DNA条形码试剂盒检测技术在大小蠹属种类鉴定中的应用

Application of DNA barcoding identification kit to identify bark beetles of the genus Dendroctonus Erichson（Coleoptera： Curculionidae： Scolytinae）

【目的】DNA条形码技术已成为生物分类鉴定的有力工具。DNA条形码技术的相关问题,如物种种内和种间的遗传距离出现重叠区域,将直接影响到物种鉴定的准确性。我们应用DNA条形码试剂盒检测技术来快速、准确地鉴定口岸截获的检疫性大小蠹属种类。【方法】针对大小蠹昆虫设计引物以提高PCR扩增效率。运用自主研发的基因条码分析软件找出基因片段上区分每个物种的多态位点规律,作为该物种的鉴定特征并建立数据库,应用于物种鉴定。【结果】使用针对大小蠹属昆虫设计的引物成功扩增出325 bp的COI基因片段。将大小蠹属12种昆虫的COI基因片段上的核苷酸诊断位点的组合作为物种的鉴定特征,可以准确地区分近似种。通过比对植物检疫鉴定系统数据库里的鉴定特征,将6个大小蠹属的未知样品成功鉴定到种（核苷酸序列一致性为100%）,与形态鉴定结果一致。【结论】结果表明DNA条形码试剂盒检测技术可以准确鉴定大小蠹属的种类。该检测技术可以应用于其他经济重要性有害生物的检测鉴定。

【Aim】DNA barcoding has become a powerful tool in taxonomic identification. However,potential issues associated with DNA barcoding, such as overlap of genetic distances between intraspecific and interspecific variation,would directly affect the accuracy of identification. To accurately identify economically important Dendroctonus spp. intercepted at ports of entry,we employed a suite of nucleotide diagnostic sites as the identification characteristics（ ICs）. Each species is represented by a unique IC. 【Methods】We designed genus-specific primers to improve amplification efficiency in Dendroctonus. Web-based software Gene Barcoding Analysis Software（ GBAS） was designed to extract the IC from molecular sequences and a database was built specifically for species identification. Six unidentified specimens of Dendroctonus were also sampled to validate our IC approach. 【Results】We obtained 325 bp of COI gene fragment（ position 889- 1 213 bp on the COI gene） using genus-specific primers. A combination of nucleotide diagnostic sites from the mitochondrial COI region of 12 species of Dendroctonus provides the IC,which is unique for each species,and thus can be used to differentiate closely related species. The ICs of six unidentified samples of Dendroctonus were compared to those of all invasive insects stored in the Plant Quarantine Organisms Identification System. The nucleotide sequence identity between the query sequence of Dendroctonus and IC database is 100% in all cases.【Conclusion】The results demonstrated that the IC approach can accurately identify the species of Dendroctonus. Our approach may be extended to barcode other economically important organisms and identify unknown samples.