Construction And Characterization Of A BAC Library For Carassius Auratus Gibelio

A genomic BAC library was constructed for Carassius auratus gibelio. The library contains 79 104 clones. A total of 160 BAC clones were randomly sampled, and the extracted DNAs were digested with NotI and separated by pulse-field gel electrophoresis on 1% agarose gels. 95% of the inserts range from 80 kb to 194 kb and 73% of the inserts are more than 100kb. The average insert size is about 104kb. The haploid genome size of gibel carp is estimated to be about 1600 Mb based on previous study by flow cytometry. Based on the clone number, the average insert size and the genome size, the gibel carp BAC library is calculated to represent approximately 5-fold haploid genome equivalent. The stability of the library was also assayed. Six BAC clones with insert size larger than 100 kb were sampled from the BAC library, and assayed by serial culture of more than 100 generations for 5 days. Each passage was considered to represent about 20 generations. Each day a sample was taken for clone stability analysis by NotI digestion and by pulse-field gel electrophoresis. The electrophoretic patterns revealed that no apparent rearrangements were observed and the gibel carp inserts in the BAC clones were stable in the vector for long culture. Subsequently, PCR-based screening BAC library was carried out using gene-specific primers. Several genes that play crucial role during oogenesis and embryogenesis were screened from the BAC library. These genes include C-lectin, 138, vasa, DAZL, sox3, interferon, and others.