珍稀植物杨叶肖槿ISSR体系建立及检测

针对珍稀植物杨叶肖槿ISSR反应的特点,建立了适用于杨叶肖槿遗传多样性研究的ISSR最适反应体系,具体包括:2.0μL 10×Buffer,27.5ng的模板DNA,2.0μL的dNTP,1U的Pyrobest DNA酶,1.25μmol/L的引物;最佳反应程序为94℃预变性5min,然后94℃变性1min,49℃退火45s,72℃延伸1min,35个循环;最后72℃延伸10min,4℃终止反应。应用该优化的反应体系筛选出了10条稳定性强、清晰度高而且表现出一定多态性的ISSR引物,并对杨叶肖槿进行了检测,获得了清晰稳定的扩增图谱。

Detection and Establishment of ISSR System for Rare Species Thespesia populnea

To seek a standardizing program of ISSR technique for genetic diversity analysis of Thespesia populnea,a single factor experiment was designed to optimize ISSR-PCR amplification system.The suitable reaction system was obtained,that is 20 μL reaction system containing 2.0 μL of 10×Buffer,27.5 ng of total DNA,2.0 μL of dNTP,1 U Pyrobest DNA polymerase and 1.25 μmol/L of ISSR primer.The profile of ISSR-PCR was an initial denaturation step for 5 min at 94℃,follow by 35 cycles of 1 min at 94℃,45 s at annealing temperature 49℃,1 min at 72℃,and a final elongation 10 min at 72℃ for one cycle,then termination reaction at 4℃.Ten polymorphic primers were screened using this reaction system.Stable and clear amplification patterns were obtained,indicating the ISSR-PCR amplification system was feasible.The factors affecting the amplification of genome DNA of Thespesia populnea were also discussed in the paper.It provides the basis on studies of germplasm resources and genetic diversity of T.populnea.