Single nucleotide polymorphisms for DNA typing in the domestic horse.

Genetic markers are important resources for individual identification and parentage assessment. Although short tandem repeats (STRs) have been the traditional DNA marker, technological advances have led to single nucleotide polymorphisms (SNPs) becoming an attractive alternative. SNPs can be highly multiplexed and automatically scored, which allows for easier standardization and sharing among laboratories. Equine parentage is currently assessed using STRs. We obtained a publicly available SNP dataset of 729 horses representing 32 diverse breeds. A proposed set of 101 SNPs was analyzed for DNA typing suitability. The overall minor allele frequency of the panel was 0.376 (range 0.304-0.419), with per breed probability of identities ranging from 5.6 × 10-35 to 1.86 × 10-42 . When one parent was available, exclusion probabilities ranged from 0.9998 to 0.999996, although when both parents were available, all breeds had exclusion probabilities greater than 0.9999999. A set of 388 horses from 35 breeds was genotyped to evaluate marker performance on known families. The set included 107 parent-offspring pairs and 101 full trios. No horses shared identical genotypes across all markers, indicating that the selected set was sufficient for individual identification. All pairwise comparisons were classified using ISAG rules, with one or two excluding markers considered an accepted parent-offspring pair, two or three excluding markers considered doubtful and four or more excluding markers rejecting parentage. The panel had an overall accuracy of 99.9% for identifying true parent-offspring pairs. Our developed marker set is both present on current generation SNP chips and can be highly multiplexed in standalone panels and thus is a promising resource for SNP-based DNA typing.

Feed restriction, but not l-carnitine infusion, alters the liver transcriptome by inhibiting sterol synthesis and mitochondrial oxidative phosphorylation and increasing gluconeogenesis in mid-lactation dairy cows.

Abomasal carnitine infusion during acute feed restriction increases hepatic fatty acid oxidation and decreases liver lipid in dairy cows. Eight mid-lactation Holstein cows were used in a replicated 4×4 Latin square design with 14-d periods. A 2×2 factorial arrangement was used to determine the effects of water infusion+ad libitum dry matter intake (DMI), water infusion+restricted DMI (50% of previous 5-d average), l-carnitine infusion (20 g/d)+ad libitum DMI, or l-carnitine infusion+restricted DMI. Liver RNA from 7 healthy cows was used for transcriptome profiling using a bovine microarray. An ANOVA with a false discovery rate was used to identify treatment and interaction effects. A substantial transcriptome change was observed only with DMI restriction, resulting in 312 (155 downregulated, 157 upregulated) differentially expressed genes. Quantitative PCR was performed to verify microarray data and measure expression of additional genes not present on the microarray. The quantitative PCR data confirmed the effect of feed restriction but not of l-carnitine treatment. Feed restriction increased expression of GPX3 and of genes associated with gluconeogenesis (PC, PDK4), inflammation (SAA3), and signaling (ADIPOR2). In contrast, feed restriction downregulated BBOX, a key for l-carnitine biosynthesis, and the transcription factor HNF4A. The bioinformatics functional analysis of genes affected by DMI restriction uncovered biosynthesis of cholesterol and energy generation by mitochondrial respiration as the most relevant and inhibited functions. The data also indicated an increase of flux toward gluconeogenesis. We interpreted those results as a likely response of the liver to spare energy and provide glucose for the lactating mammary gland during feed deprivation.

Level of nutrient intake affects mammary gland gene expression profiles in preweaned Holstein heifers.

