A porcine BAC library with tenfold genome coverage: a resource for physical and genetic map integration.

Recent advances in porcine genomics have identified quantitative trait loci (QTL) that influence pork production traits such as carcass traits, meat quality, and reproductive efficiency (Rohrer 2000; Cassady 1999). The low resolution to which most of these loci have been defined precludes the accurate application of markerassisted selection (MAS) strategies for increasing production efficiency. Large-insert genomic libraries are an excellent resource for marker development aimed at increasing the resolution of QTL and for the development of contiguous physical maps (contigs) of the chromosomal regions containing them. To facilitate applications requiring genomic clones, several porcine yeast artificial chromosome (YAC) libraries have been developed representing onefold (Leeb et al. 1995), threefold (Rogel-Gaillard et al. 1997), and 5.5-fold (Alexander et al. 1997) coverage of the pig genome. These YAC libraries have been valuable resource owing to their large insert size. However, marker isolation from YAC libraries is confounded by the equimolar representation of the complex yeast genome and YAC DNA, susceptibility to insert rearrangement, and a relatively high degree of chimerism. The development of bacterial artificial chromosome (BAC) libraries (Shizuya et al. 1992; Ioannou et al. 1994) represents a compromise between insert size, stability, and ease of clone DNA isolation. BACs are capable of stably maintaining insert sizes exceeding 200 kb and can be easily isolated by standard alkaline lysis from bacterial genomic DNA by virtue of their closed-circular conformation (Shizuya et al. 1992; Ioannou et al. 1994). The ease of BAC DNA isolation allows for efficient restriction analysis, subcloning, and direct BAC DNA sequencing, procedures invaluable for marker isolation and the development of contiguous physical maps. Recently BAC libraries representing livestock species, including cattle, sheep, and pigs (Cai et al. 1995; Warren et al. 2000; Vaiman et al. 1999; Suzuki et al. 2000; Rogel-Gaillard et al. 1999) have been developed. Among these are two porcine BAC libraries representing fourfold (Suzuki et al. 2000) and fivefold (RogelGaillard et al. 1999) coverage of the porcine genome. We undertook the development of a publicly available pig BAC library, RPCI-44, representing at least a tenfold coverage of the pig genome as a resource for physical map development and pig genome sequencing. High-molecular-weight genomic DNA from four crossbred male pigs (breed composition: 37.5% Yorkshire, 37.5% Landrace, and 25% Meishan) was isolated as previously described (Osoegawa et al. 1998), partially digested with EcoRI (New England Biolabs, Beverly, MA) and EcoRI methylase (New England Biolabs), and fractionated by clamped homogeneous electrical field (CHEF; Chu et al. 1986) electrophoresis (BioRad, Hercules, CA). Digested genomic DNA in the range of 150–200 kpb was gelisolated by electroelution and ligated to EcoRI-cut pTARBAC2 (Wang et al., unpublished; http://www.chori.org/bacpac/ vectorframe.htm). Ligated DNA was drop-dialyzed on floating membranes, first against sterile water, followed by 0.5 × TE containing 30% PEG8000, and introduced by electroporation into DH1OB electrocompetent cells (Life Technologies, Rockville, MD). Transformed bacterial cells were allowed to recover in SOC medium for 1 h at 37°C, and were spread on LB agar plates containing 20 mg/ml chloramphenicol and 5% sucrose. Individual colonies were picked with an automatic colony-picking robot (Qbot; Genetix, Stony Brook, NY) and arrayed into 528 individual 384-well microtiter dishes (Genetix) with LB medium containing 7.5% glycerol and 20 mg/ml chloramphenicol. The library was divided into two segments and gridded onto 11 high-density replica filters, each containing up to 18,432 independent clones that have been spotted in duplicate. To serve as anchor spots, an endsequenced BAC clone (1A1) from a Caenorhabditis brigsea BAC library (RPCI-94), containing DNA apparently unique to nematode genomes, was gridded on the corner of each of the six fields that comprise the high-density filters. The inclusion of a probe directed against the anchor spots can facilitate orientation of hybridized filters. The predicted coverage of RPCI-44 is a function of the total number of insert-containing clones and the size of the genomic DNA fragments they contain. From a total of 202,752 clones picked, 1,764 (segment 1, 1.9%) and 1,519 (segment 2, 1.4%) clones failed to grow, leaving a total of 199,469 clones. Non-recombinant clones containing the original vector were identified by high-density colony filter hybridization with the vector as a probe. The probing identifies two classes of clones with strong versus weak hybridization signals corresponding to the presence or absence of the original vector stuffer fragment (“pUClink”), which contains a high-copy-number replicon. Recombinant clones lack the stuffer fragment, resulting in much weaker hybridization signals. In total, 119 clones (0.06%) were identified as containing intact vector by this approach. The plate and well addresses of these are available at http://www.chori.org/framenonrec.htm. To evaluate the average insert size and insert size distribution in the library, randomly selected clones from each segment were subjected to restriction analysis with Not1, insert size determination by CHEF electrophoresis, and comparison with molecular weight standards (Osoegawa et al. 2000). Segment 1 was composed of 90,396 clones containing inserts averaging 157 kpb * Present address: University of Minnesota, Department of Animal Science, 495 An Sci/Vet Med, 1988 Fitch Ave., St. Paul, MN 55108, USA.