Abstract: Medicago ruthenica genomic DNA,extracted by modified CTAB method Orthogonal design was applied to optimize ISSR-PCR amplification system of Medicago ruthenica in five factors such as Mg2+,dNTPs,primer,TaqDNA polymerase and template DNA at four levels.The result of PCR was analyzed by tuitive analysis and variance analysis.An optimal reaction system was established,in a total volume of 25 μL ISSR-PCR system,it contained TaqDNA polymerase 1.5 U,10×Buffer(Mg2+)2.0 mmol/L,template DNA 0.5 ns/μL,dNTP 0.6 mmol/L and primer 0.9 μmol/L.The annealing temperature was also investigated.52.3℃ was the most suitable annealing temperature of HZD09211 primer.20 Medicago ruthenica germplasm materials were used to test the stabilization of the optimized reaction system.The results indicated that the optimized reaction system was very stable.

Key words: Medicago ruthenica, ISSR, Orthogonal design, Reaction system, Optimization

CLC Number: 

• S541.9