Identification of regions of the genome that contain genes foreconomically important traits in livestock species and isolation ofthose genes so that they can be utilized in breeding programsrequires high-density genome maps. Large-insert clones such asyeast artificial chromosomes (YAC), P1-derived artificial chromo-somes (PAC), and bacterial artificial chromosomes (BAC) can beused to efficiently obtain accurate, high-resolution physical mapsof eukaryotic genomes (Cai et al. 1995). YAC libraries containingclones with inserts up to 1 Mb in size have been constructed forhumans (Albertsen et al. 1990), mice (Larin et al. 1993), rats (Caiet al. 1997), cattle (Libert et al. 1993), sheep (Broom and Hill1994), and pigs (Rogel-Gaillard et al. 1997). However, YACclones are often chimeric, consisting of DNA from different partsof the genome that have accidentally been combined into a singleclone (Libert et al. 1993). This is a serious hindrance to physicalmapping and chromosome walking because the true location of thegene of interest cannot be readily determined.BAC libraries have been constructed for a number of plantspecies as well as for humans (Kim et al. 1996), mice (ResearchGenetics, Inc., Huntsville, AL), cattle (Cai et al. 1995), chickens(Zimmer and Gibbons 1997), goats (Schibler et al. 1998) andhorses (Godard et al. 1998) with cloning systems based on theEscherichia coli F factor. BAC clones are stable, easy to manipu-late, and are rarely chimeric (Shizuya et al. 1992; Cai et al. 1995).The only disadvantage of the BAC technology for chromosomewalking is that the DNA inserts are somewhat smaller (100–300kb) than those maintained by YAC clones. Despite this, BACclones are becoming the vector of choice for physically mappinggenes to specific chromosomal locations and for isolating genes bypositional cloning.In this paper, we present the construction and characterizationof an ovine BAC library containing 59,904 clones with an averageinsert size of 103 kb, which corresponds to about two genomeequivalents. The quality of the library was determined by PCR-based screening and fluorescence in situ hybridization (FISH). ABAC library covering the ovine genome will be a key resource forcomparative gene mapping studies, for identifying quantitativetrait loci (QTL) by positional cloning, and for functional studies tounderstand gene expression and regulation.High-molecular-weight DNA for BAC library constructionwas prepared by encapsulating white blood cells from a purebredSuffolk ram in agarose microbeads which were digested withHindIII and separated by PFGE as described by Cai et al. (1995).To get a majority of DNA fragments in the size range of 50–250kb, two-thirds of the microbeads used for library construction weredigested with 0.75 U HindIII, and the remainder were digestedwith1UHindIII. This restriction enzyme was selected to avoid thebulk of the repetitive centromeric DNA that is resistant to diges-tion with HindIII and remained in the compression band when thefragments were separated by CHEF gel electrophoresis.The partial digests with 0.75 U and 1 U HindIII were pooled ina single lane for size selection by gel electrophoresis. The CHEFgel for size selection was electrophoresed in1×TAErather than0.5 × TBE because borate ions inhibit ligation. The pulse time wasalso altered to 90-s because a 50-s pulse time previously had beenfound to adversely affect the ligation efficiency of the fragments,presumably because the 58 overhang or phosphate group was lost.The lambda ladder, however, was not resolved when a 90-s pulsetime was used. Consequently, the spread of fragments from 50 to250 kb were excised from the low-melting-point agarose gel basedon the mobility in the previous CHEF gel that used a 50-s pulsetime. This problem is likely to account for the smaller (∼100 kb)than expected average library insert sizes (∼150 kb).The excised gel was cut into four pieces. DNA from the threegel pieces containing the largest fragments was ligated topBeloBAC11 and transformed by electroporation into DH10b E.coli. Colonies produced from each ligation were sized, and it wasdetermined that the DNA in the second largest gel piece would beused for library construction. Many of the inserts obtained from thelargest gel piece were actually smaller than those from the secondlargest piece and may indicate that the majority of fragments inthat gel slice exceeded the cloning capability of the BAC vector.The transformation efficiency for the BAC clones was 1.5 × 10