Study On DNA Barcoding Of Scolytidae Species Intercepted From Ports

Scolytidae insects, one group of the most important forest pests, are widely distributed, withmultiple varities and heavily destructive and are often intercepted from wood and woodpackaging materials in imports and exports. However, as many intercepted insects are in egg orjuvenile stage and sometimes mutilated, there are some difficulties and limitations inidentification by traditional morphology in plant quarantine. Therefore, new methods to solve theproblem is urgently needed.With molecular biological techniques widely developing in insect taxonomy, evolution andother aspects, DNA barcoding technique based on mitochondrial COⅠ gene has become a newtechnology in identification in recent years. To explore the application of DNA barcoding in themolecular identification of the Scolytidae insects intercepted in plant quarantine, genomic DNAextraction method, as well as DNA barcoding technique based on mitochondrial COⅠ gene andgenetic barcode library construction were studied, shown as following:1. Four different methods including classical phenol/chloroform extracting method,improved cetyltrimethyl ammonium bromide (CTAB) method, the sodium dodecyl sulfate (SDS)method and animal tissue/cell genomic DNA extraction kit (i.e. paramagnetic particle method)were used in genomic DNA extraction of bark beetles, before treated with photometric analysisand COⅠgene amplification. The results showed that four methods were effective in total DNAextraction. For the specimen soaked in75%ethanol for2years, these four methods were allapplicable. However, for specimen having been soaked in75%ethanol over two years, the DNAextraction was effective only by using the paramagnetic particle method. Moreover, theparamagnetic particle method was also applicable for the total DNA extraction from microscaletissue.2. By analyzing the amplified sequence, a pair of primers was specifically designed for theScolytidae insects amplification. Combining the primer with the universal primer, nested-PCRwas used in genomic DNA amplification.82bark beetles successful amplification indicated thatthe primer was versatile in the genome DNA amplification of the bark beetles.3. Researching on the Ips, Dendroctonus and Xylebrous insects, COⅠ gene of the sameposition fragment was amplified and the relation between the genetic distance and systemevolutionary were studied for molecular identificatin. The results showed that the intraspecificgenetic distance was lower than interspecies, the same species were in the same position of thephylogenetic tree, which verified the effectiveness of DNA barcoding in bark beetles molecularidentification.4. According to the new method of DNA barcoding for classification and identification,COⅠ gene of the intercepted bark beetles were amplified, gene barcode library was built from142bark beetles of82species,22genera in three subfamilies of Scolytidae, which providedtheory and data foundation for the molecular identification of bark beetles in plant quarantine.