[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 ge...[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.展开更多
Late blight caused by the oomycete Phytophthora infestans Montagne de Bary is a devastating disease on potato production. A total of six parameters affecting the PCR system in P. infestans were investigated based on t...Late blight caused by the oomycete Phytophthora infestans Montagne de Bary is a devastating disease on potato production. A total of six parameters affecting the PCR system in P. infestans were investigated based on the template DNA of the isolate HH06-23 and EST-SSR primer pair Pi08N. The results showed that the optimal annealing temperature was 63℃, and the optimum PCR system of EST-SSR was 25 ng template DNA, 0.5 mmol·L-1 dNTPs, 2 μL 10×Buffer (Mg 2+ free), 1.75 mmol·L-1 MgCl 2 , 15 pmol primer, and 1.2 U Taq DNA polymerase in total 20 μL reaction system. The PCR program was initial denaturation at 94℃ for 2 min, followed by 35 cycles of 94℃ for 30 s, 63℃ for 30 s, and 72℃ for 30 s, then a final extension step was 72℃ for 7 min, and held at 4℃. In addition, using the optimal PCR system, a total of 20 isolates of P. infestans were used for testing the stability and polymorphism of the PCR amplification. The clarity and abundant polymorphism indicated that this system was stable and suitable for researching the genetic diversity of P. infestans population.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 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.
基金Supported by Natural Science Foundation of Heilongjiang Province of China (C200622)
文摘Late blight caused by the oomycete Phytophthora infestans Montagne de Bary is a devastating disease on potato production. A total of six parameters affecting the PCR system in P. infestans were investigated based on the template DNA of the isolate HH06-23 and EST-SSR primer pair Pi08N. The results showed that the optimal annealing temperature was 63℃, and the optimum PCR system of EST-SSR was 25 ng template DNA, 0.5 mmol·L-1 dNTPs, 2 μL 10×Buffer (Mg 2+ free), 1.75 mmol·L-1 MgCl 2 , 15 pmol primer, and 1.2 U Taq DNA polymerase in total 20 μL reaction system. The PCR program was initial denaturation at 94℃ for 2 min, followed by 35 cycles of 94℃ for 30 s, 63℃ for 30 s, and 72℃ for 30 s, then a final extension step was 72℃ for 7 min, and held at 4℃. In addition, using the optimal PCR system, a total of 20 isolates of P. infestans were used for testing the stability and polymorphism of the PCR amplification. The clarity and abundant polymorphism indicated that this system was stable and suitable for researching the genetic diversity of P. infestans population.
