From RFLP, specific primer to DNA barcoding: preliminary study on molecular identification of common stored product psocid

Abstract

Stored product psocids (Insecta: Psocoptera) is one kind of common storage insect pests in the world and is difficult to be identified morphologically. The molecular identification methods, from Restriction Fragment Length Polymorphism (RFLP), specific primer, to DNA Barcoding, were studied gradually in this paper with international collaboration and based on sequence of 16S rDNA gene of the common species of stored psocids such as Liposcelis. bostrychophila, L. brunnea, L. corrodens, L. decolor, L. entomophila, L. mendax, L. paeta, L. pearmani, L. rufa, and L. tricolor. The samples of different geographical populations in this study were from Peoples Republic of China, Czech Republic, United States of America, Croatia, Portugal and Denmark respectively. The result presented that it was successful to accomplish the sequencing of 16S rDNA gene from single individual of above psocids with one pair of primers (16Sar and 16Sbr) and our methods of DNA extraction, Polymerase Chain Reaction (PCR) reaction mix and condition. Twenty-two sequences were submitted to GenBank and the accession numbers were allocated (Table 1). According to the related sequences, RFLP method was studied firstly and one restrictive endonuclease (DraI) was selected to discriminate the most common four species such as L. bostrychophila, L. entomophila, L. decolor, and L. paeta. The method of specific primer was researched in order to determine L. corrodens which species had not occurred in China. Two pairs of specific primers were designed and selected for the identification of L. corrodens with specific bands from other species of common stored psocids. As an applied example, the samples of Liposcelis captured from imported graze seeds of Denmark by Chinese plant quarantine agency were identified with method of DNA Barcoding. The results showed that Denmark sample shared 98.94% sequence similarity with L. corrodens, and the maximum-likelihood (ML), neighbor-joining (NJ), and maximum parsimonious (MP) phylogenetic analysis indicated that Denmark sample and L. corrodens were in the same subgroup in the phylogenetic relationship tree. For further development, we are researching the DNA Barcoding with mtDNA COI (mitochondrial cytochrome oxidase I) gene of stored psocids as the prototype of other stored pests. More collaboration from the world is regarded as the key point for the successful research of molecular identification of stored insect pests.