C. A. Gill

Whole genome linkage disequilibrium maps in cattle

BackgroundBovine whole genome linkage disequilibrium maps were constructed for eight breeds of cattle. These data provide fundamental information concerning bovine genome organization which will allow the design of studies to associate genetic variation with economically important traits and also provides background information concerning the extent of long range linkage disequilibrium in cattle.ResultsLinkage disequilibrium was assessed using r2 among all pairs of syntenic markers within eight breeds of cattle from the Bos taurus and Bos indicus subspecies. Bos taurus breeds included Angus, Charolais, Dutch Black and White Dairy, Holstein, Japanese Black and Limousin while Bos indicus breeds included Brahman and Nelore. Approximately 2670 markers spanning the entire bovine autosomal genome were used to estimate pairwise r2 values. We found that the extent of linkage disequilibrium is no more than 0.5 Mb in these eight breeds of cattle.ConclusionLinkage disequilibrium in cattle has previously been reported to extend several tens of centimorgans. Our results, based on a much larger sample of marker loci and across eight breeds of cattle indicate that in cattle linkage disequilibrium persists over much more limited distances. Our findings suggest that 30,000–50,000 loci will be needed to conduct whole genome association studies in cattle.

High-resolution haplotype block structure in the cattle genome

BackgroundThe Bovine HapMap Consortium has generated assay panels to genotype ~30,000 single nucleotide polymorphisms (SNPs) from 501 animals sampled from 19 worldwide taurine and indicine breeds, plus two outgroup species (Anoa and Water Buffalo). Within the larger set of SNPs we targeted 101 high density regions spanning up to 7.6 Mb with an average density of approximately one SNP per 4 kb, and characterized the linkage disequilibrium (LD) and haplotype block structure within individual breeds and groups of breeds in relation to their geographic origin and use.ResultsFrom the 101 targeted high-density regions on bovine chromosomes 6, 14, and 25, between 57 and 95% of the SNPs were informative in the individual breeds. The regions of high LD extend up to ~100 kb and the size of haplotype blocks ranges between 30 bases and 75 kb (10.3 kb average). On the scale from 1–100 kb the extent of LD and haplotype block structure in cattle has high similarity to humans. The estimation of effective population sizes over the previous 10,000 generations conforms to two main events in cattle history: the initiation of cattle domestication (~12,000 years ago), and the intensification of population isolation and current population bottleneck that breeds have experienced worldwide within the last ~700 years. Haplotype block density correlation, block boundary discordances, and haplotype sharing analyses were consistent in revealing unexpected similarities between some beef and dairy breeds, making them non-differentiable. Clustering techniques permitted grouping of breeds into different clades given their similarities and dissimilarities in genetic structure.ConclusionThis work presents the first high-resolution analysis of haplotype block structure in worldwide cattle samples. Several novel results were obtained. First, cattle and human share a high similarity in LD and haplotype block structure on the scale of 1–100 kb. Second, unexpected similarities in haplotype block structure between dairy and beef breeds make them non-differentiable. Finally, our findings suggest that ~30,000 uniformly distributed SNPs would be necessary to construct a complete genome LD map in Bos taurus breeds, and ~580,000 SNPs would be necessary to characterize the haplotype block structure across the complete cattle genome.

Haplotype variation in bovine Toll-like receptor 4 and computational prediction of a positively selected ligand-binding domain

Toll-like receptor 4 (TLR4) is a cell-surface receptor that activates innate and adaptive immune responses. Because it recognizes a broad class of pathogen-associated molecular patterns presented by lipopolysaccharides and lipoteichoic acid, TLR4 is a candidate gene for resistance to a large number of diseases. In particular, mouse models suggest TLR4 as a candidate gene for resistance to major agents in bovine respiratory disease and Johnes disease. The coding sequence of bovine TLR4 is divided into three exons, with intron/exon boundaries and intron sizes similar to those of human TLR4 transcript variant 1. We amplified each exon in 40 individuals from 11 breeds and screened the sequence for single-nucleotide polymorphisms (SNPs). We identified 32 SNPs, 28 of which are in the coding sequence, for an average of one SNP per 90 bp of coding sequence. Eight SNPs were nonsynonymous and potentially alter specificity of pathogen recognition or efficiency of signaling. To evaluate the functional importance of these SNPs, we used codon-substitution models to detect diversifying selection in an extracellular region that may physically interact with ligands. One nonsynonymous SNP is located within this region, and other substitutions are in adjacent regions that may interact with coreceptor molecules. The 32 SNPs were found in 20 haplotypes that can be assigned to geographic ranges of origin. Haplotype-tagging SNP analysis indicated that 12 SNPs need to be genotyped to distinguish these 20 haplotypes. These data provide a basic understanding of bovine TLR4 sequence variation and supply haplotype markers for disease association studies.

