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Reproductive success is an important component of commercial beef cattle production, and identification of DNA markers with predictive merit for reproductive success would facilitate accurate prediction of mean daughter pregnancy rate, enabling effective selection of bulls to improve female fertility. A previous study identified SNP associated with beef cattle reproductive efficiency based on a genomewide association analysis approach using genotyping multiple-animal pools of DNA to increase the number of animals that could be genotyped with available resources. For the current study, we expand on this previous study by individually genotyping cattle from the pooling study for 89 SNP that were associated with female pregnancy rate. The aims of the study were to confirm the results of the pooling study and, more specifically, identify modes of gene action and DNA variations such as haplotypes that would not be possible with pooled genotyping. Eighty-nine SNP selected from the pooling study were evaluated using the Sequenom MassARRAY system to individually genotype animals from populations evaluated in the pooling study, including both and breeds. From this research, regions on chromosomes 5 (26.3-48.1 Mb; UMD3.1 assembly) and 9 (37,436,575 bp; UMD3.1 assembly), first identified in the previous pooling study, were shown through individual genotyping to harbor genetic variation ( < 0.05 genomewide significance) affecting reproductive efficiency in interspecific crosses ( and ) of cattle. Each of these markers exhibited additive (vs. dominant) gene action. Additionally, a haplotype block harboring an allele of origin with negative effects on reproduction was identified on chromosome 5 in interspecific composite breeds of × composites.

The promise of genomic selection is accurate prediction of the genetic potential of animals from their genotypes. Simple DNA tests might replace low-accuracy predictions for expensive or lowly heritable measures of puberty and fertility based on performance and pedigree. Knowing with some certainty which DNA variants (e.g., SNP) affect puberty and fertility is the best way to fulfill the promise. Several SNP from the BovineSNP50 assay have tentatively been associated with reproductive traits including age at puberty, antral follicle count, and pregnancy observed on different sets of heifers. However, sample sizes are too small and SNP density is too sparse to definitively determine genomic regions harboring causal variants affecting reproductive success. Additionally, associations between individual SNP and similar phenotypes are inconsistent across data sets, and genomic predictions do not appear to be globally applicable to cattle of different breeds. Discrepancies may be a result of different QTL segregating in the sampled populations, differences in linkage disequilibrium (LD) patterns such that the same SNP are not correlated with the same QTL, and spurious correlations with phenotype. Several approaches can be used independently or in combination to improve detection of genomic factors affecting heifer puberty and fertility. Larger samples and denser SNP will increase power to detect real associations with SNP having more consistent LD with underlying QTL. Meta-analysis combining results from different studies can also be used to effectively increase sample size. High-density genotyping with heifers pooled by pregnancy status or early and late puberty can be a cost-effective means to sample large numbers. Networks of genes, implicated by associations with multiple traits correlated with puberty and fertility, could provide insight into the complex nature of these traits, especially if corroborated by functional annotation, established gene interaction pathways, and transcript expression. Example analyses are provided to demonstrate how integrating information about gene function and regulation with statistical associations from whole-genome SNP genotyping assays might enhance knowledge of genomic mechanisms affecting puberty and fertility, enabling reliable DNA tests to guide heifer selection decisions.

