胡麻专化型尖孢镰刀菌ISSR-PCR最佳反应体系的建立

Optimization of ISSR-PCR Reaction System for Fusarium oxysporum Schl.

采用正交试验设计原理,对尖孢镰刀菌ISSR-PCR反应体系中的5种主要因素进行优化筛选,确立了适合尖孢镰刀菌ISSR分析的反应体系,即25μL PCR反应体积中含有20 ng模板DNA、1 UTaq酶、0.4μmol/L引物、0.2 mmol/LdNTPs、4.0 mmol/L Mg2+和2.5μL10×buffer。PCR反应最佳退火温度根据引物而定,在此基础上筛选出12条扩增稳定、多态性丰富的ISSR引物。此研究为今后利用ISSR技术分析尖孢镰刀菌遗传多样性和群体结构奠定了基础。

An Orthogonal design is applied to the optimization of ISSR-PCR reaction system for Fusarium oxysporum Schl.The effect of the five main factors was tested.A most suitable ISSR-PCR reaction system for Fusarium oxysporum Schl.was established.The optimized reaction system consists of 20 ng template DNA,1 U Taq DNA polymerase,0.4 μmol/L primer,0.2 mmol/L dNTPs,4.0 mmol/L Mg2＋ and 2.5 μL 10× buffer.Optimal annealing temperature was decided by different primers.12 primers with stable amplification and rich polymorphism for ISSR were selected.This study laid the foundation for the analysis of diversity and group structure by using ISSR technique for Fusarium oxysporum Schl.in the future.