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The attainment of a specific mature body size is one of the most fundamental differences among species of mammals. Moreover, body size seems to be the central factor underlying differences in traits such as growth rate, energy metabolism and body composition. An important proportion of this variability is of genetic origin. The goal of the genetic analysis of animal growth is to understand its "genetic architecture", that is the number and position of loci affecting the trait, the magnitude of their effects, allele frequencies and types of gene action. In this review, the different strategies developed to identify and characterize genes involved in the regulation of growth in the mouse are described, with emphasis on the methods developed to map loci contributing to the regulation of quantitative traits (QTLs).

Despite regulatory elements such as long non - coding RNAs representing most of the transcriptome, the functional understanding of long non - coding RNAs in relation to major health conditions including bovine mastitis is limited. This study examined the milk somatic cell transcriptome from udder quarters of 6 Holstein dairy cows to identify differentially expressed long non - coding RNAs using RNA - Sequencing. Ninety - four differentially expressed long non - coding RNAs are identified, 5 of which are previously annotated for gene name and length, 11 are annotated for gene name and 78 are novel, having no gene name or length previously annotated. Significant inflammatory response and regulation of immune response pathways (false discovery rate < 0.05) are associated with the differentially expressed long non - coding RNAs. QTL annotation analysis revealed 31 QTL previously annotated in the genomic regions of the 94 differentially expressed long non - coding RNAs, and the majority are associated with milk traits. This research provides a better understanding of long non - coding RNAs regulatory elements in milk somatic cells, which may enhance current breeding strategies for more adaptable or high mastitis resistant cattle., (© 2024. The Author(s).)

Anti-Müllerian hormone (AMH) is proposed as a biomarker for fertility in cattle, yet this associative relationship appears to be influenced by heat stress (HS). The objective was to test serum AMH and AMH-related single nucleotide polymorphisms (SNPs) as markers potentially predictive of reproductive traits in dairy cows experiencing HS. The study included 300 Holstein cows that were genotyped using BovineSNP50 (54,000 SNP). A genome-wide association study was then executed. Nine intragenic SNPs within the pathways that influence the AMH gene were found important with multiple comparisons adjustment tests ( p < 1.09 × 10 -6 ). A further validation study was performed in an independent Holstein cattle population, which was divided into moderate (MH; n = 152) and severe heat-stressed (SH; n = 128) groups and then subjected to a summer reproductive management program. Serum AMH was confirmed as a predictor of fertility measures ( p < 0.05) in MH but not in the SH group. Cows were genotyped, which revealed four SNPs as predictive markers for serum AMH ( p < 0.01), reproductive traits ( p < 0.01), and additional physiological variables ( p < 0.05). These SNPs were in the genes AMH , IGFBP1 , LGR5 , and TLR4 . In conclusion, serum AMH concentrations and AMH polymorphisms are proposed as predictive markers that can be used in conjunction with genomic breeding value approaches to improve reproductive performance in Holstein cows exposed to summer HS conditions.

Dairy production in Holstein cows in a semiarid environment is challenging due to heat stress. Under such conditions, genetic selection for heat tolerance appears to be a useful strategy. The objective was to validate molecular markers associated with milk production and thermotolerance traits in Holstein cows managed in a hot and humid environment. Lactating cows ( n = 300) exposed to a heat stress environment were genotyped using a medium-density array including 53,218 SNPs. A genome-wide association study (GWAS) detected six SNPs associated with total milk yield (MY305) that surpassed multiple testing ( p < 1.14 × 10 -6 ). These SNPs were further validated in 216 Holstein cows from two independent populations that were genotyped using the TaqMan bi-allelic discrimination method and qPCR. In these cows, only the SNPs rs8193046, rs43410971, and rs382039214, within the genes TLR4 , GRM8 , and SMAD3 , respectively, were associated ( p < 0.05) with MY305, rectal temperature (RT), and respiratory rate. Interestingly, these variables improved as the number of favorable genotypes of the SNPs increased from 0 to 3. In addition, a regression analysis detected RT as a significant predictor (R 2 = 0.362) for MY305 in cows with >1 favorable genotype, suggesting this close relationship was influenced by genetic markers. In conclusion, SNPs in the genes TLR4 , GRM8 , and SMAD3 appear to be involved in the molecular mechanism that regulates milk production in cows under heat-stressed conditions. These SNPs are proposed as thermotolerance genetic markers for a selection program to improve the milk performance of lactating Holstein cows managed in a semiarid environment.

We investigated the possibility of single-step genome editing in small ruminants by CRISPR-Cas9 zygote electroporation. We targeted SOCS2 and PDX1 in sheep embryos and OTX2 in goat embryos, utilizing a dual sgRNA approach. Gene editing efficiency was compared between microinjection and three different electroporation settings performed at four different times of embryo development. Electroporation of sheep zygotes 6 h after fertilization with settings that included short high-voltage (poring) and long low-voltage (transfer) pulses was efficient at producing SOCS2 knock-out blastocysts. The mutation rate after CRISPR/Cas9 electroporation was 95.6% ± 8%, including 95.4% ± 9% biallelic mutations; which compared favorably to 82.3% ± 8% and 25% ± 10%, respectively, when using microinjection. We also successfully disrupted the PDX1 gene in sheep and the OTX2 gene in goat embryos. The biallelic mutation rate was 81 ± 5% for PDX1 and 85% ± 6% for OTX2. In conclusion, using single-step CRISPR-Cas9 zygote electroporation, we successfully introduced biallelic deletions in the genome of small ruminant embryos.

Background: Mastitis is a very common disease in the dairy industry that producers encounter daily. Transcriptomics, using RNA-Sequencing (RNA - Seq) technology, can be used to study the functional aspect of mastitis resistance to identify animals that have a better immune response to mastitis. When the cow has mastitis, not only genes but also specific mRNA isoforms generated via alternative splicing (AS) could be differentially expressed (DE), leading to the phenotypic variation observed. Therefore, the objective of this study was to use large gap read mapping to identify mRNA isoforms DE between healthy and mastitic milk somatic cell samples (N = 12). These mRNA isoforms were then categorized based on being 1) annotated mRNA isoforms for gene name and length, 2) annotated mRNA isoforms with different transcript length and 3) novel mRNA isoforms of non - annotated genes., Results: Analysis identified 333 DE transcripts (with at least 2 mRNA isoforms annotated, with at least one being DE) between healthy and mastitic samples corresponding to 303 unique genes. Of these 333 DE transcripts between healthy and mastitic samples, 68 mRNA isoforms are annotated in the bovine genome reference (ARS.UCD.1.2), 249 mRNA isoforms had novel transcript lengths of known genes and 16 were novel transcript lengths of non - annotated genes in the bovine genome reference (ARS.UCD.1.2). Functional analysis including gene ontology, gene network and metabolic pathway analysis was performed on the list of 288 annotated and unique DE mRNA isoforms. In total, 67 significant metabolic pathways were identified including positive regulation of cytokine secretion and immune response. Additionally, numerous DE novel mRNA isoforms showed potential involvement with the immune system or mastitis. Lastly, QTL annotation analysis was performed on coding regions of the DE mRNA isoforms, identifying overlapping QTLs associated with clinical mastitis and somatic cell score., Conclusion: This study identified novel mRNA isoforms generated via AS that could lead to differences in the immune response of Holstein dairy cows and be potentially implemented in future breeding programs., (© 2022. The Author(s).)

