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作 者 ：张龙进;白成科;*

期 刊 ：植物研究 2011年 31卷 1期 页码：105-108

关键词：北重楼；ISSR-PCR；分子标记；正交优化；

Keywords:Paris vertcillata, ISSR-PCR, molecular markers, orthogonal optimization,

摘 要 ：利用正交试验设计L₉(3⁴)的方法，最终建立了北重楼ISSR-PCR的优化反应体系，即20 μL反应体系中包含1×buffer，dNTP 175 μmol·L^-1，Primer 0.8 μmol·L^-1，Mg²⁺ 1.4 mmol·L^-1，Taq酶2.5 U及模板DNA 80 ng。适宜的扩增程序为95℃预变性5 min；94℃变性40 sec，55℃退火45 sec，72℃延伸90 sec，40个循环；72℃延伸7 min，4℃保存。该优化体系的建立，将为北重楼及其近缘种的系统学和种质资源鉴定及遗传多样性的研究奠定基础。

Abstract:The optimal ISSR-PCR system for Paris vertcillata was established with orthogonal design.A total of 20 μL ISSR-PCR system contains 1×buffer, dNTP 175 μmol·L^-1，Primer 0.6 μmol·L^-1，Mg²⁺ 1.4 mmol·L^-1，Taq DNA polymerase 2.5 U and 80 ng template DNA.The suitable PCR procedure is one cycle denaturing at 95℃ for 5 min;40 cycles each involved denaturing at 94℃for 40 s,annealing at 55℃ for 45 s,extending at 72℃ for 90 s,one cycle extending at 72℃ for 7 min,and then remaining at 4℃.This optimized system would play an important role in further research of systematology on P.vertcillata and its related species, as well as identification on germplasm, and genetic diversity by ISSR molecular marker.

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