Establishment and Optimization of ISSR Reaction System for Elymus sibiricus L.

[Objective] This study aimed to establish and optimize the ISSR-PCR reaction system and amplification process for Elymus sibiricus L.,to provide scientific basis for exploring the genetic diversity of E.sibiricus germplasm resources.[Method] Orthogonal design and single factor test were applied to establish the ISSR-PCR reaction system of E.sibiricus,optimize the affecting factors including Taq DNA polymerase,template DNA concentration,Mg2+,dNTP,primer concentration,and screen the annealing temperature,number of cycles and extension time.[Result] The optimal reaction system for ISSR analysis contains 0.2 mmol/L dNTPs,0.2 μmol/L ISSR primers,1.5 U of Taq DNA polymerase,2.5 μl of 10×PCR Buffer,1.5 mmol/L MgCl2 and 40 ng of template DNA in 25 μl total volume;the amplification was conducted with 35 cycles and extension time of 90 s.[Conclusion] ISSR-PCR reaction system for E.sibiricus L.was established and optimized in this study,which was verified using two E.sibiricus germplasms,results show that the ISSR-PCR reaction system is stable and can be used for the genetic analysis of E.sibiricus.