A comprehensive annotation of transcript isoforms in domesticated species is lacking. Especially considering that transcriptome complexity and splicing patterns are not well-conserved between species, this presents a substantial obstacle to genomic selection programs that seek to improve production, disease resistance, and reproduction. Recent advances in long-read sequencing technology have made it possible to directly extrapolate the structure of full-length transcripts without the need for transcript reconstruction. In this study, we demonstrate the power of long-read sequencing for transcriptome annotation by coupling Oxford Nanopore Technology (ONT) with large-scale multiplexing of 93 samples, comprising 32 tissues collected from adult male and female Hereford cattle. More than 30 million uniquely mapping full-length reads were obtained from a single ONT flow cell, and used to identify and characterize the expression dynamics of 99,044 transcript isoforms at 31,824 loci. Of these predicted transcripts, 21% exactly matched a reference transcript, and 61% were novel isoforms of reference genes, substantially increasing the ratio of transcript variants per gene, and suggesting that the complexity of the bovine transcriptome is comparable to that in humans. Over 7,000 transcript isoforms were extremely tissue-specific, and 61% of these were attributed to testis, which exhibited the most complex transcriptome of all interrogated tissues. Despite profiling over 30 tissues, transcription was only detected at about 60% of reference loci. Consequently, additional studies will be necessary to continue characterizing the bovine transcriptome in additional cell types, developmental stages, and physiological conditions. However, by here demonstrating the power of ONT sequencing coupled with large-scale multiplexing, the task of exhaustively annotating the bovine transcriptome - or any mammalian transcriptome - appears significantly more feasible., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Halstead, Islas-Trejo, Goszczynski, Medrano, Zhou and Ross.)

Gene regulatory elements are central drivers of phenotypic variation and thus of critical importance towards understanding the genetics of complex traits. The Functional Annotation of Animal Genomes consortium was formed to collaboratively annotate the functional elements in animal genomes, starting with domesticated animals. Here we present an expansive collection of datasets from eight diverse tissues in three important agricultural species: chicken (Gallus gallus), pig (Sus scrofa), and cattle (Bos taurus). Comparative analysis of these datasets and those from the human and mouse Encyclopedia of DNA Elements projects reveal that a core set of regulatory elements are functionally conserved independent of divergence between species, and that tissue-specific transcription factor occupancy at regulatory elements and their predicted target genes are also conserved. These datasets represent a unique opportunity for the emerging field of comparative epigenomics, as well as the agricultural research community, including species that are globally important food resources.

Background: Although considerable progress has been made towards annotating the noncoding portion of the human and mouse genomes, regulatory elements in other species, such as livestock, remain poorly characterized. This lack of functional annotation poses a substantial roadblock to agricultural research and diminishes the value of these species as model organisms. As active regulatory elements are typically characterized by chromatin accessibility, we implemented the Assay for Transposase Accessible Chromatin (ATAC-seq) to annotate and characterize regulatory elements in pigs and cattle, given a set of eight adult tissues., Results: Overall, 306,304 and 273,594 active regulatory elements were identified in pig and cattle, respectively. 71,478 porcine and 47,454 bovine regulatory elements were highly tissue-specific and were correspondingly enriched for binding motifs of known tissue-specific transcription factors. However, in every tissue the most prevalent accessible motif corresponded to the insulator CTCF, suggesting pervasive involvement in 3-D chromatin organization. Taking advantage of a similar dataset in mouse, open chromatin in pig, cattle, and mice were compared, revealing that the conservation of regulatory elements, in terms of sequence identity and accessibility, was consistent with evolutionary distance; whereas pig and cattle shared about 20% of accessible sites, mice and ungulates only had about 10% of accessible sites in common. Furthermore, conservation of accessibility was more prevalent at promoters than at intergenic regions., Conclusions: The lack of conserved accessibility at distal elements is consistent with rapid evolution of enhancers, and further emphasizes the need to annotate regulatory elements in individual species, rather than inferring elements based on homology. This atlas of chromatin accessibility in cattle and pig constitutes a substantial step towards annotating livestock genomes and dissecting the regulatory link between genome and phenome.

The cattle reference genome assembly has underpinned major innovations in beef and dairy genetics through genome-enabled selection, including removal of deleterious recessive variants and selection for favorable alleles affecting quantitative production traits. The initial reference assemblies, up to and including UMD3.1 and Btau4.1, were based on a combination of clone-by-clone sequencing of bacterial artificial chromosome clones generated from blood DNA of a Hereford bull and whole-genome shotgun sequencing of blood DNA from his inbred daughter/granddaughter named L1 Dominette 01449 (Dominette). The approach introduced assembly gaps, misassemblies, and errors, and it limited the ability to assemble regions that undergo rearrangement in blood cells, such as immune gene clusters. Nonetheless, the reference supported the creation of genotyping tools and provided a basis for many studies of gene expression. Recently, long-read sequencing technologies have emerged that facilitated a re-assembly of the reference genome, using lung tissue from Dominette to resolve many of the problems and providing a bridge to place historical studies in common context. The new reference, ARS-UCD1.2, successfully assembled germline immune gene clusters and improved overall continuity (i.e., reduction of gaps and inversions) by over 250-fold. This reference properly places nearly all of the legacy genetic markers used for over a decade in the industry. In this review, we discuss the improvements made to the cattle reference; remaining issues present in the assembly; tools developed to support genome-based studies in beef and dairy cattle; and the emergence of newer genome assembly methods that are producing even higher-quality assemblies for other breeds of cattle at a fraction of the cost. The new frontier for cattle genomics research will likely include a transition from the individual Hereford reference genome, to a "pan-genome" reference, representing all the DNA segments existing in commonly used cattle breeds, bringing the cattle reference into line with the current direction of human genome research., (Copyright © 2020 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Background: Major advances in selection progress for cattle have been made following the introduction of genomic tools over the past 10-12 years. These tools depend upon the Bos taurus reference genome (UMD3.1.1), which was created using now-outdated technologies and is hindered by a variety of deficiencies and inaccuracies., Results: We present the new reference genome for cattle, ARS-UCD1.2, based on the same animal as the original to facilitate transfer and interpretation of results obtained from the earlier version, but applying a combination of modern technologies in a de novo assembly to increase continuity, accuracy, and completeness. The assembly includes 2.7 Gb and is >250× more continuous than the original assembly, with contig N50 >25 Mb and L50 of 32. We also greatly expanded supporting RNA-based data for annotation that identifies 30,396 total genes (21,039 protein coding). The new reference assembly is accessible in annotated form for public use., Conclusions: We demonstrate that improved continuity of assembled sequence warrants the adoption of ARS-UCD1.2 as the new cattle reference genome and that increased assembly accuracy will benefit future research on this species., (© The Author(s) 2020. Published by Oxford University Press.)

Inorganic arsenic oxides have been identified as carcinogens in several human tissues, including epidermis. Due to the chemical similarity between trivalent inorganic arsenic (arsenite) and antimony (antimonite), we hypothesized that common intracellular targets lead to similarities in cellular responses. Indeed, transcriptional and proteomic profiling revealed remarkable similarities in differentially expressed genes and proteins resulting from exposure of cultured human epidermal keratinocytes to arsenite and antimonite in contrast to comparisons of arsenite with other metal compounds. These data were analyzed to predict upstream regulators and affected signaling pathways following arsenite and antimonite treatments. A majority of the top findings in each category were identical after treatment with either compound. Inspection of the predicted upstream regulators led to previously unsuspected roles for oncostatin M, corticosteroids and ephrins in mediating cellular response. The influence of these predicted mediators was then experimentally verified. Together with predictions of transcription factor effects more generally, the analysis has led to model signaling networks largely accounting for arsenite and antimonite action. The striking parallels between responses to arsenite and antimonite indicate the skin carcinogenic risk of exposure to antimonite merits close scrutiny.

