咖啡ISSR与RAPD-PCR反应体系优化

Optimization of RAPD and ISSR-PCR Reaction Systems for Coffee

以中粒种咖啡部分种质为试材,采用U25(55)均匀设计表,对影响ISSR和RAPD标记的模板DNA、Mg2+、dNTPs、TaqDNA聚合酶和引物浓度5个因素进行优化,分别建立适合于中粒种咖啡ISSR-PCR和RAPD-PCR标记的反应体系。结果表明:2种标记的反应体系相似,20μL的反应体系中含DNA模板20 ng,Mg2+1.5 mmol/L,dNTPs 0.3 mmol/L,Taq酶1.25 U,引物0.6μmol/L。利用所确立的体系对13份中粒种咖啡种质进行扩增,结果条带清晰明亮,多态性好。

The uniform design was used to optimize ISSR and RAPD reaction system for C. canephora, five influencing factors on ISSR and RAPD, including template DNA, Mg2＋, dNTPs, TaqDNA polymerase and primer, concentration were tested by uniform design. The suitable ISSR-PCR and RAPD-PCR reaction systems for Coffea canephora were developed as follows： 20μL PCR reaction volume containing 20 ng templates DNA, 1.5 mmol/L Mg2., 0.3 mmol/L dNTPs, 0.6μmol/L primer, 1.25 U TaqDNA polymerase. Under this optimum system amplifications carried out on 13 gentypes of Coffea canephora produced clear polymorphic patterns.