The three objectives of this study were to 1) test for the existence of beef tenderness markers in the CAPN1 gene segregating in Brahman cattle; 2) test existing CAPN1 tenderness markers in indicus-influenced crossbred cattle; and 3) produce a revised marker system for use in cattle of all subspecies backgrounds. Previously, two SNP in the CAPN1 gene have been described that could be used to guide selection in Bos taurus cattle (designated Markers 316 and 530), but neither marker segregates at high frequency in Brahman cattle. In this study, we examined three additional SNP in CAPN1 to determine whether variation in this gene could be associated with tenderness in a large, multisire American Brahman population. One marker (termed 4751) was associated with shear force on postmortem d 7 (P < 0.01), 14 (P = 0.015), and 21 (P < 0.001) in this population, demonstrating that genetic variation important for tenderness segregates in Bos indicus cattle at or near CAPN1. Marker 4751 also was associated with shear force (P < 0.01) in the same large, multisire population of cattle of strictly Bos taurus descent that was used to develop the previously reported SNP (referred to as the Germplasm Evaluation [GPE] Cycle 7 population), indicating the possibility that one marker could have wide applicability in cattle of all subspecies backgrounds. To test this hypothesis, Marker 4751 was tested in a third large, multisire cattle population of crossbred subspecies descent (including sire breeds of Brangus, Beefmaster, Bonsmara, Romosinuano, Hereford, and Angus referred to as the GPE Cycle 8 population). The highly significant association of Marker 4751 with shear force in this population (P < 0.001) confirms the usefulness of Marker 4751 in cattle of all subspecies backgrounds, including Bos taurus, Bos indicus, and crossbred descent. This wide applicability adds substantial value over previously released Markers 316 and 530. However, Marker 316, which had previously been shown to be associated with tenderness in the GPE Cycle 7 population, also was highly associated with shear force in the GPE Cycle 8 animals (P < 0.001). Thus, Marker 316 may continue to be useful in a variety of populations with a high percentage of Bos taurus backgrounds. An optimal marker strategy for CAPN1 in many cases will be to use both Markers 316 and 4751.

Background: Existing linkage maps of the bovine genome primarily contain anonymous microsatellite markers. These maps have proved valuable for mapping quantitative trait loci (QTL) to broad regions of the genome, but more closely spaced markers are needed to fine-map QTL, and markers associated with genes and annotated sequence are needed to identify genes and sequence variation that may explain QTL., Results: Bovine expressed sequence tag (EST) and bacterial artificial chromosome (BAC)sequence data were used to develop 918 single nucleotide polymorphism (SNP) markers to map genes on the bovine linkage map. DNA of sires from the MARC reference population was used to detect SNPs, and progeny and mates of heterozygous sires were genotyped. Chromosome assignments for 861 SNPs were determined by twopoint analysis, and positions for 735 SNPs were established by multipoint analyses. Linkage maps of bovine autosomes with these SNPs represent 4585 markers in 2475 positions spanning 3058 cM. Markers include 3612 microsatellites, 913 SNPs and 60 other markers. Mean separation between marker positions is 1.2 cM. New SNP markers appear in 511 positions, with mean separation of 4.7 cM. Multi-allelic markers, mostly microsatellites, had a mean (maximum) of 216 (366) informative meioses, and a mean 3-lod confidence interval of 3.6 cM Bi-allelic markers, including SNP and other marker types, had a mean (maximum) of 55 (191) informative meioses, and were placed within a mean 8.5 cM 3-lod confidence interval. Homologous human sequences were identified for 1159 markers, including 582 newly developed and mapped SNP., Conclusion: Addition of these EST- and BAC-based SNPs to the bovine linkage map not only increases marker density, but provides connections to gene-rich physical maps, including annotated human sequence. The map provides a resource for fine-mapping quantitative trait loci and identification of positional candidate genes, and can be integrated with other data to guide and refine assembly of bovine genome sequence. Even after the bovine genome is completely sequenced, the map will continue to be a useful tool to link observable phenotypes and animal genotypes to underlying genes and molecular mechanisms influencing economically important beef and dairy traits.

Micromolar calcium activated neutral protease (CAPN1) was evaluated as a candidate gene for a quantitative trait locus (QTL) on BTA29 affecting meat tenderness by characterization of nucleotide sequence variation in the gene. Single-nucleotide polymorphisms (SNP) were identified by sequencing all 22 exons and 19 of the 21 introns in two sires (Piedmontese x Angus located at the U.S. Meat Animal Research Center in Clay Center, NE; Jersey x Limousin located at AgResearch in New Zealand) of independent resource populations previously shown to be segregating meat tenderness QTL on BTA29. The majority of the 38 SNP were found in introns or were synonymous substitutions in the coding regions, with two exceptions. Exons 14 and 9 contained SNP that were predicted to alter the protein sequence by the substitution of isoleucine for valine in Domain III of the protein, and alanine for glycine in Domain II of the protein. The resource populations were genotyped for these two SNP in addition to six intronic polymorphisms and two silent substitutions. Analysis of genotypes and shear force values in both populations revealed a difference between paternal CAPN1 alleles in which the allele encoding isoleucine at position 530 and glycine at position 316 associated with decreased meat tenderness (increased shear force values) relative to the allele encoding valine at position 530 and alanine at position 316 (P < 0.05). The association of maternal alleles with meat tenderness phenotypes is consistent with the hypothesis of CAPN1 as the gene underlying the QTL effect in two independent resource populations and presents the possibility of using these markers for selective breeding to reduce the numbers of animals with unfavorable meat tenderness traits.

