Screening And Identification Of Sex-Specific DNA Fragments In Aristichthys Nobilis

Scientists have been intrested in the research of animal sex. Many heartening achievements have been made in mammals. Fishes play an important role in the evolution of vertebrate .They are also the most varied in species and most vast in distribution among vertebrate, possessing rich biological characteristics and significant economic value. Hence, it is of great significance to study the sex determination mechanism of fishes. The sex chromosome is in the primitive state of evolution in fish, so that it is hard to distinguish from shape between sex chromosome and autosome. At present there are only a small prorortion fish which have been found. It is difficult to detect the difference between male and female in fish genomics by current cytogenetic techniques. with the rapid development of biological molecular technology, sex-specific fragments were cloned by molecular genetic markers in several species,and laid a foundation for identifying the genetic gender in fishes.In the present work, Aristichthys nobilis, as representative anmial, was studied on some aspects of molecular cytogenetics by using RAPD,ISSR and Southern blot hybridization methods. Terch belong to Cypriniformes,Cyprinidae, hypopHthalmichthyinae, Aristichthys. The species is widely distributed in east of Asia and manly in basin and downstream of yangtse river. The terch, as one of the four freshwater aquaculture in china, is an important economic fish and have significant economic value in Cyprinidae. This study was the first looked for terch's sex-specific fragments by molecular genetic markers .In this study, terch from Henan institute zhandian-based aquaculture, as the experimental material,we selected 420 long-10bp random primers for the random amplified polymorpHic DNA-polymerase chain reaction (RAPD-PCR) and 77 piece of inter-simple sequence repeat primers for scaning with the genomic DNA from two sex individuals as template. When using the S1269(RAPD)and ISSR-65(ISSR) primer, a novel 600bp and 754bp sex-specific PCR product was amplified from known female fish. Then the two fragments of DNA were cloned and sequenced. According to the two fragments we have respectively designed a pair of primers, and did the PCR amplification with the template of male and female individuals genome, at the same time with the housekeeping geneβ-actin as positive control. The results indicated that the ISSR-65 was the female-specific primer but the S1269 not,which was in every individuals.The ISSR-65 PCR products in the gel were then transferred onto nylon membranes and hybridized with a DIG-labeled probe of the cloned female-specific DNA fragment. Clear hybridization signals were found only in all of the female terch. The same result was obtained from dot blotting hybridization. We have not found any homologous sequence by retrieval.These results show that we have successfully isolated a female-specific DNA fragment of terch. So that it can be used to rapidly and accurately identify the genetic gender of terch, and laid the foundation for studying the mechanism of chromosome evolution in fishes.