的研究。

T o obtain an optimal cpDNA-PCR system for Lilium pumilum DC., single-factor test and L16（45）orthogonal test were used to 6 populations which collected from southeast region of Qinghai. Concentration of Mg2＋, d NTPs, primers, Taq polymerase and DNA template were screened at four levels. As a result, the best cpDNA-PCR amplification system for Lilium pumilum was Mg2＋2 mmol/L, d NTPs 0.2 mmol/L, Primer 0.2 μmol/L,Taq polymerase 0.25 U, and DNA template 50 ng in a 25 μL reaction system. Annealing temperature was 53.1℃.The optimized cpDNA-PCR system was validated on 6 populations, by which the high tability and repeatability of this system was confirmed. The establishment of the reaction conditions could be further used in study of genetic diversity and phylogeography of Lilium pumilum DC..