苏铁属植物RAPD反应体系的优化及部分种类亲缘关系的探索

通过优化苏铁属植物的RAPD反应体系,进而探讨苏铁属部分种类的亲缘关系。结果表明,Mg~(2+)、dNTP、Taq酶及随机引物浓度在RAPD反应中有重要影响,而模板DNA浓度有一个很大的适应范围。适合苏铁属植物RAPD分析的反应体系:20μL反应体系中,含有10 mmol/L Tris-HCl(pH8.3)、50 mmol/L KCl、2.0mmol/L MgCl_2、200μmol/L dNTP、0.3μmol/L随机引物、模板DNA 50ng、Taq酶1.0 U。聚类分析结果基本反应了苏铁属各个种间的亲缘关系,证明了RAPD适用于苏铁属种间亲缘关系分析。RAPD聚类分析结合形态学研究表明:海南苏铁、台湾苏铁、广东苏铁、滇南苏铁和仙湖苏铁之间的亲缘关系较远,支持各自成为独立的种。

Improving of RAPD reaction system in Cycas and studies on the genetic relationships of some species

In this paper,the reaction system of RAPD in Cycas had been established and applied to discuss the relationships among C.hainanensis,C.taiwaniana,C.guangdongensis C.diannanensis and C.fairylakea. The results showed that RAPD was affected by the concentrations of four components i.e.Mg2+,dNTP,Taq DNA polymerase and primers. The optimal reaction system was as follows:20 μL reaction volume,including 10 mmol/L Tris-HCl(pH 8.3),50 mmol/L KCl,2.0 mmol/L MgCl2,200 μmol/L dNTP,0.3 μmol/L primers,50 ng template DNA,1.0 U Taq DNA polymerase. The reflection of RAPD cluster to the genetic relationship of all the species showed that RAPD was suitable for analysis of interspecific genetic relationship in Cycas. Genetic relationship analysis by RAPD markers showed that five defined species had far genetic relationships each other,combining with the analysis of morphological traits,it's concluded that they were five independent genetic units at species level.