狭边大叶藓（Rhodobryum ontariense）ISSR-PCR反应体系的建立与优化

研究目的是获得适用于狭边大叶藓（Rhodobryum ontariense）遗传多样性研究的ISSR—PCR反应标准化程序。通过单因素试验设计，对Mg^2＋、dNTPs、Taq DNA聚合酶、模板、引物浓度，以及退火温度、循环次数等影响ISSR扩增的主要因素进行了研究和优化。结果表明，UBC808、UBC811、UBC812、UBC825、UBC826、UBC841、UBC888及UBC891引物适用于该研究；20μL ISSR—PCR最适反应体系包括：6ngDNA模板、0．4μmol／L引物、2．25mmoL／LMg^2＋、0．6U Taq DNA聚合酶、0．4mmol／LdNTPs。扩增程序为：94℃预变性4min；然后94℃变性1min，48～50℃（根据不同引物确定）复性2min，72℃延伸1min，共进行40个循环；最后，72℃延伸7min，4℃保存。

Optimization of ISSR-PCR Reaction System in a Rare Medicinal Plant Rhodobryum ontariense (Bryaceae)

To seek a standardizing program of ISSR technique for the genetic diversity analysis of a rare medicinal plant Rhodobryum ontariense, a single-factor experiment was designed to optimize ISSR-PCR amplification system.A suitable ISSR reaction system was obtained,that is:20 μL reaction system containing 6 ng DNA template,0.4 μmol/L primer,2.25 mmol/L Mg~(2+),0.6 U Taq DNA polymerase,0.4 mmol/L dNTPs.The profile of ISSR amplification was 4 min at 94℃, followed by 40 cycles of denaturation for 1 min at 94℃,annealing for 1 min at 48～50℃ (according to specific primer),and extension for 1 min at 72℃,and ending with a final extension for 7 min at 72℃,stored at 4℃.