AgAnimalGenomes: browsers for viewing and manually annotating farm animal genomes.

Current genome sequencing technologies have made it possible to generate highly contiguous genome assemblies for non-model animal species. Despite advances in genome assembly methods, there is still room for improvement in the delineation of specific gene features in the genomes. Here we present genome visualization and annotation tools to support seven livestock species (bovine, chicken, goat, horse, pig, sheep, and water buffalo), available in a new resource called AgAnimalGenomes. In addition to supporting the manual refinement of gene models, these browsers provide visualization tracks for hundreds of RNAseq experiments, as well as data generated by the Functional Annotation of Animal Genomes (FAANG) Consortium. For species with predicted gene sets from both Ensembl and RefSeq, the browsers provide special tracks showing the thousands of protein-coding genes that disagree across the two gene sources, serving as a valuable resource to alert researchers to gene model issues that may affect data interpretation. We describe the data and search methods available in the new genome browsers and how to use the provided tools to edit and create new gene models.

Genome-wide association with footrot in hair and wool sheep.

Ovine footrot is an infectious disease with important contributions from Dichelobacter nodosus and Fusobacterium necrophorum. Footrot is characterized by separation of the hoof from underlying tissue, and this causes severe lameness that negatively impacts animal wellbeing, growth, and profitability. Large economic losses result from lost production as well as treatment costs, and improved genetic tools to address footrot are a valuable long-term goal. Prior genetic studies had examined European wool sheep, but hair sheep breeds such as Katahdin and Blackbelly have been reported to have increased resistance to footrot, as well as to intestinal parasites. Thus, footrot condition scores were collected from 251 U.S. sheep including Katahdin, Blackbelly, and European-influenced crossbred sheep with direct and imputed genotypes at OvineHD array (>500,000 single nucleotide polymorphism) density. Genome-wide association was performed with a mixed model accounting for farm and principal components derived from animal genotypes, as well as a random term for the genomic relationship matrix. We identified three genome-wide significant associations, including SNPs in or near GBP6 and TCHH. We also identified 33 additional associated SNPs with genome-wide suggestive evidence, including a cluster of 6 SNPs in a peak near the genome-wide significance threshold located near the glutamine transporter gene SLC38A1. These findings suggest genetic susceptibility to footrot may be influenced by genes involved in divergent biological processes such as immune responses, nutrient availability, and hoof growth and integrity. This is the first genome-wide study to investigate susceptibility to footrot by including hair sheep and also the first study of any kind to identify multiple genome-wide significant associations with ovine footrot. These results provide a foundation for developing genetic tests for marker-assisted selection to improve resistance to ovine footrot once additional steps like fine mapping and validation are complete.

An improved ovine reference genome assembly to facilitate in-depth functional annotation of the sheep genome.

BACKGROUND: The domestic sheep (Ovis aries) is an important agricultural species raised for meat, wool, and milk across the world. A high-quality reference genome for this species enhances the ability to discover genetic mechanisms influencing biological traits. Furthermore, a high-quality reference genome allows for precise functional annotation of gene regulatory elements. The rapid advances in genome assembly algorithms and emergence of sequencing technologies with increasingly long reads provide the opportunity for an improved de novo assembly of the sheep reference genome. FINDINGS: Short-read Illumina (55× coverage), long-read Pacific Biosciences (75× coverage), and Hi-C data from this ewe retrieved from public databases were combined with an additional 50× coverage of Oxford Nanopore data and assembled with canu v1.9. The assembled contigs were scaffolded using Hi-C data with Salsa v2.2, gaps filled with PBsuitev15.8.24, and polished with Nanopolish v0.12.5. After duplicate contig removal with PurgeDups v1.0.1, chromosomes were oriented and polished with 2 rounds of a pipeline that consisted of freebayes v1.3.1 to call variants, Merfin to validate them, and BCFtools to generate the consensus fasta. The ARS-UI_Ramb_v2.0 assembly is 2.63 Gb in length and has improved continuity (contig NG50 of 43.18 Mb), with a 19- and 38-fold decrease in the number of scaffolds compared with Oar_rambouillet_v1.0 and Oar_v4.0. ARS-UI_Ramb_v2.0 has greater per-base accuracy and fewer insertions and deletions identified from mapped RNA sequence than previous assemblies. CONCLUSIONS: The ARS-UI_Ramb_v2.0 assembly is a substantial improvement in contiguity that will optimize the functional annotation of the sheep genome and facilitate improved mapping accuracy of genetic variant and expression data for traits in sheep.

