Construction of an American mink Bacterial Artificial Chromosome (BAC) library and sequencing candidate genes important for the fur industry

Abstract

Background

Bacterial artificial chromosome (BAC) libraries continue to be invaluable tools for the genomic analysis of complex organisms. Complemented by the newly and fast growing deep sequencing technologies, they provide an excellent source of information in genomics projects.

Results

Here, we report the construction and characterization of the CHORI-231 BAC library constructed from a Danish-farmed, male American mink ( Neovison vison). The library contains approximately 165,888 clones with an average insert size of 170 kb, representing approximately 10-fold coverage. High-density filters, each consisting of 18,432 clones spotted in duplicate, have been produced for hybridization screening and are publicly available. Overgo probes derived from expressed sequence tags (ESTs), representing 21 candidate genes for traits important for the mink industry, were used to screen the BAC library. These included candidate genes for coat coloring, hair growth and length, coarseness, and some receptors potentially involved in viral diseases in mink. The extensive screening yielded positive results for 19 of these genes. Thirty-five clones corresponding to 19 genes were sequenced using 454 Roche, and large contigs (184 kb in average) were assembled. Knowing the complete sequences of these candidate genes will enable confirmation of the association with a phenotype and the finding of causative mutations for the targeted phenotypes.

Additionally, 1577 BAC clones were end sequenced; 2505 BAC end sequences (80% of BACs) were obtained. An excess of 2 Mb has been analyzed, thus giving a snapshot of the mink genome.

Conclusions

The availability of the CHORI-321 American mink BAC library will aid in identification of genes and genomic regions of interest. We have demonstrated how the library can be used to identify specific genes of interest, develop genetic markers, and for BAC end sequencing and deep sequencing of selected clones. To our knowledge, this is the first report of 454 sequencing of selected BAC clones in mammals and re-assures the suitability of this technique for obtaining the sequence information of genes of interest in small genomics projects. The BAC end sequences described in this paper have been deposited in the GenBank data library [HN339419-HN341884, HN604664-HN604702]. The 454 produced contigs derived from selected clones are deposited with reference numbers [GenBank: JF288166-JF288183 &JF310744].