| Orthoptera which has attracted much attention by most researchers is the largest group in the incomplete metamorphosis insect, with more than 23000 known species world wide and more than 1500 species found in China. Traditional taxonomy is mainly based on orthopteroid morphological characters, which shows certain shortage. With the rapid development in molecular biotechnique, some DNA-Based technology has been increasingly used for species identification. In order to meet the requirement of rapid assessment and inventory of the global biodiversity, some scientists propose that it is possible to use a short fragment of DNA sequence from a given region of the genome as the specific species tag, i.e. DNA barcode, which is a newest species identification technology. DNA microarrays as a recent development technology with fast and high throughput characteristic, have become crucial and effective tools in species diagnostic applications.The present work has made a preliminary study on Acridoidea and Tettigonioidea species of China by DNA barcoding and phylochips using self-sequenced 658bp standardized barcode sequence of mitochondrial cytochrome c oxidase subunitâ… gene (COâ… ) and related sequences from GenBank. A total of 300 species and 643 individuals belonging to 7 families in Acridoidea and 160 species and 548 individuals belonging to 9 families in Tettigonioidea were studied. Intra- and interspecific pairwise Kimura-2-parameter distance were analyzed in detail, and the threshold value for species identification was established. All samples were performed for NJ tree cluster analysis and Blast analysis. Based on all barcode sequences,303 genus specific or species specific probes were designed for virtual hybrid to identify species. The main results of the present study are as follows:1. COâ… barcodes is suitable to molecular identification of Orthoptera at the species level. Intraspecific genetic distances variation levels in Acridoidea are 0-2.96%. An average of genetic distances within intraspecific are ranging from 0.18% to 1.31%, which is obviously less than 3%, the divergence of interspecific comparisons are 4.09-20.69%. Interspecific variation levels in Tettigonioidea is 0-2.65%, An average of genetic distances within intraspecific are ranging from 0 to 1.54%. Interspecific comparisons divergence are 4.90-23.90%. It's rational and feasiblel to set 3% as the DNA barcodes threshold value to distinguish Orthoptera species.2. CO I-based BLAST search is a better molecular species identification approach than tree-based ones. Although NJ tree cluster analysis could effectively distinguish a single morphological species, tree-based method shows limitation in calculating procedure. In this case, CO I-based BLAST search is a fine molecular species identification approach than tree-based ones.3. The probability of coamplification of nuclear mitochondrial pseudogenes (numts) is very low in Orthoptera using self-designed primer pair.4.685bp-length CO I barcodes sequence is not suitable for designing specific probes in Orthoptera. In this study,97 (about 61%) species of 160 species in Tettigonioidea and 168 (56%) species of 300 species in Acridoidea could be selected for specific probes. Virtual hybrid results show that 71 (about 73%) target species and 86 (about 51%) target species could be indentified in Tettigonioidea and Acridoidea, respectively. In a word, COI barcodes sequence is unsuitable for developing phylochip to identify Orthoptera species.
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