Abstract: 【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.  

Key words: Bark beetle, invasive species, DNA barcoding, COI, identification characteristics, GBAS