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 influencing factors including Taq DNA polymerase, DNA template 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 0.2mmol/L dNTPs, 0.2μmol/L ISSR primers, 1.5U of Taq DNA polymerase, 2.5μl of 10×PCR Buffer, 1.5mmol/L MgCl2 and 40ng of template DNA in 25μl total volume; the amplification was conducted with 35 cycles and extension time of 90s.[Conclusion] ISSR-PCR reaction system for E. sibiricus was established and optimized, and then verified using two E. sibiricus germplasms, demonstrating that the ISSR-PCR reaction system is stable and can be used for the genetic analysis of E. sibiricus.