Using whole genome sequence to compare variant callers and breed differences of US sheep.

As whole genome sequence (WGS) data sets have become abundant and widely available, so has the need for variant detection and scoring. The aim of this study was to compare the accuracy of commonly used variant calling programs, Freebayes and GATK HaplotypeCaller (GATK-HC), and to use U.S. sheep WGS data sets to identify novel breed-associated SNPs. Sequence data from 145 sheep consisting of 14 U.S. breeds were filtered and biallelic single nucleotide polymorphisms (SNPs) were retained for genotyping analyses. Genotypes from both programs were compared to each other and to genotypes from bead arrays. The SNPs from WGS were compared to the bead array data with breed heterozygosity, principal component analysis and identifying breed associated SNPs to analyze genetic diversity. The average sequence read depth was 2.78 reads greater with 6.11% more SNPs being identified in Freebayes compared to GATK-HC. The genotype concordance of the variant callers to bead array data was 96.0% and 95.5% for Freebayes and GATK-HC, respectively. Genotyping with WGS identified 10.5 million SNPs from all 145 sheep. This resulted in an 8% increase in measured heterozygosity and greater breed separation in the principal component analysis compared to the bead array analysis. There were 1,849 SNPs identified in only the Romanov sheep where all 10 rams were homozygous for one allele and the remaining 135 sheep from 13 breeds were homozygous for the opposite allele. Both variant calling programs had greater than 95% concordance of SNPs with bead array data, and either was suitably accurate for ovine WGS data sets. The use of WGS SNPs improved the resolution of PCA analysis and was critical for identifying Romanov breed-associated SNPs. Subsets of such SNPs could be used to estimate germplasm composition in animals without pedigree information.

Characterizing Genetic Regulatory Elements in Ovine Tissues.

The Ovine Functional Annotation of Animal Genomes (FAANG) project, part of the broader livestock species FAANG initiative, aims to identify and characterize gene regulatory elements in domestic sheep. Regulatory element annotation is essential for identifying genetic variants that affect health and production traits in this important agricultural species, as greater than 90% of variants underlying genetic effects are estimated to lie outside of transcribed regions. Histone modifications that distinguish active or repressed chromatin states, CTCF binding, and DNA methylation were used to characterize regulatory elements in liver, spleen, and cerebellum tissues from four yearling sheep. Chromatin immunoprecipitation with sequencing (ChIP-seq) was performed for H3K4me3, H3K27ac, H3K4me1, H3K27me3, and CTCF. Nine chromatin states including active promoters, active enhancers, poised enhancers, repressed enhancers, and insulators were characterized in each tissue using ChromHMM. Whole-genome bisulfite sequencing (WGBS) was performed to determine the complement of whole-genome DNA methylation with the ChIP-seq data. Hypermethylated and hypomethylated regions were identified across tissues, and these locations were compared with chromatin states to better distinguish and validate regulatory elements in these tissues. Interestingly, chromatin states with the poised enhancer mark H3K4me1 in the spleen and cerebellum and CTCF in the liver displayed the greatest number of hypermethylated sites. Not surprisingly, active enhancers in the liver and spleen, and promoters in the cerebellum, displayed the greatest number of hypomethylated sites. Overall, chromatin states defined by histone marks and CTCF occupied approximately 22% of the genome in all three tissues. Furthermore, the liver and spleen displayed in common the greatest percent of active promoter (65%) and active enhancer (81%) states, and the liver and cerebellum displayed in common the greatest percent of poised enhancer (53%), repressed enhancer (68%), hypermethylated sites (75%), and hypomethylated sites (73%). In addition, both known and de novo CTCF-binding motifs were identified in all three tissues, with the highest number of unique motifs identified in the cerebellum. In summary, this study has identified the regulatory regions of genes in three tissues that play key roles in defining health and economically important traits and has set the precedent for the characterization of regulatory elements in ovine tissues using the Rambouillet reference genome.

