Construction and characterization of a bovine BAC library with four genome-equivalent coverage

Abstract

-A bovine artificial chromosome (BAC) library of 105 984 clones has been constructed in the vector pBeloBAC11 and organized in 3-dimension pools and high density membranes for screening by PCR and hybridization. The average insert size, determined after analysis of 388 clones, was estimated at 120 kb corresponding to a four genome coverage. Given the fact that a male was used to construct the library, the probability of finding any given autosomal and X or Y locus is respectively 0.98 and 0.86. The library was screened for 164 microsatellite markers and an average of 3.9 superpools was positive for each PCR system. None of the 50 or so BAC clones analysed by FISH was chimeric. This BAC library increases the international genome coverage for cattle to around 28 genome equivalents and extends the coverage of the ruminant genomes available at the Inra resource center to 15 genome equivalents. bovine / BAC library / mapping

Fine Mapping and Physical Characterization of Two Linked Quantitative Trait Loci Affecting Milk Fat Yield in Dairy Cattle on BTA26

“…Physical map construction: As mentioned previously, all the microsatellite markers considered in this study were screened for in the INRA BAC library (Eggen et al 2001). Together with previous physical and comparative map information for BTA26 Schibler et al 2004), these data allowed us to anchor each marker into the INRA first-generation bovine physical map (http:/ /locus.jouy.inra.fr/fpc/cattle).…”

“…PCR primers were designed for the 16 remaining and first tested to screen the BAC library (Eggen et al 2001) to identify or confirm the BAC clone(s) from which the microsatellite markers were isolated.…”

“…Some of these genes also represented functional candidate genes. The INRA BAC library was PCR screened as previously described (Eggen et al 2001) for the gene PRKG1, using primers deriving from the sequence Y08961 (PRKG1F, GCTCCAGGA GAAGATCGAGGA and PRKG1R, GCTGAGATCCTGGATGT CAAA). Although this gene could not be mapped directly on the BTA26 radiation hybrid (RH) map due to strong sequence similarities among bovine primers and hamster genomic sequences, comparative mapping results indicate that it represents a good candidate to refine the boundaries between the two blocks of conserved synteny called HB4 and HB5 in one of our previous publications .…”

A first generation bovine BAC-based physical map

“…A PCR-based screening was performed on the Inra bovine BAC library as previously described [14]. Briefly, primer pairs for markers were obtained from the BOVMAP database (http://locus.jouy.inra.fr).…”

“…Two libraries were used: the Inra BAC library [14] with 105 984 clones and part of the CHORI-240 (www.chori.org/bacpac) BAC library (26 500 clones). About 9% of the clones were lost during the fingerprinting process, due to poor DNA preparation or low DNA yield (7%), injection failure or damaged capillaries (2%).…”