A small genetic region near the telomere of ovine chromosome 18 was previously shown to carry the mutation causing the callipyge muscle hypertrophy phenotype in sheep. Expression of this phenotype is the only known case in mammals of paternal polar overdominance gene action. A region surrounding two positional candidate genes was sequenced in animals of known genotype. Mutation detection focused on an inbred ram of callipyge phenotype postulated to have inherited chromosome segments identical-by-descent with exception of the mutated position. In support of this hypothesis, this inbred ram was homozygous over 210 Kb of sequence, except for a single heterozygous base position. This single polymorphism was genotyped in multiple families segregating the callipyge locus (CLPG), providing 100% concordance with animals of known CLPG genotype, and was unique to descendants of the founder animal. The mutation lies in a region of high homology among mouse, sheep, cattle, and humans, but not in any previously identified expressed transcript. A substantial open reading frame exists in the sheep sequence surrounding the mutation, although this frame is not conserved among species. Initial functional analysis indicates sequence encompassing the mutation is part of a novel transcript expressed in sheep fetal muscle we have named CLPG1.

High-throughput genotyping of swine populations is a potentially efficient method for establishing animal lineage and identification of loci important to animal health and efficient pork production. Markers were developed based upon single nucleotide polymorphisms (SNPs), which are abundant and amenable to automated genotyping platforms. The focus of this research was SNP discovery in expressed porcine genes providing markers to develop the porcine/human comparative map. Locus specific amplification (LSA) and comparative sequencing were used to generate PCR products and allelic information from parents of a swine reference family. Discovery of 1650 SNPs in 403 amplicons and strategies for optimizing LSA-based SNP discovery using alternative methods of PCR primer design, data analysis, and germplasm selection that are applicable to other populations and species are described. These data were the first large-scale assessment of frequency and distribution of porcine SNPs.

DNA marker technology represents a promising means for determining the genetic identity and kinship of an animal. Compared with other types of DNA markers, single nucleotide polymorphisms (SNPs) are attractive because they are abundant, genetically stable, and amenable to high-throughput automated analysis. In cattle, the challenge has been to identify a minimal set of SNPs with sufficient power for use in a variety of popular breeds and crossbred populations. This report describes a set of 32 highly informative SNP markers distributed among 18 autosomes and both sex chromosomes. Informativity of these SNPs in U.S. beef cattle populations was estimated from the distribution of allele and genotype frequencies in two panels: one consisting of 96 purebred sires representing 17 popular breeds, and another with 154 purebred American Angus from six herds in four Midwestern states. Based on frequency data from these panels, the estimated probability that two randomly selected, unrelated individuals will possess identical genotypes for all 32 loci was 2.0 x 10(-13) for multi-breed composite populations and 1.9 x 10(-10) for purebred Angus populations. The probability that a randomly chosen candidate sire will be excluded from paternity was estimated to be 99.9% and 99.4% for the same respective populations. The DNA immediately surrounding the 32 target SNPs was sequenced in the 96 sires of the multi-breed panel and found to contain an additional 183 polymorphic sites. Knowledge of these additional sites, together with the 32 target SNPs, allows the design of robust, accurate genotype assays on a variety of high-throughput SNP genotyping platforms.