Global Analysis of Transcription Start Sites in the New Ovine Reference Genome (Oar rambouillet v1.0).

The overall aim of the Ovine FAANG project is to provide a comprehensive annotation of the new highly contiguous sheep reference genome sequence (Oar rambouillet v1.0). Mapping of transcription start sites (TSS) is a key first step in understanding transcript regulation and diversity. Using 56 tissue samples collected from the reference ewe Benz2616, we have performed a global analysis of TSS and TSS-Enhancer clusters using Cap Analysis Gene Expression (CAGE) sequencing. CAGE measures RNA expression by 5' cap-trapping and has been specifically designed to allow the characterization of TSS within promoters to single-nucleotide resolution. We have adapted an analysis pipeline that uses TagDust2 for clean-up and trimming, Bowtie2 for mapping, CAGEfightR for clustering, and the Integrative Genomics Viewer (IGV) for visualization. Mapping of CAGE tags indicated that the expression levels of CAGE tag clusters varied across tissues. Expression profiles across tissues were validated using corresponding polyA+ mRNA-Seq data from the same samples. After removal of CAGE tags with <10 read counts, 39.3% of TSS overlapped with 5' ends of 31,113 transcripts that had been previously annotated by NCBI (out of a total of 56,308 from the NCBI annotation). For 25,195 of the transcripts, previously annotated by NCBI, no TSS meeting stringent criteria were identified. A further 14.7% of TSS mapped to within 50 bp of annotated promoter regions. Intersecting these predicted TSS regions with annotated promoter regions (±50 bp) revealed 46% of the predicted TSS were "novel" and previously un-annotated. Using whole-genome bisulfite sequencing data from the same tissues, we were able to determine that a proportion of these "novel" TSS were hypo-methylated (32.2%) indicating that they are likely to be reproducible rather than "noise". This global analysis of TSS in sheep will significantly enhance the annotation of gene models in the new ovine reference assembly. Our analyses provide one of the highest resolution annotations of transcript regulation and diversity in a livestock species to date.

Identification of genes directly responding to DLK1 signaling in Callipyge sheep.

BACKGROUND: In food animal agriculture, there is a need to identify the mechanisms that can improve the efficiency of muscle growth and protein accretion. Callipyge sheep provide excellent machinery since the up-regulation of DLK1 and RTL1 results in extreme postnatal muscle hypertrophy in distinct muscles. The aim of this study is to distinguish the genes that directly respond to DLK1 and RTL1 signaling from the genes that change as the result of muscle specific effects. RESULTS: The quantitative PCR results indicated that DLK1 expression was significantly increased in hypertrophied muscles but not in non-hypertrophied muscles. However, RTL1 was up-regulated in both hypertrophied and non-hypertrophied muscles. Five genes, including PARK7, DNTTIP1, SLC22A3, METTL21E and PDE4D, were consistently co-expressed with DLK1, and therefore were possible transcriptional target genes responding to DLK1 signaling. Treatment of myoblast and myotubes with DLK1 protein induced an average of 1.6-fold and 1.4-fold increase in Dnttip1 and Pde4d expression respectively. Myh4 expression was significantly elevated in DLK1-treated myotubes, whereas the expression of Mettl21e was significantly increased in the DLK1-treated myoblasts but reduced in DLK1-treated myotubes. DLK1 treatment had no impact on Park7 expression. In addition, Park7 and Dnttip1 increased Myh4 and decreased Myh7 promoter activity, resemble to the effects of Dlk1. In contrast, expression of Mettl21e increased Myh7 and decreased Myh4 luciferase activity. CONCLUSION: The study provided additional supports that RTL1 alone was insufficient to induce muscle hypertrophy and concluded that DLK1 was likely the primary effector of the hypertrophy phenotype. The results also suggested that DNTTIP1 and PDE4D were secondary effector genes responding to DLK1 signaling resulting in muscle fiber switch and muscular hypertrophy in callipyge lamb.

