Construction And Characterization Of Bacterial Artificial Chromosome Libraries For Zhikong Scallop, Chlamys Farreri And Pacific White Shrimp, Litopenaeus Vannamei

Large-insert bacterial artificial chromosome (BAC) libraries are necessary for advanced genetics and genomics research. To facilitate gene cloning and characterization, genome analysis and physical mapping of Zhikong Scallop, Chlamys farreri Jones et Preston and Pacific white shrimp, Litopenaeus vannamei, two BAC libraries were constructed from nuclear DNA of the two species, respectively. The scallop BAC libraries were constructed in the BamHI and MboI sites of the vector pECBAC1, respectively. The BamHI library consists of 73,728 clones, and approximately 99 % of the clones contain scallop nuclear DNA inserts with an average size of 110 kb, covering 8.0 x haploid genome equivalents. Similarly, the MboI library consists of 7,680 clones, with an average insert size of 145 kb and no insert-empty clones, thus providing a genome coverage of 1.1 x. The combined libraries collectively contain a total of 81,408 BAC clones arrayed in 212 384-well microtiter plates, representing 9.1 x haploid genome equivalents and having a probability of greater than 99% of discovering at least one positive clone with a single-copy sequence. High-density clone filters prepared from a subset of the two libraries were screened with nine pairs of Overgos designed from the cDNA or DNA sequences of six genes involved in the innate immune system of mollusks. Positive clones were identified for every gene, with an average of 7.3 BAC clones per gene probe. These results suggest that the two scallop BAC libraries provide useful tools for gene cloning, genome physical mapping and large-scale sequencing in the species.The shrimp BAC libraries were constructed in the HindIII site of the vectors pECBAC1 and pCLD04541, respectively. The combined libraries consists of 102,528 clones arrayed in 267 384-well microtiter plates, and approximately 95 % of the clones contain shrimp nuclear DNA inserts with an average size of 101 kb, covering 5.0 x haploid genome equivalents. High-density clone filters prepared from a subset of the pECBAC1 library were screened with Overgos designed from the cDNA sequences of six genes involved in immune system and sex determination related factor. Positive clones were identified for every gene, with an average of 3.3 BAC clones per gene probe. These results suggest that the shrimp BAC libraries provide useful tools for gene cloning, genome physical mapping and large-scale sequencing in the species.