利用Indel标记鉴定太白贝母及其近缘种

Molecular Identification of Fritillaria taipaiensis and Its Relatives Based on Indel Marker

目的 建立基于插入缺失(Indel)标记的常规聚合酶链反应(PCR)方法对太白贝母及其近缘种开展分子鉴定研究。方法 从GenBank下载103条来自不同贝母来源植物的叶绿体基因组序列,序列比对筛选太白贝母的特异性Indel标记。在该Indel标记的上下游保守区中设计引物,通过常规PCR与凝胶电泳方法对不同贝母来源植物开展鉴定,并考察方法的特异性、灵敏性与最适反应温度。结果 太白贝母在accD-psaI基因间隔区具有一段长度为137 bp的缺失,可以作为太白贝母的特异性Indel标记。建立的常规PCR与凝胶电泳方法对太白贝母有良好的特异性,仅有太白贝母产生302 bp的条带,从而与暗紫贝母等其余12种不同贝母来源植物明显区分。该方法对贝母DNA模板的检测下限为0.239 ng·μL^(-1),最适熔解温度(T_(m))值为58℃。结论 该检测方法具有特异性高、快捷与成本低等特点,不仅能够实现对太白贝母及其近缘种的准确鉴定,还能够为其他重要来源植物的分子鉴定提供开发思路。

OBJECTIVE To construct molecularidentification of Fritillaria taipaiensis and its relatives based on Indel marker.METHODS One hundred and three chloroplast genomes of 13 Fritillaria species were downloaded from GenBank and were subsequently used to screen Indel marker based on sequence alignment.Then,in accordance with the conserved upstream and downstream of Indel marker,a pair of specific primers was designed accordingly,by which a method including routine PCR and electrophoresis was constructed.Furthermore,the specificity,sensitivity,and optimal reaction temperature of the method were assessed.RESULTS One deletion of 137 bp was found in the accD-psaI spacer region of F.taipaiensis chloroplast genome according to the multiple sequence alignments,which was defined as the Indel marker for F.taipaiensis.The constructed method exhibited high specificity due to the fact that only F.taipaiensis exhibited a band of 302 bp after routine PCR and electrophoresis.The limit of detection for F.taipaiensis DNA template was 0.239 ng·μL~(-1),and the optimal melting temperature(T_m) value was 58 ℃.CONCLUSION This accurate,quick and cost-affordable method provides not only identification of F.taipaiensis and its related species,but also technical reference for identification of other traditional medicines.