结缕草属植物RAPD反应体系的优化

Optimalization of RAPD Reaction System for Zoysia

为建立一个稳定的结缕草属植物RAPD-PCR反应体系,以结缕草种源Z111(Zoysia japonicaSteud.)为实验材料,研究了模板DNA浓度、Taq酶、Mg2+浓度、dNTP浓度、引物及扩增缓冲液浓度等各个主要因素对结缕草RAPD-PCR反应的影响,并分别对各项单因子进行优化。结果表明:总反应体积为20μL时,各反应物的适宜用量分别为模板DNA浓度30 ng、Taq酶1.0 U、Mg2+2.5 mmol/L、dNTP 0.19 mmol/L、引物0.5μmol/L、10×buffer2.0μL;5种、1变种共20份结缕草种源材料验证,显示该体系扩增条带多、清晰结果稳定,表明该体系是一个适合结缕草属植物RAPD-PCR反应的体系。

The fresh leaves of zoysiagrass （Z111） were used to study the effect of the primary factors including template DNA, Taq DNA polymerase, Mg2＋ , dNTP, random primers, and buffer on the RAPD-PCR reaction system. The results show that the reaction materials of the optimized RAPD- PCR reaction system were as follows. 20 μL total volume of 30 ng template DNA, Taq DNA polymerase at 1.0U, Mg2＋ at 2.5 mmol/L, dNTP at 0.19 mmol/L, random primers at 0.5 μmol/L, and 10× buffer 2.0 μL. The optimized system was tested with 20 accessions of zoysiagrass including five species and one variety, and the results indicate that the reaction system of RAPD could be used in molecular marking of Zoysia Willd.