[Objective] This study aimed to optimize the SCoT-PCR system for Citrus.Under the optimized SCoT-PCR amplification conditions, the genetic DNA of Youzishatangju and Wuzishatangju were amplified. [Method] Orthogonal de...[Objective] This study aimed to optimize the SCoT-PCR system for Citrus.Under the optimized SCoT-PCR amplification conditions, the genetic DNA of Youzishatangju and Wuzishatangju were amplified. [Method] Orthogonal design was adopted to optimize the five influencing factors on amplification effect of SCoT-PCR,including the DNA template, Mg2＋, primers, dNTPs and Taq DNA polymerse concentrations. With the optimized SCoT-PCR system, the genomic DNA of Youzishatangju and Wuzishatangju were amplified by 60 SCoT primers, respectively, and the specific bands for Youzishatangju or Wuzishatangju were selected for SCAR transformation. [Result] The optimized SCoT-PCR reaction system（20 μl） was as follows： Mg2＋1.5 mmol/L, d NTPs 0.35 mmol/L, primer 0.25 μmol/L, Taq enzyme 0.5 U, DNA template 30 ng. The optimum annealing temperature was 50.6 °C. With the optimized SCoT-PCR system, the genomic DNA of Youzishatangju and Wuzishatangju were amplified respectively by 60 SCoT primers, and a total of 42 pairs of primers were screened. Among the 42 pairs of primers, only one primer amplified different band pattern between Youzishatangju and Wuzishatangju. [Conclusion] It indicates that there is certain difference between Youzishatangju and Wuzishatangju at genomic DNA level.展开更多
基金Supported by Foundation for Distinguished Young Talents in Higher Education of Guangdong,China(2012LM0135)Scientific and Technological Innovation Project of Zhaoqing City,Guangdong Province(2012G21)Guangdong Provincial Natural Science Foundation(S2013010015195)~~
文摘[Objective] This study aimed to optimize the SCoT-PCR system for Citrus.Under the optimized SCoT-PCR amplification conditions, the genetic DNA of Youzishatangju and Wuzishatangju were amplified. [Method] Orthogonal design was adopted to optimize the five influencing factors on amplification effect of SCoT-PCR,including the DNA template, Mg2＋, primers, dNTPs and Taq DNA polymerse concentrations. With the optimized SCoT-PCR system, the genomic DNA of Youzishatangju and Wuzishatangju were amplified by 60 SCoT primers, respectively, and the specific bands for Youzishatangju or Wuzishatangju were selected for SCAR transformation. [Result] The optimized SCoT-PCR reaction system（20 μl） was as follows： Mg2＋1.5 mmol/L, d NTPs 0.35 mmol/L, primer 0.25 μmol/L, Taq enzyme 0.5 U, DNA template 30 ng. The optimum annealing temperature was 50.6 °C. With the optimized SCoT-PCR system, the genomic DNA of Youzishatangju and Wuzishatangju were amplified respectively by 60 SCoT primers, and a total of 42 pairs of primers were screened. Among the 42 pairs of primers, only one primer amplified different band pattern between Youzishatangju and Wuzishatangju. [Conclusion] It indicates that there is certain difference between Youzishatangju and Wuzishatangju at genomic DNA level.