Impacts of the Callipyge mutation on ovine plasma metabolites and muscle fibre type.

The ovine Callipyge mutation causes postnatal muscle hypertrophy localized to the pelvic limbs and torso, as well as body leanness. The mechanism underpinning enhanced muscle mass is unclear, as is the systemic impact of the mutation. Using muscle fibre typing immunohistochemistry, we confirmed muscle specific effects and demonstrated that affected muscles had greater prevalence and hypertrophy of type 2X fast twitch glycolytic fibres and decreased representation of types 1, 2C, 2A and/or 2AX fibres. To investigate potential systemic effects of the mutation, proton NMR spectra of plasma taken from lambs at 8 and 12 weeks of age were measured. Multivariate statistical analysis of plasma metabolite profiles demonstrated effects of development and genotype but not gender. Plasma from Callipyge lambs at 12 weeks of age, but not 8 weeks, was characterized by a metabolic profile consistent with contributions from the affected hypertrophic fast twitch glycolytic muscle fibres. Microarray analysis of the perirenal adipose tissue depot did not reveal a transcriptional effect of the mutation in this tissue. We conclude that there is an indirect systemic effect of the Callipyge mutation in skeletal muscle in the form of changes of blood metabolites, which may contribute to secondary phenotypes such as body leanness.

Genes contributing to genetic variation of muscling in sheep.

Selective breeding programs aiming to increase the productivity and profitability of the sheep meat industry use elite, progeny tested sires. The broad genetic traits of primary interest in the progeny of these sires include skeletal muscle yield, fat content, eating quality, and reproductive efficiency. Natural mutations in sheep that enhance muscling have been identified, while a number of genome scans have identified and confirmed quantitative trait loci (QTL) for skeletal muscle traits. The detailed phenotypic characteristics of sheep carrying these mutations or QTL affecting skeletal muscle show a number of common biological themes, particularly changes in developmental growth trajectories, alterations of whole animal morphology, and a shift toward fast twitch glycolytic fibers. The genetic, developmental, and biochemical mechanisms underpinning the actions of some of these genetic variants are described. This review critically assesses this research area, identifies gaps in knowledge, and highlights mechanistic linkages between genetic polymorphisms and skeletal muscle phenotypic changes. This knowledge may aid the discovery of new causal genetic variants and in some cases lead to the development of biochemical and immunological strategies aimed at enhancing skeletal muscle.

A gene network switch enhances the oxidative capacity of ovine skeletal muscle during late fetal development.

BACKGROUND: The developmental transition between the late fetus and a newborn animal is associated with profound changes in skeletal muscle function as it adapts to the new physiological demands of locomotion and postural support against gravity. The mechanisms underpinning this adaption process are unclear but are likely to be initiated by changes in hormone levels. We tested the hypothesis that this developmental transition is associated with large coordinated changes in the transcription of skeletal muscle genes. RESULTS: Using an ovine model, transcriptional profiling was performed on Longissimus dorsi skeletal muscle taken at three fetal developmental time points (80, 100 and 120 d of fetal development) and two postnatal time points, one approximately 3 days postpartum and a second at 3 months of age. The developmental time course was dominated by large changes in expression of 2,471 genes during the interval between late fetal development (120 d fetal development) and 1-3 days postpartum. Analysis of the functions of genes that were uniquely up-regulated in this interval showed strong enrichment for oxidative metabolism and the tricarboxylic acid cycle indicating enhanced mitochondrial activity. Histological examination of tissues from these developmental time points directly confirmed a marked increase in mitochondrial activity between the late fetal and early postnatal samples. The promoters of genes that were up-regulated during this fetal to neonatal transition were enriched for estrogen receptor 1 and estrogen related receptor alpha cis-regulatory motifs. The genes down-regulated during this interval highlighted de-emphasis of an array of functions including Wnt signaling, cell adhesion and differentiation. There were also changes in gene expression prior to this late fetal--postnatal transition and between the two postnatal time points. The former genes were enriched for functions involving the extracellular matrix and immune response while the latter principally involved functions associated with transcriptional regulation of metabolic processes. CONCLUSIONS: It is concluded that during late skeletal muscle development there are substantial and coordinated changes in the transcription of a large number of genes many of which are probably triggered by increased estrogen levels. These changes probably underpin the adaption of muscle to new physiological demands in the postnatal environment.

