[Advanced methods of preparing pachytene bivalents and high resolution multiple bands of zebrafish (Danio rerio)]

The pachytene bivalents with high-resolution multiple G bands of zebrafish were obtained after the treatment with alkaline hypotonic solution and high concentration of chloroform fixative solution. When comparing six group chromosomes from different pachytene specimens, the characteristic and the number of bands were well matched. In order to systematize this technique and get stable result, we summarize the preparation procedure of the zebrafish bivalents. The 6-month-old to one-year-old zebrafish whose spermary appears ivory-white and opaque, is good material. The whole testis should be treated with hypotonic solution for 1.5 approximately 2 h at room temperature. Then, the testes were fixed for 20 min in high chloroform fixative solution (chloroform: methanol: acetic acid, 3: 6:1), and fixed in Carnoy's solution (methanol: acetic acid, 3:1) for two times. In addition, with the treatment of restriction endonuclease Alu I directed in situ nick translation, we successfully obtained well-resolved restrictive endonuclease banding of zebrafish bivalents, which was considered as G-like band patterns. The aging of the specimen is also important factor, should let them dry at room temperature for one week. The application of these methods in cytogenetics research of zebrafish and other fish can be expected. Construction of the steady technique system to prepare high resolution banding bivalents and idiogram of zebrafish is the basement to found stable and accurate framework for physical map.