Reactive Nitrogen Species (RNS) are a group of bactericidal molecules produced by macrophages in response to pathogens in a process called oxidative burst. Nitric oxide (NO - ) is a member of RNS produced from arginine by inducible Nitric Oxide Synthase (iNOS) enzyme. The activity of iNOS and production of NO - by macrophages following stimulation is one of the indicators of macrophage polarization towards M1/proinflammatory. Production of NO - by bovine monocyte-derived macrophage (MDM) and mouse peritoneal macrophages has been shown to be strongly associated with host genetic with the heritability of 0.776 in bovine MDM and 0.8 in mouse peritoneal macrophages. However, the mechanism of genetic regulation of macrophage response has remained less explored. In the current study, the transcriptome of bovine MDMs was compared between two extreme phenotypes that had been classified as high and low responder based on NO - production. The results showed that 179 and 392 genes were differentially expressed (DE) between high and low responder groups at 3 and 18 hours after exposure to Escherichia coli, respectively. A set of 11 Transcription Factors (TFs) (STAT1, IRF7, SPI1, STAT4, IRF1, HIF1A, FOXO3, REL, NFAT5, HIC1, and IRF4) at 3 hours and a set of 13 TFs (STAT1, IRF1, HIF1A, STAT4, ATF4, TP63, EGR1, CDKN2A, RBL1, E2F1, PRDM1, GATA3, and IRF4) at 18 hours after exposure to E. coli were identified to be differentially regulated between the high and low responder phenotypes. These TFs were found to be divided into two clusters of inflammatory- and hypoxia-related TFs. Functional analysis revealed that some key canonical pathways such as phagocytosis, chemotaxis, antigen presentation, and cell-to-cell signalling are enriched among the over-expressed genes by high responder phenotype. Based on the results of this study, it was inferred that the functional characteristics of bovine MDMs are associated with NO-based classification. Since NO - production is strongly associated with host genetics, this study for the first time shows the distinct proinflammatory profiles of macrophages are controlled by the natural genetic polymorphism in an outbred population. In addition, the results suggest that genetics can be considered as a new dimension in the current model of macrophage polarization which is currently described by the combination of stimulants, only.

Maternal recognition of pregnancy (MRP) in the mare is not well defined. In a non-pregnant mare, prostaglandin F 2α (PGF) is released on day 14 post-ovulation (PO) to cause luteal regression, resulting in loss of progesterone production. Equine MRP occurs prior to day 14 to halt PGF production. Studies have failed to identify a gene candidate for MRP, so attention has turned to small, non-coding RNAs. The objective of this study was to evaluate small RNA (<200 nucleotides) content in endometrium during MRP. Mares were used in a cross-over design with each having a pregnant and non-mated cycle. Each mare was randomly assigned to collection day 11 or 13 PO ( n = 3/day) and endometrial biopsies were obtained. Total RNA was isolated and sequencing libraries were prepared using a small RNA library preparation kit and sequenced on a HiSeq 2000. EquCab3 was used as the reference genome and DESeq2 was used for statistical analysis. On day 11, 419 ncRNAs, representing miRNA, snRNA, snoRNA, scaRNA, and vaultRNA, were different between pregnancy statuses, but none on day 13. Equine endometrial ncRNAs with unknown structure and function were also identified. This study is the first to describe ncRNA transcriptome in equine endometrium. Identifying targets of these ncRNAs could lead to determining MRP.

Mastitis is a very costly and common disease in the dairy industry. The study of the transcriptome from healthy and mastitic milk somatic cell samples using RNA-Sequencing technology can provide measurements of transcript levels associated with the immune response to the infection. The objective of this study was to characterize the Holstein milk somatic cell transcriptome from 6 cows to determine host response to intramammary infections. RNA-Sequencing was performed on 2 samples from each cow from 2 separate quarters, one classified as healthy (n = 6) and one as mastitic (n = 6). In total, 449 genes were differentially expressed between the healthy and mastitic quarters (false discovery rate <0.05, fold change >±2). Among the differentially expressed genes, the most expressed genes based on reads per kilobase per million mapped reads (RPKM) in the healthy group were associated with milk components (CSN2 and CSN3), and in the mastitic group they were associated with immunity (B2M and CD74). In silico functional analysis was performed using the list of 449 differentially expressed genes, which identified 36 significantly enriched metabolic pathways (false discovery rate <0.01), some of which were associated with the immune system, such as cytokine-cytokine interaction and cell adhesion molecules. Seven functional candidate genes were selected, based on the criteria of being highly differentially expressed between healthy and mastitic groups and significantly enriched in metabolic pathways that are relevant to the inflammatory process (GLYCAM1, B2M, CD74, BoLA-DRA, FCER1G, SDS, and NFKBIA). Last, we identified the differentially expressed genes that are located in quantitative trait locus regions previously known to be associated with mastitis, specifically clinical mastitis, somatic cell count, and somatic cell score. It was concluded that multiple genes within quantitative trait locus regions could potentially affect host response to mastitis-causing agents, making some cows more susceptible to intramammary infections. The identification of potential candidate genes with functional, statistical, biological, and positional relevance associated with host defense to infection will contribute to a better understanding of the underlying genetic architecture associated with mastitis. This in turn will improve the sustainability of agricultural practices by facilitating the selection of cows with improved host defense leading to increased resistance to mastitis., (Copyright © 2019 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Equine maternal recognition of pregnancy (MRP) is a process whose signal remains unknown. During MRP the conceptus and endometrium communicate to attenuate prostaglandin F 2α (PGF) secretion, sparing the corpus luteum and maintaining progesterone production. Recognition of a mobile conceptus by the endometrium is critical by days 14-16 post-ovulation (PO), when endometrium produces PGF, initiating luteolysis. The objective of this study was to evaluate endometrial gene expression changes based upon pregnancy status via RNA sequencing. This experiment utilized a cross-over design with each mare serving as both a pregnant and non-mated control on days nine, 11, and 13 PO ( n = 3/status/day). Mares were randomly assigned to collection day and pregnancy confirmed by terminal uterine lavage at the time of endometrial biopsy. Total RNA was isolated and libraries prepared using Illumina TruSeq RNA sample preparation kit. Reads were mapped and annotated using HISAT2 and Stringtie. Expression values were evaluated with DESEQ2 ( P ≤ 0.05 indicated significance). On day nine, 11, and 13 there were 1435, 1435 and 916 significant transcripts, respectively. Multiple genes with splice variants had different expression patterns within the same day. These are the first data to evaluate the endometrial transcriptome during MRP on days nine, 11, and 13.

Embryonic stem cells (ESCs) are derived from the inner cell mass of preimplantation blastocysts. For decades, attempts to efficiently derive ESCs in animal livestock species have been unsuccessful, but this goal has recently been achieved in cattle. Together with the recent reconstitution of the germ cell differentiation processes from ESCs in mice, these achievements open new avenues for the development of promising technologies oriented toward improving health, animal production, and the environment. In this article, we present a strategy that will notably accelerate genetic improvement in livestock populations by reducing the generational interval, namely in vitro breeding (IVB). IVB combines genomic selection, a widely used strategy for genetically improving livestock, with ESC derivation and in vitro differentiation of germ cells from pluripotent stem cells. We also review the most recent findings in the fields on which IVB is based. Evidence suggests this strategy will be soon within reach., (© The Author(s) 2018. Published by Oxford University Press on behalf of Society for the Study of Reproduction.)

