ISSR-PCR And Heredity Analysis Among Three Demodex Mites

Objective To optimize conditions and screen primers for inter-simple sequence repeat-anchored (ISSR-PCR) of Demodex, using genome DNA of Demodex caprae (D.c.) as the template. The best annealing temperature of each of screened primers is selected by gradient polymerase chain reaction (PCR). ISSR-PCR amplifications are conducted using genomic DNA of Demodex folliculorum(D.f.), Demodex brevis (D.b.) and D.c., which prepare for the genetic diversity research of Demodex with ISSR mark. The results of genetic diversity and molecular genetics classification of D.f., D.b. and D.c. come from the ISSR-PCR amplifications.Methods Collected enough D.f. and D.b. using apparatus made by ourselves. Got D.c. from the goat’s skin nodules. Under the dissecting microscope, the mites were cleansed and isolated from the sebum with a special needle made by ourselves, and then grinded them with grindingrod. The genome DNA of D.f. and D.b. were extracted with a kit for trace genomic DNA samplet, the genome DNA of D.c. were extracted with Ezup pillar type animal genome a kit, then screened primers were carried out using the DNA of D.c. as the template and90items as the primer that were designed by Canada British Columbia University. Six primers were selected, which produced clear bands, good polymorphism and high repetition. Finally, the best annealing temperature of each of screened primers is selected by gradient PCR.ISSR-PCR was carried out respetyely plate, using selected primers and genome DNA of D.f., D.b. and Dc as a template.the results are shown using agar-agar gel electrophoresis, the staistical analysis of data of experiment were carried out.ted primers and genome DNA ofResults The plenty of D.f., D.b. and D.c. was collected. The pretreatmented method of the demodex mites was improved. Genome DNA of D.f., D.b. and D.c were extracted respectively with kits. After an optimal reaction system of ISSR-PCR D.c. was established,6optimal primer was screened out and then the ISSR-PCR amplifications of D.f., D.b. and D.c. were carried out. The results showed that106bands of ISSR markers were amplified with6suitable primers, and the length of DNA fragment were ranged from250to2000bp, and83bands were polymorphic, and the percentage of polymorphism bands reached78%. Genetic similarity coefficient of D.b. and D.c., D.f.and D.b, D.f. and D.c were respectively0.335、0.298、0.208. The genetic distance of them (GD) was respectively0.665、0.702and0.792. The result shown that there was the closest relationship between D.b. and D.c., that of D.f and D.b. was median, the relationship of D.b.and D.f is the farthest.Conclusion Genomic DNA of D.f., D.b. and D.c. were extracted. The optimal conditions of ISSR-PCR and primers of genome DNA of Demodex were screened. The relationships among three demodex mites were analyzed with the results of ISSR-PCR. The result and experiment skill are the basis of farther molecular biological research of demodex mites.