The imprinted retrotransposon-like gene PEG11 (RTL1) is expressed as a full-length protein in skeletal muscle from Callipyge sheep.

Members of the Ty3-Gypsy retrotransposon family are rare in mammalian genomes despite their abundance in invertebrates and some vertebrates. These elements contain a gag-pol-like structure characteristic of retroviruses but have lost their ability to retrotranspose into the mammalian genome and are thought to be inactive relics of ancient retrotransposition events. One of these retrotransposon-like elements, PEG11 (also called RTL1) is located at the distal end of ovine chromosome 18 within an imprinted gene cluster that is highly conserved in placental mammals. The region contains several conserved imprinted genes including BEGAIN, DLK1, DAT, GTL2 (MEG3), PEG11 (RTL1), PEG11as, MEG8, MIRG and DIO3. An intergenic point mutation between DLK1 and GTL2 causes muscle hypertrophy in callipyge sheep and is associated with large changes in expression of the genes linked in cis between DLK1 and MEG8. It has been suggested that over-expression of DLK1 is the effector of the callipyge phenotype; however, PEG11 gene expression is also strongly correlated with the emergence of the muscling phenotype as a function of genotype, muscle type and developmental stage. To date, there has been no direct evidence that PEG11 encodes a protein, especially as its anti-sense transcript (PEG11as) contains six miRNA that cause cleavage of the PEG11 transcript. Using immunological and mass spectrometry approaches we have directly identified the full-length PEG11 protein from postnatal nuclear preparations of callipyge skeletal muscle and conclude that its over-expression may be involved in inducing muscle hypertrophy. The developmental expression pattern of the PEG11 gene is consistent with the callipyge mutation causing recapitulation of the normal fetal-like gene expression program during postnatal development. Analysis of the PEG11 sequence indicates strong conservation of the regions encoding the antisense microRNA and in at least two cases these correspond with structural or functional domains of the protein suggesting co-evolution of the sense and antisense genes.

Effect of DLK1 and RTL1 but not MEG3 or MEG8 on muscle gene expression in Callipyge lambs.

Callipyge sheep exhibit extreme postnatal muscle hypertrophy in the loin and hindquarters as a result of a single nucleotide polymorphism (SNP) in the imprinted DLK1-DIO3 domain on ovine chromosome 18. The callipyge SNP up-regulates the expression of surrounding transcripts when inherited in cis without altering their allele-specific imprinting status. The callipyge phenotype exhibits polar overdominant inheritance since only paternal heterozygous animals have muscle hypertrophy. Two studies were conducted profiling gene expression in lamb muscles to determine the down-stream effects of over-expression of paternal allele-specific DLK1 and RTL1 as well as maternal allele-specific MEG3, RTL1AS and MEG8, using Affymetrix bovine expression arrays. A total of 375 transcripts were differentially expressed in callipyge muscle and 25 transcripts were subsequently validated by quantitative PCR. The muscle-specific expression patterns of most genes were similar to DLK1 and included genes that are transcriptional repressors or affect feedback mechanisms in beta-adrenergic and growth factor signaling pathways. One gene, phosphodiesterase 7A had an expression pattern similar to RTL1 expression indicating a biological activity for RTL1 in muscle. Only transcripts that localize to the DLK1-DIO3 domain were affected by inheritance of a maternal callipyge allele. Callipyge sheep are a unique model to study over expression of both paternal allele-specific genes and maternal allele-specific non-coding RNA with an accessible and nonlethal phenotype. This study has identified a number of genes that are regulated by DLK1 and RTL1 expression and exert control on postnatal skeletal muscle growth. The genes identified in this model are primary candidates for naturally regulating postnatal muscle growth in all meat animal species, and may serve as targets to ameliorate muscle atrophy conditions including myopathic diseases and age-related sarcopenia.