Realized deviations from the expected Mendelian inheritance of alleles from heterozygous parents have been previously reported in a broad range of organisms (i.e., transmission ratio distortion; TRD). Various biological mechanisms affecting gametes, embryos, fetuses, or even postnatal offspring can produce patterns of TRD. However, knowledge about its prevalence and potential causes in livestock species is still scarce. Specific Bayesian models have been recently developed for the analyses of TRD for biallelic loci, which accommodated a wide range of population structures, enabling TRD investigation in livestock populations. The parameterization of these models is flexible and allows the study of overall (parent-unspecific) TRD and sire- and dam-specific TRD. This research aimed at deriving Bayesian models for fitting TRD on the basis of haplotypes, testing the models for both haplotype- and SNP-based methods in simulated data and actual Holstein genotypes, and developing a specific software for TRD analyses. Results obtained on simulated data sets showed that the statistical power of the analysis increased with sample size of trios (n), proportion of heterozygous parents, and the magnitude of the TRD. On the other hand, the statistical power to detect TRD decreased with the number of alleles at each loci. Bayesian analyses showed a strong Pearson correlation coefficient (≥0.97) between simulated and estimated TRD that reached the significance level of Bayes factor ≥10 for both single-marker and haplotype analyses when n ≥ 25. Moreover, the accuracy in terms of the mean absolute error decreased with the increase of the sample size and increased with the number of alleles at each loci. Using real data (55,732 genotypes of Holstein trios), SNP- and haplotype-based distortions were detected with overall TRD, sire-TRD, or dam-TRD, showing different magnitudes of TRD and statistical relevance. Additionally, the haplotype-based method showed more ability to capture TRD compared with individual SNP. To discard possible random TRD in real data, an approximate empirical null distribution of TRD was developed. The program TRDscan v.1.0 was written in Fortran 2008 language and provides a powerful statistical tool to scan for TRD regions across the whole genome. This developed program is freely available at http://www.casellas.info/files/TRDscan.zip., (Copyright © 2019 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

The identification of biological processes related to the regulation of complex traits is a difficult task. Commonly, complex traits are regulated through a multitude of genes contributing each to a small part of the total genetic variance. Additionally, some loci can simultaneously regulate several complex traits, a phenomenon defined as pleiotropy. The lack of understanding on the biological processes responsible for the regulation of these traits results in the decrease of selection efficiency and the selection of undesirable hitchhiking effects. The identification of pleiotropic key-regulator genes can assist in developing important tools for investigating biological processes underlying complex traits. A multi-breed and multi-OMICs approach was applied to study the pleiotropic effects of key-regulator genes using three independent beef cattle populations evaluated for fertility traits. A pleiotropic map for 32 traits related to growth, feed efficiency, carcass and meat quality, and reproduction was used to identify genes shared among the different populations and breeds in pleiotropic regions. Furthermore, data-mining analyses were performed using the Cattle QTL database (CattleQTLdb) to identify the QTL category annotated in the regions around the genes shared among breeds. This approach allowed the identification of a main gene network (composed of 38 genes) shared among breeds. This gene network was significantly associated with thyroid activity, among other biological processes, and displayed a high regulatory potential. In addition, it was possible to identify genes with pleiotropic effects related to crucial biological processes that regulate economically relevant traits associated with fertility, production and health, such as MYC, PPARG, GSK3B, TG and IYD genes. These genes will be further investigated to better understand the biological processes involved in the expression of complex traits and assist in the identification of functional variants associated with undesirable phenotypes, such as decreased fertility, poor feed efficiency and negative energetic balance., Competing Interests: The authors have declared that no competing interests exist.

Background: Numerous long non-coding RNAs (lncRNAs) have been identified and their roles in gene regulation in humans, mice, and other model organisms studied; however, far less research has been focused on lncRNAs in farm animal species. While previous studies in chickens, cattle, and pigs identified lncRNAs in specific developmental stages or differentially expressed under specific conditions in a limited number of tissues, more comprehensive identification of lncRNAs in these species is needed. The goal of the FAANG Consortium (Functional Annotation of Animal Genomes) is to functionally annotate animal genomes, including the annotation of lncRNAs. As one of the FAANG pilot projects, lncRNAs were identified across eight tissues in two adult male biological replicates from chickens, cattle, and pigs., Results: Comprehensive lncRNA annotations for the chicken, cattle, and pig genomes were generated by utilizing RNA-seq from eight tissue types from two biological replicates per species at the adult developmental stage. A total of 9393 lncRNAs in chickens, 7235 lncRNAs in cattle, and 14,429 lncRNAs in pigs were identified. Including novel isoforms and lncRNAs from novel loci, 5288 novel lncRNAs were identified in chickens, 3732 in cattle, and 4870 in pigs. These transcripts match previously known patterns of lncRNAs, such as generally lower expression levels than mRNAs and higher tissue specificity. An analysis of lncRNA conservation across species identified a set of conserved lncRNAs with potential functions associated with chromatin structure and gene regulation. Tissue-specific lncRNAs were identified. Genes proximal to tissue-specific lncRNAs were enriched for GO terms associated with the tissue of origin, such as leukocyte activation in spleen., Conclusions: LncRNAs were identified in three important farm animal species using eight tissues from adult individuals. About half of the identified lncRNAs were not previously reported in the NCBI annotations for these species. While lncRNAs are less conserved than protein-coding genes, a set of positionally conserved lncRNAs were identified among chickens, cattle, and pigs with potential functions related to chromatin structure and gene regulation. Tissue-specific lncRNAs have potential regulatory functions on genes enriched for tissue-specific GO terms. Future work will include epigenetic data from ChIP-seq experiments to further refine these annotations.

The liver plays a central role in metabolism and produces important hormones. Hepatic estrogen receptors and the release of insulin-like growth factor 1 (IGF1) are critical links between liver function and the reproductive system. However, the role of liver in pubertal development is not fully understood. To explore this question, we applied transcriptomic analyses to liver samples of pre- and post-pubertal Brahman heifers and identified differentially expressed (DE) genes and genes encoding transcription factors (TFs). Differential expression of genes suggests potential biological mechanisms and pathways linking liver function to puberty. The analyses identified 452 DE genes and 82 TF with significant contribution to differential gene expression by using a regulatory impact factor metric. Brain-derived neurotrophic factor was observed as the most down-regulated gene ( P = 0.003) in post-pubertal heifers and we propose this gene influences pubertal development in Brahman heifers. Additionally, co-expression network analysis provided evidence for three TF as key regulators of liver function during pubertal development: the signal transducer and activator of transcription 6, PBX homeobox 2, and polybromo 1. Pathway enrichment analysis identified transforming growth factor-beta and Wnt signaling pathways as significant annotation terms for the list of DE genes and TF in the co-expression network. Molecular information regarding genes and pathways described in this work are important to further our understanding of puberty onset in Brahman heifers.