An assessment of population structure in eight breeds of cattle using a whole genome SNP panel

BackgroundAnalyses of population structure and breed diversity have provided insight into the origin and evolution of cattle. Previously, these studies have used a low density of microsatellite markers, however, with the large number of single nucleotide polymorphism markers that are now available, it is possible to perform genome wide population genetic analyses in cattle. In this study, we used a high-density panel of SNP markers to examine population structure and diversity among eight cattle breeds sampled from Bos indicus and Bos taurus.ResultsTwo thousand six hundred and forty one single nucleotide polymorphisms (SNPs) spanning all of the bovine autosomal genome were genotyped in Angus, Brahman, Charolais, Dutch Black and White Dairy, Holstein, Japanese Black, Limousin and Nelore cattle. Population structure was examined using the linkage model in the program STRUCTURE and Fst estimates were used to construct a neighbor-joining tree to represent the phylogenetic relationship among these breeds.ConclusionThe whole-genome SNP panel identified several levels of population substructure in the set of examined cattle breeds. The greatest level of genetic differentiation was detected between the Bos taurus and Bos indicus breeds. When the Bos indicus breeds were excluded from the analysis, genetic differences among beef versus dairy and European versus Asian breeds were detected among the Bos taurus breeds. Exploration of the number of SNP loci required to differentiate between breeds showed that for 100 SNP loci, individuals could only be correctly clustered into breeds 50% of the time, thus a large number of SNP markers are required to replace the 30 microsatellite markers that are currently commonly used in genetic diversity studies.

A physical map of the bovine genome

BackgroundCattle are important agriculturally and relevant as a model organism. Previously described genetic and radiation hybrid (RH) maps of the bovine genome have been used to identify genomic regions and genes affecting specific traits. Application of these maps to identify influential genetic polymorphisms will be enhanced by integration with each other and with bacterial artificial chromosome (BAC) libraries. The BAC libraries and clone maps are essential for the hybrid clone-by-clone/whole-genome shotgun sequencing approach taken by the bovine genome sequencing project.ResultsA bovine BAC map was constructed with HindIII restriction digest fragments of 290,797 BAC clones from animals of three different breeds. Comparative mapping of 422,522 BAC end sequences assisted with BAC map ordering and assembly. Genotypes and pedigree from two genetic maps and marker scores from three whole-genome RH panels were consolidated on a 17,254-marker composite map. Sequence similarity allowed integrating the BAC and composite maps with the bovine draft assembly (Btau3.1), establishing a comprehensive resource describing the bovine genome. Agreement between the marker and BAC maps and the draft assembly is high, although discrepancies exist. The composite and BAC maps are more similar than either is to the draft assembly.ConclusionFurther refinement of the maps and greater integration into the genome assembly process may contribute to a high quality assembly. The maps provide resources to associate phenotypic variation with underlying genomic variation, and are crucial resources for understanding the biology underpinning this important ruminant species so closely associated with humans.

Promoter I of the ovine acetyl-CoA carboxylase-alpha gene: an E-box motif at -114 in the proximal promoter binds upstream stimulatory factor (USF)-1 and USF-2 and acts as an insulin-response sequence in differentiating adipocytes.

Acetyl-CoA carboxylase-alpha (ACC-alpha) plays a central role in co-ordinating de novo fatty acid synthesis in animal tissues. We have characterized the regulatory region of the ovine ACC-alpha gene. Three promoters, PI, PII and PIII, are dispersed throughout 50 kb of genomic DNA. Expression from PI is limited to adipose tissue and liver. Sequence comparison of the proximal promoters of ovine and mouse PIs demonstrates high nucleotide identity and that they are characterized by a TATA box at -29, C/EBP (CCAAT enhancer-binding protein)-binding motifs and multiple E-box motifs. A 4.3 kb ovine PI-luciferase reporter construct is insulin-responsive when transfected into differentiated ovine adipocytes, whereas when this construct is transfected into ovine preadipocytes and HepG2 cells the construct is inactive and is not inducible by insulin. By contrast, transfection of a construct corresponding to 132 bp of the proximal promoter linked to a luciferase reporter is active and inducible by insulin in all three cell systems. Insulin signalling to the -132 bp construct in differentiated ovine adipocytes involves, in part, an E-box motif at -114. Upstream stimulatory factor (USF)-1 and USF-2, but not sterol regulatory element-binding protein 1 (SREBP-1), are major components of protein complexes that bind this E-box motif. Activation of the 4.3 kb PI construct in differentiated ovine adipocytes is associated with endogenous expression of PI transcripts throughout differentiation; PI transcripts are not detectable by RNase-protection assay in ovine preadipocytes, HepG2 cells or 3T3-F442A adipocytes. These data indicate the presence of repressor motifs in PI that are required to be de-repressed during adipocyte differentiation to allow induction of the promoter by insulin.

