Fosmid Library Construction And Initial Analysis In Female Half-smooth Tongue Sole (Cynoglossus Semilaevis)

Half-smooth tongue sole (Cynoglossus semilaevis) is an important marine flatfish in north China and its aquaculture industry has been booming quickly in recent years. However, the research on its genome is underdeveloped compared with the study of its culture and breeding technology. Genomic studies require the availability of large-insert genomic libraries, such as cosmid, fosmid, bacterial artificial chromosome (BAC), and yeast artificial chromosome (YAC) libraries. The fosmid system developed in 1992 has some significant advantages. First, fosmid clones are easier to handle than BAC and YAC for their smaller insert size. Second, the insert fragments were prepared by random shearing without specific restriction endonuclease digestion and thus there is no potential bias of insert DNA. Third, the fosmid vector allows maintenance and storage as single copy for better stability of cloned inserts while the copy number also can be induced to 10～50 copies per cell to improve DNA yields for applications. In this thesis, the first female Half-smooth tongue sole fosmid library construction and initial Analysis has been presented.The major results are as follows:1. Construction and characterization of female Half-smooth tongue sole fosmid libraryA female half-smooth tongue sole fosmid library has been constructed in this study. The DNA content of female half-smooth tongue sole blood cell has been determined to be 0.620±0.044pg via the flow cytometry method and the chicken (Gallus domesticus) erythrocyte (1C = 1.25 pg) was selected as the internal reference standard. The genome size of female half-smooth tongue sole was then calculated to be about 606.36 Mb. The genomic DNA was prepared from different tissues using different methods and the ones prepared from muscle via the phenol-chloroform procedure showed the best fragment size and DNA purity. The prepared DNA was end-repaired and then the size selected fragments which lager than 36kb were recovered from low melting point gel using different agarose enzyme. The ones using GelaseTM appeared to have better result. The collected fragments were ligated into the fosmid vectors and then packaged to construct the library. The final fosmid library consists of 49,920 clones with an average insert size of 39.2kb kb, amounting to 3.23 genome equivalents. Insert fragments assay showed 3 of 96 clones selected randomly have no insert DNA and the frequency of recombinant clones was calculated to be 96.88%.The reason for lower recombinant frequency might be cell contamination. The insert DNA may have restriction enzymes sites which resulted in 8 of the random 96 clones showed two DNA fragments after NotI digestion. The probability of recovering any specific sequence of interest was 95.6% and the fosmid stability assays indicated that inserted DNA was stable during propagation. The fosmid library will serve as a useful resource for end sequences, physical mapping, and positional cloning of half-smooth tongue sole. It provides a base for further genetic analysis in this species and will be important for a better understanding of the genome.2. Analysis of fosmid end sequencesIn the present study, 1,152 individual clones have been sequenced, resulting in 2,247sequences with the total of 1,921,341 bp. These 2,247sequences represent approximately 3.17‰of the female Half-smooth tongue sole fosmid library and 1,383 sequences in them has been found to significantly hit to sequences in GenBank database using BLASTN and BLASTX (E < e-5). BLASTN searches on the nr databases, EST databases and BLASTX searches on the nr database resulted 232, 232 and 384 significant hits, respectively.3. Isolation and characterization of microsatellite markers from female half-smooth tongue sole fosmid libraryThree hundred and eighty-four fosmid clones were randomly selected to sequence (double strand reading), and 168 sequences in one hundred and forty-three clones were found to contain microsatellites. Among 101 primer pairs which were designed, 64 gave polymorphic PCR products. Based on characterization with 36 individuals, the number of alleles ranged from two to nine. The values of observed and expected heterozygosities varied from 0.06 to 1.00 and from 0.05 to 0.94, respectively. These markers have the potential for population structure evaluation, ecological analyses and linkage map construction. 4. Isolation of sex-related sequence from female half-smooth tongue sole fosmid libraryThe sequence results of 384 fosmid clones were radomly selected to design primer pairs with PRIMER PREMIER version 5.0 and 374 pairs were finally produced. Among theses 374 pairs, 1 pairs amplifyed sex-related sequence after a series of PCR screens by using female DNA pool and male DNA pool as templates. This primer pair came from fosmid clone B169-47.The lenth of sex specific sequence is 291bp.BLASTN searches on the nr databases, EST databases and BLASTX searches on the nr database had no significant hit of this sequence.