Background: The individual, together with its environment, has been reported as the main force driving composition and structure of skin microbiota in healthy dogs. Therefore, one of the major concerns when analyzing canine skin microbiota is the likely influence of the environment. Despite the dense fur covering, certain skin diseases exhibit differential prevalence among skin sites, dog breeds, and individuals., Results: We have characterized the normal variability of dog skin microbiota in a well-controlled cohort of a large number of Golden-Labrador Retriever crossed dogs (N = 35) with similar ages, related genetic background, and a shared environment. We found that the individual drives the skin microbiota composition and structure followed by the skin site. The main bacterial classes inhabiting dog skin in this cohort are Gammaproteobacteria and Bacilli. We also detected bacteria associated to the environment on different dog skin sites that could be reflecting the different degrees of exposure of each skin site and each dog. Network analyses elucidated bacterial interactions within and between skin sites, especially in the chin, abdomen, axilla, and perianal region, with the highly shared interactions probably representing an anatomical, behavioral, or environmental component. When analyzing each skin site independently to assess host-specific factors, we found that temporality (season of birth and time spent in the kennel) affected all the skin sites and specially the inner pinna. The most abundant taxon driving this difference was Sphingomonas. We also found taxonomic differences among male and female dogs on the abdomen, axilla, and back., Conclusions: We observed a large inter-individual variability and differences among skin sites. Host-specific variables, such as temporality or sex, were also shaping skin microbiota of healthy dogs, even in an environmental homogenous cohort.

Fertility traits, such as heifer pregnancy, are economically important in cattle production systems, and are therefore, used in genetic selection programs. The aim of this study was to identify single nucleotide polymorphisms (SNPs) using RNA-sequencing (RNA-Seq) data from ovary, uterus, endometrium, pituitary gland, hypothalamus, liver, longissimus dorsi muscle, and adipose tissue in 62 candidate genes associated with heifer puberty in cattle. RNA-Seq reads were assembled to the bovine reference genome (UMD 3.1.1) and analyzed in five cattle breeds; Brangus, Brahman, Nellore, Angus, and Holstein. Two approaches used the Brangus data for SNP discovery 1) pooling all samples, and 2) within each individual sample. These approaches revealed 1157 SNPs. These were compared with those identified in the pooled samples of the other breeds. Overall, 172 SNPs within 13 genes (CPNE5, FAM19A4, FOXN4, KLF1, LOC777593, MGC157266, NEBL, NRXN3, PEPT-1, PPP3CA, SCG5, TSG101, and TSHR) were concordant in the five breeds. Using Ensembl's Variant Effector Predictor, we determined that 12% of SNPs were in exons (71% synonymous, 29% nonsynonymous), 1% were in untranslated regions (UTRs), 86% were in introns, and 1% were in intergenic regions. Since these SNPs were discovered in RNA, the variants were predicted to be within exons or UTRs. Overall, 160 novel transcripts in 42 candidate genes and five novel genes overlapping five candidate genes were observed. In conclusion, 1157 SNPs were identified in 62 candidate genes associated with puberty in Brangus cattle, of which, 172 were concordant in the five cattle breeds. Novel transcripts and genes were also identified.

Parameters of bone geometry such as width, length, and cross-sectional area are major determinants of bone strength. Although these traits are highly heritable, few genes influencing bone geometry have been identified. Here, we dissect a major quantitative trait locus (QTL) influencing femur size. This QTL was originally identified in an F2 cross between the C57BL/6J-hg/hg (HG) and CAST/EiJ strains and was referred to as femur length in high growth mice 2 ( Feml2 ). Feml2 was located on chromosome (Chr.) 9 at ∼20 cM. Here, we show that the HG.CAST-( D9Mit249-D9Mit133 )/Ucd congenic strain captures Feml2 In an F2 congenic cross, we fine-mapped the location of Feml2 to an ∼6 Mbp region extending from 57.3 to 63.3 Mbp on Chr. 9. We have identified candidates by mining the complete genome sequence of CAST/EiJ and through allele-specific expression (ASE) analysis of growth plates in C57BL/6J × CAST/EiJ F1 hybrids. Interestingly, we also find that the refined location of Feml2 overlaps a cluster of six independent genome-wide associations for human height. This work provides the foundation for the identification of novel genes affecting bone geometry., (Copyright © 2017 Sabik et al.)

Epizootic bovine abortion (EBA), or "foothill abortion," is the leading cause of beef cattle abortion in California and has also been reported in Nevada and Oregon. In the 1970s, the soft-shelled tick Ornithodoros coriaceus , or "pajaroello tick," was confirmed as the disease-transmitting vector. In 2005, a novel Deltaproteobacterium was discovered as the etiologic agent of EBA (aoEBA), recently named Pajaroellobacter abortibovis This organism cannot be grown in culture using traditional microbiological techniques; it can only be grown in experimentally-infected severe combined immunodeficient (SCID) mice. The objectives of this study were to perform a de novo genome assembly for P. abortibovis and identify and validate potential antigenic proteins as candidates for future recombinant vaccine development. DNA and RNA were extracted from spleen tissue collected from experimentally-infected SCID mice following exposure to P. abortibovis This combination of mouse and bacterial DNA was sequenced and aligned to the mouse genome. Mouse sequences were subtracted from the sequence pool and the remaining sequences were de novo assembled at 50x coverage into a 1.82 Mbp complete closed circular Deltaproteobacterial genome containing 2250 putative protein-coding sequences. Phylogenetic analysis of P. abortibovis predicts that this bacterium is most closely related to the organisms of the order Myxococcales, referred to as Myxobacteria. In silico prediction of vaccine candidates was performed using a reverse vaccinology approach resulting in the identification and ranking of the top 10 candidate proteins that are likely to be antigenic. Immunologic testing of these candidate proteins confirmed antigenicity of seven of the nine expressed protein candidates using serum from P. abortibovis immunized mice., (Copyright © 2017 Welly et al.)

To understand genes, pathways, and networks related to puberty, we characterized the transcriptome of two tissues: the pituitary gland and ovaries. Samples were harvested from pre- and postpubertal Brahman heifers (same age group). Brahman heifers () are older at puberty compared with , a productivity issue. With RNA sequencing, we identified differentially expressed (DEx) genes and important transcription factors (TF) and predicted coexpression networks. The number of DEx genes detected in the pituitary gland was 284 ( < 0.05), and was the most DEx gene (fold change = 4.12, = 0.01). The gene promotes bone mineralization through transforming growth factor-β (TGFβ) signaling. Further studies of the link between bone mineralization and puberty could target . In ovaries, 3,871 genes were DEx ( < 0.05). Four highly DEx genes were noteworthy for their function: (a γ-aminobutyric acid [GABA] transporter), (), and () and its receptor . These genes had higher ovarian expression in postpubertal heifers. The GABA and its receptors and transporters were expressed in the ovaries of many mammals, suggesting a role for this pathway beyond the brain. The pathway has been known to influence the timing of puberty in rats, via modulation of GnRH. The effects of at the hypothalamus, pituitary gland, and ovaries have been documented. and its receptors are known factors in the release of GnRH, similar to and GABA, although their roles in ovarian tissue are less clear. Pathways previously related to puberty such as TGFβ signaling ( = 6.71 × 10), Wnt signaling ( = 4.1 × 10), and peroxisome proliferator-activated receptor (PPAR) signaling ( = 4.84 × 10) were enriched in our data set. Seven genes were identified as key TF in both tissues: , , , , , , and a novel gene. An ovarian subnetwork created with TF and significant ovarian DEx genes revealed five zinc fingers as regulators: , , , , and . Recent work of hypothalamic gene expression also pointed to zinc fingers as TF for bovine puberty. Although some zinc fingers may be ubiquitously expressed, the identification of DEx genes in common across tissues points to key regulators of puberty. The hypothalamus and pituitary gland had eight DEx genes in common. The hypothalamus and ovaries had 89 DEx genes in common. The pituitary gland and ovaries had 48 DEx genes in common. Our study confirmed the complexity of puberty and suggested further investigation on genes that code zinc fingers.

