孙秀秀, 陈加利, 杨立荣, 等. 文昌锥SRAP−PCR体系优化及有效引物筛选[J]. 西南林业大学学报（自然科学）, 2021, 41(1): 39–46 . DOI: 10.11929/j.swfu.201909020
引用本文: 孙秀秀, 陈加利, 杨立荣, 等. 文昌锥SRAP−PCR体系优化及有效引物筛选[J]. 西南林业大学学报（自然科学）, 2021, 41(1): 39–46 . DOI: 10.11929/j.swfu.201909020
shu
Xiuxiu Sun, Jiali Chen, Lirong Yang, Yong Yun, Huasha Qi, Daojun Zheng. Optimization of SRAP−PCR System and Screening of Effective Primers for Castanopsis wenchangensis[J]. Journal of Southwest Forestry University, 2021, 41(1): 39-46. DOI: 10.11929/j.swfu.201909020
Citation: Xiuxiu Sun, Jiali Chen, Lirong Yang, Yong Yun, Huasha Qi, Daojun Zheng. Optimization of SRAP−PCR System and Screening of Effective Primers for Castanopsis wenchangensis[J]. Journal of Southwest Forestry University, 2021, 41(1): 39-46. DOI: 10.11929/j.swfu.201909020
shu

文昌锥SRAP−PCR体系优化及有效引物筛选

Optimization of SRAP−PCR System and Screening of Effective Primers for Castanopsis wenchangensis

    摘要
  • 摘要: 通过单因素试验和正交试验相结合，分析DNA、引物、dNTPs、Taq DNA聚合酶4种因素对文昌锥SRAP−PCR扩增结果的影响，优化建立文昌锥SRAP−PCR体系。结果表明：文昌锥基因组DNA浓度和dNTPs浓度过低或过高均扩增不出产物，而引物浓度和Taq酶用量的增加能提高扩增效率。在适宜浓度范围内，各个因素对文昌锥SRAP−PCR扩增影响大小依次为：引物=dNTPs>Taq酶>DNA；最优反应体系为：总体系为20 μL时，DNA 20 ng，引物0.6 μmol/L，dNTPs 0.15 mmol/L和Taq酶 4 U。经验证，该体系获得的扩增产物清晰、稳定；筛选获得45对有效性引物组合多态性好，可应用于SRAP分子标记技术在文昌锥资源研究。

     

    Abstract: In order to optimize and establish the SRAP−PCR system for Castanopsis wenchangensis, the combination of single factor test and orthogonal test were used to analysis the effects of DNA, primers, dNTPs and Taq DNA polymerase on SRAP−PCR. The results showed that the genomic DNA concentration and dNTPs concentration were too low or too high to amplify the product, and the increase of primer concentration and Taq DNA polymerase could increase the amplification efficiency. Within the appropriate concentration range, the order of influence on the SRAP−PCR system for C. wenchangensis was: primer = dNTPs > Taq > DNA. The optimal reaction system was: when the total system was 20 μL, the DNA 20 ng, the primer 0.6 μmol/L, the dNTPs 0.15 mmol/L and Taq 4 U. It has been verified that the amplification products obtained by this system were clear and stable, 45 pairs of effective primer combination polymorphisms were obtained by screening, which can be applied to the research of SRAP molecular marker in C. wenchangensis resources.

     

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