Cytogenetic anchoring of radiation hybrid and virtual maps of sheep chromosome X and comparison of X chromosomes in sheep, cattle, and human.

A comprehensive physical map was generated for Ovis aries chromosome X (OARX) based on a cytogenomics approach. DNA probes were prepared from bacterial artificial chromosome (BAC) clones from the CHORI-243 sheep library and were assigned to G-banded metaphase spreads via fluorescence in-situ hybridization (FISH). A total of 22 BACs gave a single hybridization signal to the X chromosome and were assigned out of 32 tested. The positioned BACs contained 16 genes and a microsatellite marker which represent new cytogenetically mapped loci in the sheep genome. The gene and microsatellite loci serve to anchor between the existing radiation hybrid (RH) and virtual sheep genome (VSG) maps to the cytogenetic OARX map, whilst the BACs themselves also serve as anchors between the VSG and the cytogenetic maps. An additional 17 links between the RH and cytogenetic maps are provided by BAC end sequence (BES) derived markers that have also been positioned on the RH map. Comparison of the map orders for the cytogenetic, RH, and virtual maps reveals that the orders for the cytogenetic and RH maps are most similar, with only one locus, represented by BAC CH243-330E18, mapping to relatively different positions. Several discrepancies, including an inverted segment are found when comparing both the cytogenetic and RH maps with the virtual map. These discrepancies highlight the value of using physical mapping methods to inform the process of future in silico map construction. A detailed comparative analysis of sheep, human, and cattle mapping data allowed the construction of a comparative map that confirms and expands the knowledge about evolutionary conservation and break points between the X chromosomes of the three mammalian species.

Comparative genomics of Toll-like receptor signalling in five species.

BACKGROUND: Over the last decade, several studies have identified quantitative trait loci (QTL) affecting variation of immune related traits in mammals. Recent studies in humans and mice suggest that part of this variation may be caused by polymorphisms in genes involved in Toll-like receptor (TLR) signalling. In this project, we used a comparative approach to investigate the importance of TLR-related genes in comparison with other immunologically relevant genes for resistance traits in five species by associating their genomic location with previously published immune-related QTL regions. RESULTS: We report the genomic localisation of TLR1-10 and ten associated signalling molecules in sheep and pig using in-silico and/or radiation hybrid (RH) mapping techniques and compare their positions with their annotated homologues in the human, cattle and mouse whole genome sequences. We also report medium-density RH maps for porcine chromosomes 8 and 13. A comparative analysis of the positions of previously published relevant QTLs allowed the identification of homologous regions that are associated with similar health traits in several species and which contain TLR related and other immunologically relevant genes. Additional evidence was gathered by examining relevant gene expression and association studies. CONCLUSION: This comparative genomic approach identified eight genes as potentially causative genes for variations of health related traits. These include susceptibility to clinical mastitis in dairy cattle, general disease resistance in sheep, cattle, humans and mice, and tolerance to protozoan infection in cattle and mice. Four TLR-related genes (TLR1, 6, MyD88, IRF3) appear to be the most likely candidate genes underlying QTL regions which control the resistance to the same or similar pathogens in several species. Further studies are required to investigate the potential role of polymorphisms within these genes.

Analysis of the callipyge phenotype through skeletal muscle development; association of Dlk1 with muscle precursor cells.