Iberian pig production includes purebred (IB) and Duroc-crossbred (IBxDU) pigs, which show important differences in growth, fattening and tissue composition. This experiment was conducted to investigate the effects of genetic type and muscle (Longissimus dorsi (LD) vs Biceps femoris (BF)) on gene expression and transcriptional regulation at two developmental stages. Nine IB and 10 IBxDU piglets were slaughtered at birth, and seven IB and 10 IBxDU at four months of age (growing period). Carcass traits and LD intramuscular fat (IMF) content were measured. Muscle transcriptome was analyzed on LD samples with RNA-Seq technology. Carcasses were smaller in IB than in IBxDU neonates (p < 0.001), while growing IB pigs showed greater IMF content (p < 0.05). Gene expression was affected (p < 0.01 and Fold change > 1.5) by the developmental stage (5,812 genes), muscle type (135 genes), and genetic type (261 genes at birth and 113 at growth). Newborns transcriptome reflected a highly proliferative developmental stage, while older pigs showed upregulation of catabolic and muscle functioning processes. Regarding the genetic type effect, IBxDU newborns showed enrichment of gene pathways involved in muscle growth, in agreement with the higher prenatal growth observed in these pigs. However, IB growing pigs showed enrichment of pathways involved in protein deposition and cellular growth, supporting the compensatory gain experienced by IB pigs during this period. Moreover, newborn and growing IB pigs showed more active glucose and lipid metabolism than IBxDU pigs. Moreover, LD muscle seems to have more active muscular and cell growth, while BF points towards lipid metabolism and fat deposition. Several regulators controlling transcriptome changes in both genotypes were identified across muscles and ages (SIM1, PVALB, MEFs, TCF7L2 or FOXO1), being strong candidate genes to drive expression and thus, phenotypic differences between IB and IBxDU pigs. Many of the identified regulators were known to be involved in muscle and adipose tissues development, but others not previously associated with pig muscle growth were also identified, as PVALB, KLF1 or IRF2. The present study discloses potential molecular mechanisms underlying phenotypic differences observed between IB and IBxDU pigs and highlights candidate genes implicated in these molecular mechanisms., Competing Interests: The authors have declared that no competing interests exist, excepting that the coauthors Antonio Gonzalez-Bulnes and Cristina Óvilo are PLOS ONE Editorial Board members. However, this does not alter the authors’ adherence to all the PLOS ONE policies.

Wilson disease (WD), a genetic disorder affecting copper transport, is characterized by hepatic and neurological manifestations with variable and often unpredictable presentation. Global DNA methylation in liver was previously modified by dietary choline in tx-j mice, a spontaneous mutant model of WD. We therefore hypothesized that the WD phenotype and hepatic gene expression of tx-j offspring could be modified by maternal methyl supplementation during pregnancy. In an initial experiment, female tx-j mice or wild type mice were fed control or choline-supplemented diets 2 weeks prior to mating through embryonic day 17. Transcriptomic analysis (RNA-seq) on embryonic livers revealed tx-j-specific differences in genes related to oxidative phosphorylation, mitochondrial dysfunction, and the neurological disorders Huntington's disease and Alzheimer disease. Maternal choline supplementation restored the transcript levels of a subset of genes to wild type levels. In a separate experiment, a group of tx-j offspring continued to receive choline-supplemented or control diets, with or without the copper chelator penicillamine (PCA) for 12 weeks until 24 weeks of age. Combined choline supplementation and PCA treatment of 24-week-old tx-j mice was associated with increased liver transcript levels of methionine metabolism and oxidative phosphorylation-related genes. Sex differences in gene expression within each treatment group were also observed. These results demonstrate that the transcriptional changes in oxidative phosphorylation and methionine metabolism genes in WD that originate during fetal life are, in part, prevented by prenatal maternal choline supplementation, a finding with potential relevance to preventive treatments of WD.

Background: Changes in metabolism have been suggested to contribute to the aberrant phenotype of vascular wall cells, including fibroblasts, in pulmonary hypertension (PH). Here, we test the hypothesis that metabolic reprogramming to aerobic glycolysis is a critical adaptation of fibroblasts in the hypertensive vessel wall that drives proliferative and proinflammatory activation through a mechanism involving increased activity of the NADH-sensitive transcriptional corepressor C-terminal binding protein 1 (CtBP1)., Methods: RNA sequencing, quantitative polymerase chain reaction, 13 C-nuclear magnetic resonance, fluorescence-lifetime imaging, mass spectrometry-based metabolomics, and tracing experiments with U- 13 C-glucose were used to assess glycolytic reprogramming and to measure the NADH/NAD + ratio in bovine and human adventitial fibroblasts and mouse lung tissues. Immunohistochemistry was used to assess CtBP1 expression in the whole-lung tissues. CtBP1 siRNA and the pharmacological inhibitor 4-methylthio-2-oxobutyric acid (MTOB) were used to abrogate CtBP1 activity in cells and hypoxic mice., Results: We found that adventitial fibroblasts from calves with severe hypoxia-induced PH and humans with idiopathic pulmonary arterial hypertension (PH-Fibs) displayed aerobic glycolysis when cultured under normoxia, accompanied by increased free NADH and NADH/NAD + ratios. Expression of the NADH sensor CtBP1 was increased in vivo and in vitro in fibroblasts within the pulmonary adventitia of humans with idiopathic pulmonary arterial hypertension and animals with PH and cultured PH-Fibs, respectively. Decreasing NADH pharmacologically with MTOB or genetically blocking CtBP1 with siRNA upregulated the cyclin-dependent genes (p15 and p21) and proapoptotic regulators (NOXA and PERP), attenuated proliferation, corrected the glycolytic reprogramming phenotype of PH-Fibs, and augmented transcription of the anti-inflammatory gene HMOX1. Chromatin immunoprecipitation analysis demonstrated that CtBP1 directly binds the HMOX1 promoter. Treatment of hypoxic mice with MTOB decreased glycolysis and expression of inflammatory genes, attenuated proliferation, and suppressed macrophage numbers and remodeling in the distal pulmonary vasculature., Conclusions: CtBP1 is a critical factor linking changes in cell metabolism to cell phenotype in hypoxic and other forms of PH and a therapeutic target., Competing Interests: Disclosures: The authors have no conflict of interests., (© 2016 American Heart Association, Inc.)

Background: Next-generation sequencing projects commonly commence by aligning reads to a reference genome assembly. While improvements in alignment algorithms and computational hardware have greatly enhanced the efficiency and accuracy of alignments, a significant percentage of reads often remain unmapped., Results: We generated de novo assemblies of unmapped reads from the DNA and RNA sequencing of the Bos taurus reference individual and identified the closest matching sequence to each contig by alignment to the NCBI non-redundant nucleotide database using BLAST. As expected, many of these contigs represent vertebrate sequence that is absent, incomplete, or misassembled in the UMD3.1 reference assembly. However, numerous additional contigs represent invertebrate species. Most prominent were several species of Spirurid nematodes and a blood-borne parasite, Babesia bigemina. These species are either not present in the US or are not known to infect taurine cattle and the reference animal appears to have been host to unsequenced sister species., Conclusions: We demonstrate the importance of exploring unmapped reads to ascertain sequences that are either absent or misassembled in the reference assembly and for detecting sequences indicative of parasitic or commensal organisms.