Analysis of Integration Sites of Jaagsiekte Sheep Retrovirus in Ovine Pulmonary Adenocarcinoma

ABSTRACT Ovine pulmonary adenocarcinoma (OPA) is an infectious lung tumor of sheep caused by Jaagsiekte sheep retrovirus (JSRV). To test the hypothesis that JSRV insertional mutagenesis is involved in the oncogenesis of OPA, we cloned and characterized 70 independent integration sites from 23 cases of OPA. Multiple integration sites were identified in most tumors. BLAST analysis of the sequences did not disclose any potential oncogenic motifs or any identical integration sites in different tumors. Thirty-seven of the integration sites were mapped to individual chromosomes by PCR with a panel of sheep-hamster hybrid cell lines. Integration sites were found on 20 of the 28 sheep chromosomes, suggesting a random distribution. However, four integration sites from four different tumors mapped to chromosome 16. By Southern blot hybridization, probes derived from two of these sites mapped to within 5 kb of each other on normal sheep DNA. These sites were found within a single sheep bacterial artificial chromosome clone and were further mapped to only 2.5 kb apart, within an uncharacterized predicted gene and less than 200 kb from a mitogen-activated protein kinase-encoding gene. These findings suggest that there is at least one common integration site for JSRV in OPA and add weight to the hypothesis that insertional mutagenesis is involved in the development of this tumor.

Carcass merit between and among family groups of Bos indicus crossbred steers and heifers

Differences in live and carcass traits attributable to increasing Bos indicus breed influence were compared to the differences between families with similar proportions of B. indicus influence. Families of offspring from 1/2 Angus×1/2 B. indicus mated to Angus, B. indicus, and 1/2 Angus×1/2 B.indicus were raised under similar conditions. Average daily gain, slaughter weight, and dressing percentage were measured in addition to USDA yield and quality grade factors. Breed type did not affect average daily gain, slaughter weight, dressing percentage, carcass weight, adjusted 12th-rib fat thickness, estimated percentage kidney, pelvic, and heart fat, or carcass maturity. Predominately (3/4) Angus progeny produced greater (P<0.05) longissimus muscle areas than 3/4 B. indicus animals. Predominately Angus cattle also had greater (P<0.05) marbling scores and USDA quality grades than predominately B. indicus cattle. Families within breed types differed (P<0.05) with regard to all traits measured. This is interesting in light of the lack of differences between breeds for most traits. In some instances, the differences in marbling score and longissimus muscle area between families within a given breed type were similar or greater in magnitude than the differences observed between predominately Angus and predominately B. indicus breed types. Whereas growth and carcass traits varied between levels of B. indicus breeding, the opportunity does exists to improve these traits by selecting within specific family lines.

Construction and characterization of an ovine bacterial artificial chromosome library

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

Microsatellite variation in the equine MHC.

Genes within the major histocompatibility complex (MHC) encode proteins involved in innate and adaptive immune responses. Genetic variation in this region can influence the immune response of an individual animal to challenges from a variety of pathogens; however, a complete documentation of genetic variation in the MHC is lacking for most domestic animals, including horses. To provide additional genetic markers for study of the horse MHC, or ELA (equine lymphocyte antigen), we identified 37 polymorphic microsatellite repeats in ELA and used these variations separately and together with published SNPs to investigate linkage disequilibrium (LD) and haplotype structure in a sample of Thoroughbred horses. ELA SNPs alone detected little LD, but microsatellites, either separately or combined with SNPs, revealed substantially more LD. A subset of markers in very high LD across the breadth of ELA may be predictive of structural polymorphisms or linked epistases that are important drivers of haplotype structure in Thoroughbreds.