[ Objective ] This study aimed to establish and optimize the ISSR-PCR reaction system and amplification process for Elymus sibiricus L. , to provide sci- entific basis for exploring the genetic diversity of E. sibiric...[ Objective ] This study aimed to establish and optimize the ISSR-PCR reaction system and amplification process for Elymus sibiricus L. , to provide sci- entific 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 0.2 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 MgC12 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 was established and optimized, and then verified using two E. sibiricus germplasrns, demonstrating that the ISSR-PCR reaction system is stable and can be used for the genetic analysis of E. sibiricus.展开更多
基金Supported by Project of Collection,Cataloguing and Utilization of Perennial Forage Grass Germplasm Resources(NB2012-2130135-33)Conservation Project for Forage Grass Germplasm Resources
文摘[ Objective ] This study aimed to establish and optimize the ISSR-PCR reaction system and amplification process for Elymus sibiricus L. , to provide sci- entific 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 0.2 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 MgC12 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 was established and optimized, and then verified using two E. sibiricus germplasrns, demonstrating that the ISSR-PCR reaction system is stable and can be used for the genetic analysis of E. sibiricus.