Iberian ham production includes both purebred (IB) and Duroc-crossbred (IBxDU) Iberian pigs, which show important differences in meat quality and production traits, such as muscle growth and fatness. This experiment was conducted to investigate gene expression differences, transcriptional regulation and genetic polymorphisms that could be associated with the observed phenotypic differences between IB and IBxDU pigs. Nine IB and 10 IBxDU pigs were slaughtered at birth. Morphometric measures and blood samples were obtained and samples from Biceps femoris muscle were employed for compositional and transcriptome analysis by RNA-Seq technology. Phenotypic differences were evident at this early age, including greater body size and weight in IBxDU and greater Biceps femoris intramuscular fat and plasma cholesterol content in IB newborns. We detected 149 differentially expressed genes between IB and IBxDU neonates (p < 0.01 and Fold-Change > 1. 5). Several were related to adipose and muscle tissues development (DLK1, FGF21 or UBC). The functional interpretation of the transcriptomic differences revealed enrichment of functions and pathways related to lipid metabolism in IB and to cellular and muscle growth in IBxDU pigs. Protein catabolism, cholesterol biosynthesis and immune system were functions enriched in both genotypes. We identified transcription factors potentially affecting the observed gene expression differences. Some of them have known functions on adipogenesis (CEBPA, EGRs), lipid metabolism (PPARGC1B) and myogenesis (FOXOs, MEF2D, MYOD1), which suggest a key role in the meat quality differences existing between IB and IBxDU hams. We also identified several polymorphisms showing differential segregation between IB and IBxDU pigs. Among them, non-synonymous variants were detected in several transcription factors as PPARGC1B and TRIM63 genes, which could be associated to altered gene function. Taken together, these results provide information about candidate genes, metabolic pathways and genetic polymorphisms potentially involved in phenotypic differences between IB and IBxDU pigs associated to meat quality and production traits.

Background: The cattle (Bos taurus) genome was originally selected for sequencing due to its economic importance and unique biology as a model organism for understanding other ruminants, or mammals. Currently, there are two cattle genome sequence assemblies (UMD3.1 and Btau4.6) from groups using dissimilar assembly algorithms, which were complemented by genetic and physical map resources. However, past comparisons between these assemblies revealed substantial differences. Consequently, such discordances have engendered ambiguities when using reference sequence data, impacting genomic studies in cattle and motivating construction of a new optical map resource--BtOM1.0--to guide comparisons and improvements to the current sequence builds. Accordingly, our comprehensive comparisons of BtOM1.0 against the UMD3.1 and Btau4.6 sequence builds tabulate large-to-immediate scale discordances requiring mediation., Results: The optical map, BtOM1.0, spanning the B. taurus genome (Hereford breed, L1 Dominette 01449) was assembled from an optical map dataset consisting of 2,973,315 (439 X; raw dataset size before assembly) single molecule optical maps (Rmaps; 1 Rmap = 1 restriction mapped DNA molecule) generated by the Optical Mapping System. The BamHI map spans 2,575.30 Mb and comprises 78 optical contigs assembled by a combination of iterative (using the reference sequence: UMD3.1) and de novo assembly techniques. BtOM1.0 is a high-resolution physical map featuring an average restriction fragment size of 8.91 Kb. Comparisons of BtOM1.0 vs. UMD3.1, or Btau4.6, revealed that Btau4.6 presented far more discordances (7,463) vs. UMD3.1 (4,754). Overall, we found that Btau4.6 presented almost double the number of discordances than UMD3.1 across most of the 6 categories of sequence vs. map discrepancies, which are: COMPLEX (misassembly), DELs (extraneous sequences), INSs (missing sequences), ITs (Inverted/Translocated sequences), ECs (extra restriction cuts) and MCs (missing restriction cuts)., Conclusion: Alignments of UMD3.1 and Btau4.6 to BtOM1.0 reveal discordances commensurate with previous reports, and affirm the NCBI's current designation of UMD3.1 sequence assembly as the "reference assembly" and the Btau4.6 as the "alternate assembly." The cattle genome optical map, BtOM1.0, when used as a comprehensive and largely independent guide, will greatly assist improvements to existing sequence builds, and later serve as an accurate physical scaffold for studies concerning the comparative genomics of cattle breeds.

Background: Mouse chromosome 2 is linked to growth and body fat phenotypes in many mouse crosses. With the goal to identify the underlying genes regulating growth and body fat on mouse chromosome 2, we developed five overlapping subcongenic strains that contained CAST/EiJ donor regions in a C57BL/6J (hg/hg) background (hg is a spontaneous deletion of 500 Kb on mouse chromosome 10). To fine map QTL on distal mouse chromosome 2 a total of 1,712 F2 mice from the five subcongenic strains, plus 278 F2 mice from the HG2D founder congenic strain were phenotyped and analyzed. Interval mapping (IM) and composite IM (CIM) were performed on body weight and body fat traits on a combination of SNP and microsatellite markers, which generated a high-density genotyping panel., Results: Phenotypic analysis and interval mapping of total fat mass identified two QTL on distal mouse chromosome 2. One QTL between 150 and 161 Mb, Fatq2a, and the second between 173.3 and 175.6 Mb, Fatq2b. The two QTL reside in different congenic strains with significant total fat differences between homozygous cast/cast and b6/b6 littermates. Both of these QTL were previously identified only as a single QTL affecting body fat, Fatq2. Furthermore, through a novel approach referred here as replicated CIM, Fatq2b was mapped to the Gnas imprinted locus., Conclusions: The integration of subcongenic strains, high-density genotyping, and CIM succesfully partitioned two previously linked QTL 20 Mb apart, and the strongest QTL, Fatq2b, was fine mapped to a ~2.3 Mb region interval encompassing the Gnas imprinted locus.

There is considerable interest in the genetics of wolves (Canis lupus) because of their close relationship to domestic dogs (C. familiaris) and the need for informed conservation and management. This includes wolf populations in Southeast Alaska for which we determined genotypes of 305 wolves at 173662 single nucleotide polymorphism (SNP) loci. After removal of invariant and linked SNP, 123801 SNP were used to quantify genetic differentiation of wolves in Southeast Alaska and wolves, coyotes (C. latrans), and dogs from other areas in North America. There is differentiation of SNP allele frequencies between the species (wolves, coyotes, and dogs), although differentiation is relatively low between some wolf and coyote populations. There are varying levels of differentiation among populations of wolves, including low differentiation of wolves in interior Alaska, British Columbia, and the northern US Rocky Mountains. There is considerable differentiation of SNP allele frequencies of wolves in Southeast Alaska from wolves in other areas. However, wolves in Southeast Alaska are not a genetically homogeneous group and there are comparable levels of genetic differentiation among areas within Southeast Alaska and between Southeast Alaska and other geographic areas. SNP variation and other genetic data are discussed regarding taxonomy and management., (© The American Genetic Association 2014. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The specific genes regulating the quantitative variation in macronutrient preference and food intake are virtually unknown. We fine mapped a previously identified mouse chromosome 17 region harboring quantitative trait loci (QTL) with large effects on preferential macronutrient intake-carbohydrate (Mnic1), total kilcalories (Kcal2), and total food volume (Tfv1) using interval-specific strains. These loci were isolated in the [C57BL/6J.CAST/EiJ-17.1-(D17Mit19-D17Mit50); B6.CAST-17.1] strain, possessing a ∼ 40.1 Mb region of CAST DNA on the B6 genome. In a macronutrient selection paradigm, the B6.CAST-17.1 subcongenic mice eat 30% more calories from the carbohydrate-rich diet, ∼ 10% more total calories, and ∼ 9% more total food volume per body weight. In the current study, a cross between carbohydrate-preferring B6.CAST-17.1 and fat-preferring, inbred B6 mice was used to generate a subcongenic-derived F2 mapping population; genotypes were determined using a high-density, custom SNP panel. Genetic linkage analysis substantially reduced the 95% confidence interval for Mnic1 (encompassing Kcal2 and Tfv1) from 40.1 to 29.5 Mb and more precisely established its boundaries. Notably, no genetic linkage for self-selected fat intake was detected, underscoring the carbohydrate-specific effect of this locus. A second key finding was the separation of two energy balance QTLs: Mnic1/Kcal2/Tfv1 for food intake and a newly discovered locus regulating short term body weight gain. The Mnic1/Kcal2/Tfv1 QTL was further de-limited to 19.0 Mb, based on the absence of nutrient intake phenotypes in subcongenic HQ17IIa mice. Analyses of available sequence data and gene ontologies, along with comprehensive expression profiling in the hypothalamus of non-recombinant, cast/cast and b6/b6 F2 controls, focused our attention on candidates within the QTL interval. Zfp811, Zfp870, and Btnl6 showed differential expression and also contain stop codons, but have no known biology related to food intake regulation. The genes Decr2, Ppard and Agapt1 are more appealing candidates because of their involvement in lipid metabolism and down-regulation in carbohydrate-preferring animals.

