Fosmid Library Construction And Genomic Structure Analysis In Zhikong Scallop (Chlamys Farreri)

Zhikong scallop (Chlamys farreri) is one of the most commercially important bivalves in China, but study on its genome is underdeveloped. In this study, we constructed the first Zhikong scallop fosmid library, and analyzed the fosmid end sequences to provide a preliminary assessment of the genome. Additionally, chromosome assignment of some fosmid clones has contributed to the identification of 8 chromosomes. The major results are as follows:1. Construction and characterization of fosmid library in Zhikong scallopThe first Zhikong scallop fosmid library was constructed in this study. It consists of 133,851 clones with an average insert size of about 40 kb, amounting to 4.3 genome equivalents. Fosmid stability assays indicate that Zhikong scallop DNA was stable during propagation in the fosmid system. Library screening with two genes and seven microsatellite markers yielded between 2 and 8 positive clones, and none of those tested was absent from the library. The fosmid library will serve as a useful resource for physical mapping and positional cloning, and provide a better understanding of Zhikong scallop genome.2. Analysis of fosmid end sequences in Zhikong scallopIn the present study, 2,016 individual clones were sequenced, resulting in 3,646 sequences. A total of 2,286,986 bp of genomic sequences was generated, representing approximately 1.84‰of the Zhikong scallop genome. Using Tandem Repeats Finder (TRF) software to analyze the sequences, a total of 2,500 tandem repeats were found, including 371 microsatellites, 1,816 minisatellites and 313 satellites, accounting for 14.84%, 72.64% and 12.52% of all tandem repeats, respectively. The total length of tandem repeats was 552,558 bp, in which the length of microsatellites, minisatellites and satellites was 9,425 bp, 336,001 bp and 207,132 bp, accounting for 1.71%, 60.81% and 37.49% of the total length of tandem repeats, and 0.41%, 14.69% and 9.06% of the total length, respectively. RepeatMasker analysis showed that a total of 317 interspersed repetitive sequences were found, which account for 4.26% of the total length. There are 4 types in these sequences: DNA transposons, LTR retrotransposons, LINE retrotransposons and rolling circles. Among them, LINE retrotransposons is the most frequent, followed by LTR retrotransposons, DNA transposons and rolling circles. As to the length abundance, LTR retrotransposons rank first, followed by LINE retrotransposons, DNA transposons and rolling circles. Therefore, the percentage of retrotransposons is much higher than that of DNA transposons in Zhikong scallop.A total of 1,383 sequences were found to significantly hit to sequences in GenBank database using BLASTN and BLASTX (E < e-5). BLASTN searches on the nr and EST databases of GenBank and BLASTX searches on the nr database resulted 113, 923 and 347 significant hits, respectively.In all, survey on the characterization of all the end sequences in Zhikong scallop will provide a useful resource for physical mapping and better utilization of the existing genomic information.3. Chromosome assignment of fosmid clones in Zhikong scallopBased on the analysis of end sequences and PCR screening of the fosmid library, we selected 12 clones for FISH. Of the 8 clones which were likely to be repetitive sequences, 4 represent single locus and the other 4 showed signals on several or all chromosomes. Hybridization with the 4 clones which were likely to be low or single copy sequences was then attempted. They showed high background when tested without C0t-1 DNA, but the use of C0t-1 DNA contributed to the successful suppression of background signals from repetitive sequences within 3 clones. In all, 11 of the 12 investigated clones were successfully assigned to chromosomes finally. Except for the 3 clones which were unsuitable for chromosome identification, the remaining 8 clones realized the identification of 8 chromosomes. This will undoubtedly be useful for many aspects of cytogenetic research in Zhikong scallop including chromosomal rearrangements, chromosomal assignment of genes and development of chromosome-specific libraries and markers.