CONSTRUCTION OF A BAC-FISH EXPERIMENTAL SYSTEM FOR LOCALIZING SPECIFIC SEQUENCES ON MIRROR CARP CHROMOSOMES

An experimental system has been constructed for localizing specific genomic sequences on mirror carp (Cyprinus carpio var. specularis) chromosomes. In order to obtain individual BAC clones containing the target sequences, short genomic sequences were amplified by PCR in bacterial artificial chromosome (BAC) screening pools. BAC plasmid DNA was isolated and labeled by nick translation to prepare fluorescence in situ hybridization (FISH) probes. Via optimizing a series of protocols for chromosome slides pre-treatment, BAC plasmid probe preparation, repetitive sequences blocking by C0t-1 DNA, pre-hybridization, candidate fluorescence dyes evaluation, signal amplification and so on, the BAC-FISH system has been successfully established for mirror carp metaphase chromosomes, which is able to localize both single-copy markers and repetitive sequences. As supporting evidence to the genome duplication hypothesis of carp evolution, two microsatellite markers from zebrafish chromosome 17, namely Z6884 and Z4268, have been localized on two separate mirror carp chromosomes, with one marker on each chromosome. A male-specific marker CCmf1 from Yellow River carp has been localized on at least four chromosomes of mirror carp, suggesting that it might be a repetitive sequence useful for sex determining gene searching. The established mirror carp BAC-FISH experimental system would serve as a powerful tool for carp cytogenetic mapping, genome evolution and comparative genomics research.