目的 对铁皮石斛的目标起始密码子（start condon targeted polymorphism，SCoT）多态性反应体系进行优化，为进一步对铁皮石斛遗传多样性的SCoT分析奠定基础。方法 以铁皮石斛的新鲜叶片为试验材料，采用控制单一变量的方法分别研究模板DNA用量、引物浓度、TaqDNA聚合酶用量、dNTPs浓度、及退火温度共5个因素对SCoT-PCR扩增结果的影响。结果 铁皮石斛SCoT 标记的20 μL优化反应体系为：DNA模板20 ng、引物浓度为30 μmol/L、Taq DNA聚合酶用量为1.0 U、dNTP浓度为100 μmol/L。适宜的PCR扩增程序为95 ℃预变性4 min；95 ℃变性50 s，56.1 ℃复性退火40 s，72 ℃延伸2 min，循环36次；72 ℃延伸5 min，4 ℃保存。1.5% 琼脂糖凝胶电泳显示扩增产物在600～2 000 bp。结论 该体系的建立为铁皮石斛种质遗传多样性分析、分子标记辅助育种等研究奠定了基础。

Objective In order to optimize the reaction system of start condon targeted (SCoT) polymorphism of Dendrobium officinale, and to lay the foundation for analysis on genetic diversity of D. officinale. Methods The fresh leaves of D. officinale were used and the effects of template DNA, primer, Taq DNA polymerase, dNTPs, and annealing temperature on SCoT-PCR amplification were determined through single factor test. Results The SCoT-marked 20 μL optimized reaction system of D. officinale included 20 ng template DNA, 30 μmol/L primer, 1.0 U Taq DNA polymerase, and 100 μmol/L dNTP. The suitable PCR amplification procedure was one cycle of pre-denaturing at 94 ℃ for 4 min, 36 cycles of denaturing at 95 ℃ for 50 s, annealing at 56.1 ℃ for 40 s, and extending at 72 ℃ for 2 min, extending at 72 ℃ for 5 min, and finally, being kept at 4 ℃. PCR amplification results by 1.5% agarose gel electrophoresis showed that amplification products were mainly in the range of 600－2 000 bp. Conclusion The established SCoT-PCR reaction system could provide the reference for germplasm genetic diversity analysis and molecular marker-assisted breeding of D. officinale.

国家科技支撑计划项目（2011BAI04B06）；浙江省教育厅科研基金（71003012；Y201122310）；浙江省中医药青年基金项目（A2005Y005）