The callipyge mutation in sheep in the form of the paternal heterozygote results in skeletal muscle hypertrophy, which is most pronounced in the hindquarters. Overexpression of one of the genes in the region of the causative single-nucleotide polymorphism, Dlk1, is postulated to be a primary cause of the muscle hypertrophy although the mechanism is not clear. This study examined the expression of Dlk1 mRNA and its encoded protein in skeletal muscles of callipyge and wild-type sheep. The muscles examined included those that demonstrate hypertrophy in callipyge sheep as well as an unaffected muscle. The expression pattern of Dlk1 protein in these muscles was also measured over a developmental time course ranging from 80 days of gestation to 12 weeks after birth. Quantitative reverse transcription-polymerase chain reaction demonstrated that Dlk1 mRNA was significantly increased in affected, but not unaffected, muscles from callipyge sheep at 120 days of gestation through to 12 weeks of age. Immuno-localization of Dlk1 was pronounced in the interstitial connective tissue of fetal muscle but was less intense at later ages. No clear difference in Dlk1 immuno-localization was noted between genotypes in the fetal samples. Strong myofiber-specific Dlk1 immuno-localization was observed in hypertrophied callipyge muscles at 12 weeks of age. This staining was exclusively associated with fast type II myofibers and these had a significantly larger mean cross-sectional area, compared with fast type II myofibers in control sheep that did not overexpress Dlk1. In addition, Dlk1 immuno-localization was associated with a sub-population of Pax7-positive mononucleated cells in all skeletal muscles examined during fetal development and at birth, but this was not apparent at 12 weeks. There were no genotype-dependent alterations in the mRNA expression patterns of a number of promyogenic transcription factors indicating that the callipyge mutation was not affecting muscle cell differentiation per se. We postulate that Dlk1 is implicated in the commitment and/or proliferation of fetal myoblasts as well as in the maintenance of hypertrophy in fully differentiated myofibers.

Myoblasts isolated from hypertrophy-responsive callipyge muscles show altered growth rates and increased resistance to serum deprivation-induced apoptosis.

Back and hind limb muscles of sheep paternally heterozygous for the callipyge single nucleotide polymorphism undergo extensive hypertrophy shortly after birth. We have established cell cultures from foetal semitendinosus and longissimus dorsi muscles of normal and callipyge animals. Cultures were assessed for rates of proliferation, cell death, myogenicity and DLK1 expression. Myoblasts from callipyge semitendinosus, but not longissimus dorsi muscles, proliferated faster than myoblasts isolated from normal semitendinosus muscle, and cells isolated from either callipyge muscle were more resistant to serum deprivation-induced apoptosis than equivalent cells isolated from normal individuals. These observations indicate that there are intrinsic differences in the behaviour of isolated myoblasts, which are associated with their muscle and genotype of origin. As myoblasts are the cells responsible for hypertrophy of muscle fibres, the observed differences in cell growth may play a role in the hypertrophy of certain muscles in callipyge animals.

Using comparative genomics to reorder the human genome sequence into a virtual sheep genome.

BACKGROUND: Is it possible to construct an accurate and detailed subgene-level map of a genome using bacterial artificial chromosome (BAC) end sequences, a sparse marker map, and the sequences of other genomes? RESULTS: A sheep BAC library, CHORI-243, was constructed and the BAC end sequences were determined and mapped with high sensitivity and low specificity onto the frameworks of the human, dog, and cow genomes. To maximize genome coverage, the coordinates of all BAC end sequence hits to the cow and dog genomes were also converted to the equivalent human genome coordinates. The 84,624 sheep BACs (about 5.4-fold genome coverage) with paired ends in the correct orientation (tail-to-tail) and spacing, combined with information from sheep BAC comparative genome contigs (CGCs) built separately on the dog and cow genomes, were used to construct 1,172 sheep BAC-CGCs, covering 91.2% of the human genome. Clustered non-tail-to-tail and outsize BACs located close to the ends of many BAC-CGCs linked BAC-CGCs covering about 70% of the genome to at least one other BAC-CGC on the same chromosome. Using the BAC-CGCs, the intrachromosomal and interchromosomal BAC-CGC linkage information, human/cow and vertebrate synteny, and the sheep marker map, a virtual sheep genome was constructed. To identify BACs potentially located in gaps between BAC-CGCs, an additional set of 55,668 sheep BACs were positioned on the sheep genome with lower confidence. A coordinate conversion process allowed us to transfer human genes and other genome features to the virtual sheep genome to display on a sheep genome browser. CONCLUSION: We demonstrate that limited sequencing of BACs combined with positioning on a well assembled genome and integrating locations from other less well assembled genomes can yield extensive, detailed subgene-level maps of mammalian genomes, for which genomic resources are currently limited.

