Abstract:Species identification plays a key role in the protection of biodiversity. However, there have been great challenges in traditional morphological identification because of its innate limitations and the lack of experienced techniques. Recently thanks to the development of molecular biology, DNA-based identification is becoming a more accurate alternative method. The cytochrome oxidase I (COI) barcode is a short piece of DNA from a standard region of the mitochondrial genome, which can be used to identify and classify different species and has been proved to be highly effective in many species including fish. The advantage of COI is that it is long enough to identify variations among species yet short enough to be sequenced quickly at low cost. In this study, we analyzed DNA barcoding of Engraulidae species in order to explore the feasibility of DNA microarray technology in species identification. Oligonucleotide probes were designed based on the COI sequences of mitochondrial obtained from 101 individuals of 30 fish species in 11 genera. Forty-six specific probes obtained from 24 species were selected with online software Oligo Array 2.1, among which the probes potentially forming hairpin, stem-loop, or homo-dimers were identified with Oligonucleotide Properties Calculator and excluded. The remaining probes were virtually hybridized with the target sequences in Oligo Heat Map (OHM), and subsequently a total of 26 probes from 16 species bound to the target sequences specifically. The DNA microarray technology identified 14 species, accounting for 46.7% of the total with 100% accuracy. Therefore, DNA microarray based on COI sequences should have practical values in the identification of Engraulidae fish species. Nevertheless, this technique needs further improvement and the key step would be screening and optimization of high quality molecular probes.