The aim of the present study was twofold: first, to design a panel of 96 sires that reflects the breadth of genetic diversity in U.S. beef cattle, and second, to use this panel to discover nucleotide sequence diversity and haplotype structures of interleukin (IL)-8 in commercial populations. The latter is a requisite for epidemiological studies designed to test whether IL8 alleles are risk factors for acquiring or maintaining bacterial infections in production environments. IL-8 encodes a proinflammatory cytokine that plays a central role in cell-mediated immunity by attracting and activating neutrophils in the early stages of host defense against bacterial invasion. Seven single-nucleotide polymorphism (SNP) markers were identified by sequencing two IL8 DNA segments amplified from the panel of 17 popular cattle breeds (MARC beef cattle diversity panel, version 2.1). Assays for automated genotype scoring by matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF MS) were developed to independently verify the seven SNP alleles in the 96 bulls and 313 cattle from the MARC reference population. Five haplotype structures, spanning the two IL8 DNA segments, were unambiguously defined for the set of seven IL8 SNPs. Based on the breadth of germplasm in bovine diversity panel, the five haplotype structures for IL8 are estimated to represent >98% of those present in these DNA segments in commercial populations of U.S. beef cattle. The frequencies of the five respective haplotypes in the eight Angus sires of the diversity panel (0.75, 0.25, 0.00, 0.00. 0.00) were similar to those scored in 150 purebred Angus cattle from six herds in four Midwestern states (0.82, 0.18, 0.01, 0.00 0.00), suggesting that the diversity panel may also be useful for estimating allele frequencies in commercial populations.

DNA sequence variation provides the fundamental material for improving livestock through selection. In cattle, single nucleotide polymorphisms and small insertions/deletions (collectively referred to here as SNPs) have been identified in cytokine genes and scored in a reference population to determine linkage map positions. The aim of the present study was twofold: first, to estimate the SNP frequency in a reference population of beef cattle, and second, to determine cytokine haplotypes in a group of sires from commercial populations. Forty-five SNP markers in DNA segments from nine cytokine gene loci were analyzed in 26 reference parents. Comparison of all 52 haploid genomes at each PCR amplicon locus revealed an average of one SNP per 143 bp of sequence, whereas comparison of any two chromosomes identified heterozygous sites, on average, every 443 bp. The combination of these 45 SNP genotypes was sufficient to uniquely identify each of the 26 animals. The average number of haplotype alleles (4.4) per PCR amplicon (688 bp) and the percentage heterozygosity among founding parents (50%) were similar to those for microsatellite markers in the same population. For 49 sires from seven common breeds of beef cattle, SNP genotypes (1,225 total) were obtained by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) at three amplicon loci. All three of the amplicon haplotypes were correctly deduced for each sire without the use of parent or progeny genotypes. The latter allows a wide range of genetic studies in commercial populations of cattle where genotypic information from relatives may not be available.

Polymorphic markers at bovine gene loci facilitate the integration of cattle genetic maps with those of humans and mice. To this end, 31 single nucleotide polymorphism (SNP) markers were developed for seven bovine chemokine genes. Loci were amplified from bovine genomic DNA by the polymerase chain reaction, and candidate amplicons were sequenced to determine their identity. Amplified loci from 24 founding parents and select progeny from a beef cattle reference population were sequenced and analyzed for SNPs. SNP haplotype alleles were determined by examining segregation patterns and used to establish the locus position on the bovine linkage map. Loci for growth-related proteins (GRO3, GRO1, and GROX) were clustered with the related CXC chemokine genes, interleukin (IL) 8, and epithelial cell inflammatory protein 1, at 84 cM from the centromeric end of the bovine chromosome (BTA) 6 linkage group. Bovine loci for a cluster of IL8 receptors, a stromal cell-derived factor 1, interferon-gamma, and tumor necrosis factor-alpha were mapped at 90, 55, 59, and 34 cM, respectively, from the centromeric ends of the BTA 2, 28, 5, and 23 linkage groups. The positions of these bovine loci were compared with those of orthologous loci on the human map to refine the boundaries of conserved synteny. These seven loci provide examples of SNP development in which the efficiency was largely dependent on the availability of bovine genomic or cDNA sequence. The polymorphic nature of these SNP haplotype markers suggests that they will be useful for mapping complex traits in cattle, such as resistance to infectious disease.