| Mermithidae is a group of insect's parasitic nematode with diversified morphological characters and host specificity, and it is a valuable biological control resource. The phylogenetic relationship among different genera or species is still not clear, studying their genetic variation and phylogenetic evolution is very important for the applications of mermithidae.The method of single-enzyme amplified fragment length polymorphism (SAFLP) was optimized for Mermithidae in this study. The genetic relationships among eleven species or subspecies belonging to six genera of Mermithidae were detected using this method. Tow kinds of restriction endonucleases were used in this experiment, the result showed that EcoR I is more suitable than Mse I in SAFLP for Mermithidae. 225 bands (form 250 to 1650 bp) were amplified by three EcoR I primers with three selective bases in each primer. Nei' similarity coefficients and genetic distance (0.1980-0.4554) were calculated by NTsys-PC2.1 software and the phylogenetic tree was constructed with the method of UPGMA. The relationship indicated the eleven species or subspecies of Mermithidae could be distinguished with SAFLP markers and were grouped into two distinct clusters. Two major groupings of genera are observed in the phylogenetic tree: Romanomermis+Octomyomermis; ((Hexamermis + Ovomermis) Agamermis) Amphimermis.Comparatively, the genetic distance of different species or subspecies, which obtained from closer area in same genera, is smaller in Octomyomermis and Amphimermis. In the genus of Octomyomermis: Octomyomermis sp.A + (O. huazhongensis + Octomyomermis sp.C) , and in the genus of Amphimermis: (Amphimermis sp.B + Amphimermis sp.A) + Amphimermis sp.C. The result of this experiment is similar to the one of morphologic taxonomy system, and provides evident to the studies of phylogenetic relationship between species or genera of Mermithidae. In a conclusion, the result suggests that this optimized SAFLP was suitable for analysis of phylogenetic relationship and taxonomy among species of Mermithidae.The study of nematode mitochondrial DNA is important for the evolution of mitochondrial genome, phylogenetic development, species identification, molecular taxonomy of nematode, the study of the disease correlated to the mutation of mitochondnal genome and so on. There are 6 mermithidae (Romanomermis culicivorax, R. iyengari, R. nielseni, Thaumamermis cosgrovci, Hexamermis agrotis, Agamermis sp.) mitochondrial complete genome are clear recently. But there is not any study of mermithidae mitochondrial complete genome in china. A long-PCR amplification approach for sequencing of the entire mitochondrial genome for individual nematode was described for the first time in china. The approach was based on the special characters of mermithidae mitochondrial genome such as the variety of gene arrangement, repeating sequences and reduplicates genes, and diversity among individuals, and was suitable for amplifying the mitochondrial DNA of Ovomermis sinensis.A pair of primers was designed based on the sequences of nematodes obtained from NCBI and amplified a 4.4 Kb fragment of Ovomermis sinensis mitochondrial DNA. The fragment was sequenced and the cytochrome b (cob) gene of Ovomermis sinensis was obtained. Another pair of primers was designed based on the sequence of cob gene and an about 23Kb fragment was amplified by these primers. There are overlap area between the two fragments. The long-PCR approach require pure and integrity DNA templet. The variety of gene arrangement, repeating sequences and reduplicates genes of nematode mitochondrial genome should be advisemented when the long-PCR primers were designed. The approach described in this paper gives a key for the mermithidae mitochondrial complete genome study in our lab. |
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