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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20μL优化反应体系为:DNA模板20 ng、引物浓度为30μmol/L、Taq DNA聚合酶用量为1.0 U、dNTP浓度为100μmol/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 To optimize the SCOT polymorphism reaction system of Dendrobium candidum, which will lay the foundation for further SCoT analysis of the genetic diversity of Dendrobium candidum. Methods The fresh leaf of Dendrobium candidum was used as experimental material, and single-variable method was used to study the effect of five factors on the amplification of SCoT-PCR, including the amount of template DNA, primer concentration, Taq DNA polymerase dosage, dNTPs concentration and annealing temperature . Results The optimal reaction system for 20μL SCoT labeling was Dendrobium officinale with 20ng DNA template, primer concentration of 30μmol / L, Taq DNA polymerase concentration of 1.0 U and dNTP concentration of 100μmol / L. The optimal PCR procedure was pre-denaturation at 95 ° C for 4 min, denaturation at 95 ° C for 50 s, annealing at 56.1 ° C for 40 s, annealing at 72 ° C for 2 min, and cycling 36 times; extension at 72 ° C for 5 min and storage at 4 ° C. 1.5% agarose gel electrophoresis showed that the amplified product was between 600 and 2 000 bp. Conclusion The establishment of this system laid the foundation for the genetic diversity analysis of Dendrobium candidum and molecular marker-assisted breeding.