CAJ | 학술논문

建立并优化虎耳草ISSR—PCR反应体系和扩增程序，为探讨虎耳草种质资源遗传多样性奠定基础．采用正交设计方法和单因子试验，研究％qDNA聚合酶、dNTP、Mg^2＋、引物、模板DNA、延伸时间及循环次数对PCR扩增的影响．结果表明：虎耳草ISSR—PCR的最佳反应体系为：在25μL的反应体系中含模板DNA35ng，Mg2^＋1．25μmol／L、dNTP380μmol／L、引物1．2μmol／L、TaqDNA聚合酶1．5u．反应程序为：95℃预变性5min；94℃变性1min，50℃（据不同引物的退火温度）退火70s，72℃延伸1．5min，40个循环；72℃延伸10min，4℃保存．经过11份虎耳草种质检验．证明该体系稳定可靠，可用于虎耳草种质资源遗传多样性分析及居群鉴别的研究．
건립병우화호이초ISSR—PCR반응체계화확증정서，위탐토호이초충질자원유전다양성전정기출．채용정교설계방법화단인자시험，연구％qDNA취합매、dNTP、Mg^2＋、인물、모판DNA、연신시간급순배차수대PCR확증적영향．결과표명：호이초ISSR—PCR적최가반응체계위：재25μL적반응체계중함모판DNA35ng，Mg2^＋1．25μmol／L、dNTP380μmol／L、인물1．2μmol／L、TaqDNA취합매1．5u．반응정서위：95℃예변성5min；94℃변성1min，50℃（거불동인물적퇴화온도）퇴화70s，72℃연신1．5min，40개순배；72℃연신10min，4℃보존．경과11빈호이초충질검험．증명해체계은정가고，가용우호이초충질자원유전다양성분석급거군감별적연구．
To establish and optimize ISSR - PCR reaction system for Saxifraga stolonifera Curt. and lay foundation for its genetic diversity research. The single - factor and orthogonal design were applied for optimizing seven factors in the ISSR - PCR reaction system including Mg^2+ , dNTP, primers, Taq DNA polymerase, the template DNA, extension time and cycles. The results showed that the suitable PCR reaction system contained 35 ng template DNAI 1.25 μmol/L Mg^2+ , 380μmol/L dNTP, 1.2 μmol/L primer and 1.5U Taq DNA polymerase in total 25μL reaction solution. The suitable PCR procedure was: preliminary denaturizing at 95℃ for 5 min; denaturized at 94℃ for lmin, annealing at 50℃ for 30s, extended at 72℃ for 1.5rain, 40 cycles and a final extension at 72℃ for I0 min, then kept the temperature at 4℃. It is proved to be stable and credible for the result of 11 Saxifraga stolonifera Curt. populations. That' s mean it is suitable for the study on genetic diversity of germplasm resource and identification of Saxifraga stolonifera Curt. population.