| RAPD (random amplified polymorphic DNA), which bases on the polymerase chain reaction (PCR), is by far one of the most commonly molecular techniques to uncover DNA sequence polymorphisms. The basic priciple of this technique is that an arbitrary primer (usually lObp oligonudetide) is used to amplify random segments of DNA, and a small number of fragments will be amplified when the primer anneals on each strand over a length range. If sequence variation is present at the priming site, then a fragment may not be amplied, so the DNA polymorphic can be detected. RAPD technique has the peculiarity such as simplicity, fastness, sensitivity, cheapness and lower requirement of materials, and more important, it can be used without prior knowledge of DNA sequence. The potential use of RAPD in phylogenetic studies and population genetics has been widely documented in a large variety of organisms.Cricket is a sort of insects that are famous for their songs. In taxonomy respect, they belong to Grylloidea, Orthoptera. Much research work has been done in its systematics, but the research of molecular systematics in Grylloidea is just at the beginning. In this paper, the approach of RAPD was used to study 57 species of crickets, which belong to 26 genera, 7 families, in order to discuss their taxonomy status and gentic relationships in molecular level, and enrich the research of molecular systematics in Grylloidea.The genome DNA of the 57 species were extracted by the method of Proteinase K isolation of total DNA. l-2ul (=25-50ng) of the DNA sample was used in per amplification reaction. PCR reactions were carried out in 25 ul reaction volume. 2.5 ul of 10 x reaction buffer, 1.5 ul 25mM Mgcl2, 0.3 ul lOmM dNTP, 0.5 ul TaqDNA polymerase (5 u/ul), and lul (=20ng) of primer were used in per reaction. Each reaction was overlaid with one drop of paraffin oil. The initial denaturation step was used at 94℃ for 1 min 45 sec; then denatured at 94℃ for 30 sec, annealed at 37℃1 min, extended atIVb72℃ for 2 min and repeated the cycle 45 times, at last, extended at 72'C for lOmin. In this research, 54 arbitrary primers, each with ten nucletides in length, were used. Thermocycled PCR samples were resolved electrophoretically on 1.5% agarose gels and taken photos using a Polaroid camera. The statistical results were analyzed by the SPSS software, and the cluster figures were obtained.Conclusions could be drawn from the study as following:1) The molecular systematics of 57 species of crickets, which belong to 26 genera 7 family in Grylloidea, had been studied by the approach of RAPD. Among 54 arbitrary primers tested, only 9 primers could generate clear and reproducible fragments in 32 species belonging to 12 genera of the family Gryllidae; 9 primers in 15 species belonging to 7 genera of the family Trigonidiidae; 12 primers in 3 species belonging to the genus Oecanthus of the family Oecanthidae; 12 primers in 4 species belonging to 3 genera of the family Eneopteridae; 13 primers in 1 species belonging to the genus Homoeogryllus of the family Phalangopsidae; 13 primers in 1 species belonging to the genus Sclerogryllus of the family Sclerogryllidae; and 13 primers in 1 species belonging to the genus Eulandrevus of the family Gryllomorphidae. Only 7 primers, which are S8. S83, S142, S50, S55, S61, S379, generated clear and reproducible fragments in all 7 families of Grylloidea. And 4 primers S8, S83, S142, S55 could be applied to differentiate the sexes of crickets.2) The method of Proteinase K isolation and the method of CTAB isolation had been compared carefully while extracting the genome DNA from crickets. The result showed the method of Proteinase K isolation of total DNA was more suitable for the extraction of total DNA from insects of Grylloidea.3) Amplified DNA fragments indicated sexual and individual differences of the same species, but these differences were obviously smaller than those among species.4) The RAPD technique could be used to differentiate the sexes of species.5) RAPD was a good g...
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