Bovine mammary parenchyma (PAR) and fat pad (MFP) development are responsive to preweaning level of nutrient intake. We studied transcriptome alterations in PAR and MFP from Holstein heifer calves (n=6/treatment) fed different nutrient intakes from birth to ca. 65 d age. Conventional nutrient intake received 441 g of dry matter (DM)/d of a control milk replacer (MR) [CON; 20% crude protein (CP), 20% fat, DM basis]. Calves in the accelerated nutrition groups received 951 g/d of high-protein/low-fat MR (HPLF; 28% CP, 20% fat, DM basis), 951 g/d of high-protein/high-fat MR (HPHF; 28% CP, 28% fat, DM basis), or 1,431 g/d of HPHF (HPHF+) MR. Out of 13,000 genes evaluated, over 1,500 differentially expressed genes (DEG) were affected (false discovery rate <0.10) by level of nutrient intake in PAR or MFP. Feeding HPLF versus CON resulted in the most dramatic changes in gene expression, with 278 and 588 DEG having ≥1.5-fold change in PAR and MFP. In PAR, the most-altered molecular functions were associated with metabolism of the cell (molecular transport and lipid metabolism) with most of the genes downregulated in HPLF versus CON. In MFP, DEG also were primarily associated with metabolism but changes also occurred in genes linked to cell morphology, cell-to-cell signaling, and immune response. Compared with CON, feeding HPHF or HPHF+ did not result in substantial additional effects on DEG beyond those observed with HPLF. The pentose phosphate, mitochondrial dysfunction, and ubiquinone biosynthesis pathways were among the most enriched due to HPLF versus CON in PAR and were inhibited, whereas glycosphingolipid biosynthesis, arachidonic acid metabolism, and eicosanoid synthesis pathways were among the most enriched due to HPLF versus CON in MFP and were inhibited. These responses suggest that, in PAR, doubling nutrient intake from standard feeding rates inhibited energy metabolism and activity of oxidative pathways that partly serve to protect cells against oxidative stress. The MFP in those heifers appeared to decrease production of lipid-derived metabolites that may play roles in signaling pathways within the adipocyte. Overall, results indicated that prepubertal/preweaned mammary transcriptome is responsive to long-term enhanced nutrient supply to achieve greater growth rates before weaning. The biological significance of these results to future milk production remains to be elucidated.

Pharmacogenetic allele nomenclature: International workgroup recommendations for test result reporting.

This article provides nomenclature recommendations developed by an international workgroup to increase transparency and standardization of pharmacogenetic (PGx) result reporting. Presently, sequence variants identified by PGx tests are described using different nomenclature systems. In addition, PGx analysis may detect different sets of variants for each gene, which can affect interpretation of results. This practice has caused confusion and may thereby impede the adoption of clinical PGx testing. Standardization is critical to move PGx forward.

Bone disorders in the dog: a review of modern genetic strategies to find the underlying causes.

In man, the genetic defects of more than 600 inherited diseases, of which at least 150 skeletal diseases, have been identified as is the chromosomal location for approximately 7000 genes. This rapid progress has been made possible by the generation of a genetical and physical map of the human genome. There is no reason to believe that for the dog not a similar development may occur. This review is therefore focussed on the use of novel tools now available for comparative molecular genetic studies of skeletal dysplasias in the dog. Because the genomes of mammals at the subchromosomal level are very well conserved, likely candidate disease genes known from other species might be considered. In this review, formation of the bones and the most important canine disorders of the skeleton influencing locomotion will be discussed first. The canine disorders discussed are canine hip dysplasia, the three different forms of elbow dysplasia (fragmented coronoid process, ununited anconeal process, osteochondrosis dissecans and incongruency) and dwarfism. Where possible a link is made with similar diseases in man or mouse. Then, the molecular biological tools available to analyse the genetic defect will be reviewed and some examples discussed.

Comparative analysis of the early transcriptome of Brucella abortus--infected monocyte-derived macrophages from cattle naturally resistant or susceptible to brucellosis.

Brucellosis is a worldwide zoonotic infectious disease that has a significant economic impact on animal production and human public health. We characterized the gene expression profile of B. abortus-infected monocyte-derived macrophages (MDMs) from naïve cattle naturally resistant (R) or susceptible (S) to brucellosis using a cDNA microarray technology. Our data indicate that (1) B. abortus induced a slightly increased genome activation in R MDMs and a down-regulated transcriptome in S MDMs, during the onset of infection, (2) R MDMs had the ability to mount a type 1 immune response against B. abortus infection which was impaired in S cells, and (3) the host cell activity was not altered after 12 h post-B. abortus infection in R MDMs while the cell cycle was largely arrested in infected S MDMs at 12 h p.i. These results contribute to an improved understanding of how host responses may be manipulated to prevent infection by brucellae.

Identification of a premature stop codon in the melanocyte-stimulating hormone receptor gene (MC1R) in Labrador and Golden retrievers with yellow coat colour.