Identification of a gene network contributing to hypertrophy in callipyge skeletal muscle.

The callipyge mutation in sheep results in postnatal skeletal muscle hypertrophy in the pelvic limbs and loins with little or no effect on anterior skeletal muscles. Associated with the phenotype are changes in the expression of a number of imprinted genes flanking the site of the mutation, which lies in an intergenic region at the telomeric end of ovine chromosome 18. The manner in which these local changes in gene expression are translated into muscle hypertrophy is not known. Microarray-based transcriptional profiling was used to identify differentially expressed genes in longissimus dorsi skeletal muscle samples taken at birth and 12 wk of age from callipyge and wild-type sheep. The phenotype was only expressed at the latter developmental time and associated with decreased type 1 fibers (slow oxidative) and a shift toward type IIx and IIb fibers (fast-twitch glycolytic). We have identified 131 genes in the samples taken at 12 wk of age that were differentially expressed as a function of genotype but not due to the fiber type changes. The gene expression changes occurring as a function of genotype in the samples taken at birth indicated that the transcriptional framework underpinning the phenotype was emerging prior to expression of the phenotype. Eight genes were differentially expressed as a function of genotype at both developmental times. A model is proposed describing a core network of genes and histone epigenetic modifications that is likely to underpin the fiber type changes and muscle hypertrophy characteristic of callipyge sheep.

Determination of sheep prion gene polymorphisms from paraffin-embedded tissue.

Amino acid polymorphisms of the prion protein (PrP) greatly influence the susceptibility of sheep to scrapie. Selective breeding to increase the prevalence of PrP gene alleles associated with scrapie resistance is a flock management practice that is important for scrapie control programs. Determination of sheep PrP alleles typically has required extraction of DNA from host tissues that are freshly derived or stored frozen. We describe application of a DNA extraction procedure for formalin-fixed, paraffin-embedded tissues (PET) for the purpose of PCR amplification and nucleotide sequencing of relevant codons (136-171) of the sheep PrP gene. Tissues derived from 96 sheep were studied. The DNA sequence identity was confirmed in 87 of 94 matched samples of PET and frozen tissue specimens. DNA from brainstem PET of 2 sheep, from which fresh tissue was not available, was amplified and sequenced after formalin fixation for 7-70 days. This method will allow retrospective analysis of PrP genetics of sheep subsequent to postmortem diagnosis of scrapie when nonfixed tissue is unavailable for DNA extraction; however, it is not recommended that submission of fixed tissue supplant collection of fresh tissues for the purpose of determining PrP gene polymorphisms.

BEGAIN: a novel imprinted gene that generates paternally expressed transcripts in a tissue- and promoter-specific manner in sheep.

In this article we describe the organization of the ovine BEGAIN gene, located 138 kb proximally from the imprinted DLK1 gene and 203 kb from the CLPG mutation that causes the callipyge phenotype. We have shown that in sheep BEGAIN is ubiquitously expressed, including in skeletal muscle, throughout development. We have identified four major BEGAIN transcripts resulting from a combination of alternate promoter usage and alternative splicing. In ovine brain, kidney, liver, and skeletal muscle, these four BEGAIN transcripts exhibited paternal or biallelic expression in a tissue- and promoter-specific manner. Our results indicate that the CLPG mutation does not alter transcript levels of BEGAIN, contrary to its effect on a core cluster of genes in the DLK1-GTL2 domain. Thus, although the BEGAIN gene represents another paternally expressed gene in the ovine DLK1-GTL2 imprinted domain, its expression is not governed by the long-range regulatory element that contains the CLPG mutation.