BAC Libraries Construction And Genomic Analysis In Zhikong Scallop (Chlamys Farreri)

Zhikong scallop (Chlamys farreri) is one of the most important economicaquiculture bivalves in China, but study on its genome is underdeveloped. In thisstudy, we constructed three bacterial artificial chromosome (BAC) libraries ofC.farreri as platform for genomic research, and harvested positive BAC clonescontaining target genes, which are relevant to growth and immune function ofC.farreri. Additionally, we performed genome sequencing and comparative analysisof the C.farreri using next-generation sequencing technology, and thus made apreliminary assessment of the genome.1. Construction and characterization of BAC libraries in C.farreriWe constructed three bacterial artificial chromosome (BAC) libraries forC.farreri. The BAC libraries were generated based on three restriction enzymes,BamHI, HindIII and Sau3AI, respectively. The BamHI library consists of34,560clones, with an average insert size of98kb and no insert-empty clones. The HindIIIlibrary consists of132,480clones, with an average size of106kb, but approximately1.67%of the clones don’t contain scallop nuclear DNA inserts. The Sau3AI libraryconsists of23,040clones, with an average size of53kb, approximately3.33%of theclones don’t contain DNA inserts. The combined libraries collectively contain a totalof190,080BAC clones with an average size of98kb, representing15.4x haploidgenome equivalents. BAC stability assays show that clones are stable in the bacterialhost during propagation. Libraries were screened with ten probes designed from DNAsequences of three genes and seven microsatellite markers. Positive clones wereidentified for each gene or microsatellite marker. These results suggest that the threescallop BAC libraries will serve as an invaluable resource for genetic studies onZhikong scallop such as gene cloning, physical mapping and even large-scale sequencing of the genome.2. Application of C.farreri BAC librariesWe harvested positive BAC clones containing8candidate genes and17SNP sites,which are relevant to growth and immune function of C.farreri through4-dimensionalPCR screening system. Each gene or SNP gives three positive clones, and none ofthose was absent from the libraries. As a result,75positive BAC clones wereidentified, further verifying the genome coverage and utility of the library. Theseclones provide resources essential for research of molecular mechanismes underlyingthe growth and immune of C.farreri. Additionally, BAC clones containing fiveToll-like receptor (TLR) signaling pathway genes, including CfTLR, CfMyd88,CfTRAF6, CfNFκB and CfIκB, were physically mapped using fluorescence in situhybridisation (FISH) to C. farreri’s chromosomes. These BACs represented singlesignals respectively, and they were located on five non-homologous chromosomepairs through karyotypic analysis and cohybridization. The FISH mapping of thesekey immune genes in the Zhikong scallop will aid in the research of innate immunity,and assignment of interested genes to chromosomes.3. Survey sequencing and analysis of the C.farreri genomeUsing next-generation sequencing technology, we have successfully sequencedand de novo assembled a draft sequence of C.farreri. Illumina sequencing generated62.44Gb high quality reads, which covered the genome at50fold. The preliminaryassembly comprised870Mb, with a contig N50of1,267bp and a scaffold N50of1,517bp. The17-mer analysis suggested that the scallop genome was971Mb, andindicated high heterozygosity. The assembled scaffolds covered about57.1%of thewhole genome, and covered about88.7%of the transcriptome. Using RepeatMaskerto analyze all scaffold sequences, a total of884,644interspersed repetitive sequenceswere found, which were classified into4major types: DNA transposon, SINEretrotransposon, LINE retrotransposon and LTR retrotransposon. Among them, DNAtransposon was the most frequent type. SciRoKo analysis showed that a total of134,887simple sequence repeats (SSR) were found, among which monomeric repeat motifs were most abundant. In all, survey on the characterization of whole genomesequences in C.farreri will provide a useful resource for understanding the genomestructure, and allowing further evolutionary and biological complexity analysisof scallops.