We have examined whether black/yellow coat colour in Labrador retrievers is controlled by allelic variants at the extension locus. As the gene encoding the melanocyte-stimulating hormone receptor (MC1R) has been shown to correspond to the extension locus in several species, we have determined the genomic MC1R sequence in Labrador retrievers with black and with yellow coat colour. Using primers based on the fox (Vulpes vulpes) MC1R sequence we initially isolated and sequenced the innerpart of the canine MC1R. By means of inverse PCR we succeeded in the characterization of both flanking regions of the MC1R gene (Genbank: AF064455). Comparison of the complete MC1R sequences of a yellow and a black Labrador retriever revealed a single C-->T mutation at nucleotide position 916 in the yellow dog. This transition changed the codon for arginine at position 305 into a stop codon, resulting in the elimination of the evolutionary strongly conserved 10 carboxyterminal amino acid residues. With an allele-specific-oligonucleotide (ASO) test it was shown that the mutation cosegregated with the recessively inherited yellow coat colour in the Labrador retriever. Golden retrievers also appeared to be homozygous for the mutation. Seventeen other breeds were all negative for the mutation. Since the Labrador and Golden retriever are closely related, we suggest a common founder for the yellow coat colour in Labrador and Golden retrievers.

Isolation of DNA markers informative in purebred dog families by genomic representational difference analysis (gRDA).

Genomic Representational Difference Analysis (gRDA) is a subtractive DNA method to clone the differences between two related genomes, called tester and driver. We have evaluated this method to obtain polymorphic DNA markers for pedigree dogs. Amplified size-selected genomic restriction fragments (amplicons) of two dog littermates were repeatedly hybridized to each other in order to remove (subtract) those restriction fragments common to both sibs. Already after two rounds of subtractive hybridization, a clear enrichment of presumably tester-specific restriction fragments was observed, which was even more pronounced after the third round of subtraction. A plasmid library of 3000 recombinant clones was constructed of the second round and of the third round difference product. DNA sequence determination of randomly chosen clones of each difference product showed that approximately 1000 unique clones were obtained in the second-round difference product and approximately 500 in the third-round difference product. About half of the clones identified in the second-round difference product were also present in the third-round difference product. Of the second-round difference product, 39 different gRDA fragments could be identified, of which 21 were tester specific. In the third-round difference product, 22 different gRDA fragments were identified, of which 18 were tester specific. There were 13 fragments in common, resulting in a total of 48 different fragments. In order to establish the localization of these markers, we performed mapping using the dog radiation hybrid panel RHDF5000. Of 39 mapped clones, 29 were mapped to 20 existing RH groups, and 10 remained unlinked. It is concluded that gRDA is suitable to generate DNA markers to track disease genes within lines of pedigree dogs.

A radiation hybrid map of the X-chromosome of the dog (Canis familiaris).

The dog serves as an animal model for several human diseases including X-chromosome diseases. Although the canine X-chromosome is one of the largest chromosomes in the dog, only a few markers have been mapped to it to date. Using a commercially available canine whole genome radiation hybrid (RH) panel we have localized 14 microsatellite markers, 18 genes and 13 STSs on the canine X-chromosome, extending the total number of mapped markers to 45 covering an estimated 830 cR. Out of these 45 markers, seven distinct groups of markers could be established with an average spacing of 18.8 cR(3000) and ten markers remained unlinked. Using FISH analysis, six markers could be mapped physically to the p- or q-arm of the X-chromosome. Combined with the FISH mapping, three RH groups could be assigned to the p-arm and two RH groups to the q-arm. Comparison with the human X-chromosome map revealed conserved synteny up to 234 cR (TIMP1-ALAS2-AR-IL2RG-XIST). We show here that the similarity of the canine and human X-chromosomes is the largest for any mammalian species beyond the primates.

A male linkage map of the cattle (Bos taurus) genome.

A male linkage map of the cattle (Bos taurus) genome was constructed using nine large half-sib families. The map consists of 269 loci, of which 249 are microsatellites and 20 are structural genes. Among the 249 microsatellites, 140 are markers selected from other maps and 98 are new assignments. Chromosome assignment were established for 35 new markers by somatic cell hybrid analysis, of which 26 were confirmed by linkage analysis. Genome coverage is 1975 cM contained within terminal markers on all 29 autosomes. The average distance between adjacent loci is 9.7 cM, with 72.1% of the map intervals < or = 15 cM and 4.9% of the intervals > or = 25 cM. The inclusion of mapped markers permitted integration and comparisons with other maps, facilitating the identification of discrepancies in chromosome assignment, gene order, and map distance. The inclusion of Type I and blood group markers in the map was useful for comparative mapping, revealing possible blood group orthologies between humans and cattle. The map generated will serve as a useful tool for comparative mapping, mapping of quantitative trait loci and marker assisted selection.