| There are about 24,000 kinds of fish all over the world. Being the largest class of vertebrate animals, fish show variable biological characters and productive importance. Owning a long procedures and lots of streams of evolution, fish stand the specific situation and become ideal material to research the sex determination and differentiation of sex chromosomes of vertebrates.Among the fish which were cytogenetically studied, most of them do not possess any morphologically differentiated sex chromosomes. On the other hand, karyotypic analysis, chromosome binding, synaptonemal complex (SC) observation and comparative genomic hybridization (CGH) show in few fish species, that there exist sex chromosomes which differentiated at different levels and types. These sex chromosomes may represent the evolutional procedure of vertebrates. Therefore, the research on the fish sex chromosomes will supply important background knowledge for the exploration on the sex chromosomes evolutional history of higher vertebrates.Microdissection was brought in 80s of last century as a technique of microisolation of genetic material, and now become a normal analysis method in genetics and molecular cytogenetics, which successfully applying to genetic, evolutional and medicinal fields. Focusing on the sex chromosomes research and combined with FISH technique, a great deal of sex chromosomes libraries of plants, insects, birds and mammals were set up and gave lot of research clues. But because of the small size, high helix structure and difficultly banding of fish chromosomes, the application of microdissection was limited in fish species. Concerning microdissection on fish chromosome, few foreign reports exist and none is found in China.The spiny eel (Mastacembelus aculeatus), belonging to Perciformes, Osteichthyes, is a kind of little freshwater fish distributed in Southern China. In 1998, Our lab detected XX/XY differentiated sex chromosomes in this specie withkaryotypic analysis, and then with chromosomal binding and SC observation,we identified that its two largest metacentric chromosomes were X, Y sex chromosomes in 1999. X, Y chromosomes differ each other not only in the distribution of heterochromatin but also in their shapes, which make them could be recognized directly under the light-microscope without any banding treatment. Therefore, the sex chromosomes of spiny eel are ideal material for microdissection experiments. In present study, we microisolated several X chromosomes of spiny eel and constructed a X chromosome library. To investigate the homologue and differentiation of X and Y chromosomes of spiny eel, and to search their syntenies in other fish species, chromosomes paintings were carried out using the library probes. Furthermore, sex chromosomes specific highly repetitive sequence DNA clones of spiny eel were screened from the library too.Firstly, several whole X chromosomes were microdissected with microneedles under invert microscopy. The chromosomes were pretreated and then amplified by two rounds of DOP-PCR. The products showed a smear of DNA ranging from 200 to b800p, averaging 500bp, no emphasis amplification was detected.With T-Easy Vector?vector, electric transformation and E. coll bacterial, the sex chromosome library was constructed after the products of DOP-PCR were cloned. The library was proved to be specific to X chromosomes of spiny eel with Southern blot and FISH analysis. Every X chromosome producted about 2. 16 X 105 recombinant clones, with averaging inset fragments of 500bp, so the library included more than 98% DNA sequences of the spiny eel's X chromosome and could represent it well in FISH analysis too.To investigate the homologue between X and Y chromosomes of spiny eel, competitive FISH was performed on metaphase chromosomes of male and female individuals using the library probes. The similar hybridization signals indicated that X and Y chromosomes were homologous greatly. All the signals on X chromosomes are the same but different with those on Y. Moreover, all the signals on X a...
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