Abstract: 【Aim】 Mealybugs are one of the most important quarantine pests that injury a variety of tropical fruits and vegetables worldwide. Usually, mealybugs intercepted in the quarantine inspection include eggs, nymphs or debris, and there exist several cryptic species and small difference in related species, which make them difficult to be identified by traditional morphological identification methods correctly and timely. In this study, the validity of DNA barcoding in identification of Planococcus minor (Maskell), a potential major alien invasive species to China, was evaluated. 【Methods】 The 5′- and 3′-end of the mitochondrial cytochrome c oxidase subunit I (COI) gene and 28S ribosomal DNA (rDNA) of 36 individuals of Pl. minor intercepted during passenger inspection were amplified using universal primers, and Pl. citri (Risso), a related species of Pl. minor, from Langfang, Hebei used as a reference. The obtained partial fragment of 5′- and 3′-end of COI gene and D2-D3 fragment of 28S rDNA were sequenced. The phylogenetic tree was established by using a maximum likelihood (ML) method. The intra-and inter-species genetic distances were calculated using the Kimura-2-Parameter model. Meanwhile, the validity of Pl. minor identification based on the above three gene fragments was tested with SpeciesIdentifier software. 【Results】 When the 5′- and 3′-end of COI gene of Pl. minor were sequenced and BLAST, respectively, the nucleotide sequence identity between the fragments from GenBank and our study is 100% and 99%-100%, respectively. The nucleotide sequence identity of the 5′- and 3′-end of COI gene between Pl. minor and Pl. citri is 97%-98% and 96%-98%, respectively. There are 5 and 11 stable species-specific identification sites in the fragments of the 5′- and 3′-end of COI gene for Pl. minor and Pl. citri, respectively. Phylogenetic analysis indicated that Pl. minor intercepted during passenger inspection and those from the NCBI clustered in a clade. The sequencing results showed that the D2-D3 fragment of 28S rDNA among species belonging to the genus Planococcus has high conservation and that it could not be used to distinguish between Pl. minor and Pl. citri. The genetic distance between the two species is 0.004. The results based on the software of SpeciesIdentifier showed that the species identification based on 5′- and 3′-end of COI gene was completely correct, while that based on D2-D3 fragment of 28S rDNA resulted in 45.2%-61.9% of fuzzy identification. 【Conclusion】 Our results suggest that DNA barcoding technique based on 5′- and 3′-end of COI gene can be used to identify and detect Pl. minor rapidly and accurately. The technique will be significant in intercepting and blocking the further spreading of Pl. minor.      

Key words: Key words: Planococcus minor; Planococcus citri, DNA barcoding, COI gene, 28S rDNA, related species