The aquaculture industry continues to promote the diversification of ingredients used in aquafeed in order to achieve a more sustainable aquaculture production system. The evaluation of large numbers of diets in aquaculture species is costly and requires time-consuming trials in some species. In contrast, zebrafish (Danio rerio) can solve these drawbacks as an experimental model, and represents an ideal organism to carry out preliminary evaluation of diets. In addition, zebrafish has a sequenced genome allowing the efficient utilization of new technologies, such as RNA-sequencing and genotyping platforms to study the molecular mechanisms that underlie the organism's response to nutrients. Also, biotechnological tools like transgenic lines with fluorescently labeled neutrophils that allow the evaluation of the immune response in vivo, are readily available in this species. Thus, zebrafish provides an attractive platform for testing many ingredients to select those with the highest potential of success in aquaculture. In this perspective article aspects related to diet evaluation in which zebrafish can make important contributions to nutritional genomics and nutritional immunity are discussed.

The continuous uploading of polygenic additive mutational variability has been reported in several studies in laboratory species with an inbred genetic background. These studies have focused on the direct contribution of new mutations without considering the possibility of epistatic effects derived from the interaction of new mutations with pre-existing polymorphisms. In this work we focused on this main topic and analysed the statistical and biological relevance of the epistatic variance for 9 week body weight in two populations of inbred mice. We developed a new linear mixed model parameterization where founder-related additive genetic variability, additive mutational variability and the interaction terms between both sources of variation were accounted for under a Bayesian design and without requiring the inversion of a matrix of epistatic genetic covariances. The analyses focused on a six-generations data set from C57BL/6J mice (n = 3736) and a five-generations data set from C57BL/6J(hg/hg) mice (n = 2843). The deviance information criterion (DIC) clearly favoured the model accounting for epistatic variability with reductions larger than 50 DIC units in both populations. Modal estimates for founder related, mutational and epistatic heritabilities were 0·068, 0·011 and 0·095 in C57BL/6J and 0·060, 0·010 and 0·113 in C57BL/6J(hg/hg), ruling out any doubt about the biological relevance of epistasis originating from new mutations in mice. These results contribute new insights on the relevance of epistasis in the genetic architecture of mammals and serve as an important component of an additional source of genetic heterogeneity for inbred strains of laboratory mice.

Puberty is a complex physiological event by which animals mature into an adult capable of sexual reproduction. In order to enhance our understanding of the genes and regulatory pathways and networks involved in puberty, we characterized the transcriptome of five reproductive tissues (i.e. hypothalamus, pituitary gland, ovary, uterus, and endometrium) as well as tissues known to be relevant to growth and metabolism needed to achieve puberty (i.e., longissimus dorsi muscle, adipose, and liver). These tissues were collected from pre- and post-pubertal Brangus heifers (3/8 Brahman; Bos indicus x 5/8 Angus; Bos taurus) derived from a population of cattle used to identify quantitative trait loci associated with fertility traits (i.e., age of first observed corpus luteum (ACL), first service conception (FSC), and heifer pregnancy (HPG)). In order to exploit the power of complementary omics analyses, pre- and post-puberty co-expression gene networks were constructed by combining the results from genome-wide association studies (GWAS), RNA-Seq, and bovine transcription factors. Eight tissues among pre-pubertal and post-pubertal Brangus heifers revealed 1,515 differentially expressed and 943 tissue-specific genes within the 17,832 genes confirmed by RNA-Seq analysis. The hypothalamus experienced the most notable up-regulation of genes via puberty (i.e., 204 out of 275 genes). Combining the results of GWAS and RNA-Seq, we identified 25 loci containing a single nucleotide polymorphism (SNP) associated with ACL, FSC, and (or) HPG. Seventeen of these SNP were within a gene and 13 of the genes were expressed in uterus or endometrium. Multi-tissue omics analyses revealed 2,450 co-expressed genes relative to puberty. The pre-pubertal network had 372,861 connections whereas the post-pubertal network had 328,357 connections. A sub-network from this process revealed key transcriptional regulators (i.e., PITX2, FOXA1, DACH2, PROP1, SIX6, etc.). Results from these multi-tissue omics analyses improve understanding of the number of genes and their complex interactions for puberty in cattle.

The objective of this study was to examine five different sources of RNA, namely mammary gland tissue (MGT), milk somatic cells (SC), laser microdissected mammary epithelial cells (LCMEC), milk fat globules (MFG) and antibody-captured milk mammary epithelial cells (mMEC) to analyze the bovine mammary gland transcriptome using RNA-Sequencing. Our results provide a comparison between different sampling methods (invasive and non-invasive) to define the transcriptome of mammary gland tissue and milk cells. This information will be of value to investigators in choosing the most appropriate sampling method for different research applications to study specific physiological states during lactation. One of the simplest procedures to study the transcriptome associated with milk appears to be the isolation of total RNA directly from SC or MFG released into milk during lactation. Our results indicate that the SC and MFG transcriptome are representative of MGT and LCMEC and can be used as effective and alternative samples to study mammary gland expression without the need to perform a tissue biopsy.

THE AIM OF THIS STUDY WAS TO INVESTIGATE THE GENETIC DIVERSITY WITHIN AND AMONG THREE BREEDS OF SHEEP: Corriedale, Merino and Creole. Sheep from the three breeds (Merino n = 110, Corriedale n = 108 and Creole n = 10) were genotyped using the Illumina Ovine SNP50 beadchip(®). Genetic diversity was evaluated by comparing the minor allele frequency (MAF) among breeds. Population structure and genetic differentiation were assessed using STRUCTURE software, principal component analysis (PCA) and fixation index (FST). Fixed markers (MAF = 0) that were different among breeds were identified as specific breed markers. Using a subset of 18,181 single nucleotide polymorphisms (SNPs), PCA and STUCTURE analysis were able to explain population stratification within breeds. Merino and Corriedale divergent lines showed high levels of polymorphism (89.4% and 86% of polymorphic SNPs, respectively) and moderate genetic differentiation (FST = 0.08) between them. In contrast, Creole had only 69% polymorphic SNPs and showed greater genetic differentiation from the other two breeds (FST = 0.17 for both breeds). Hence, a subset of molecular markers present in the OvineSNP50 is informative enough for breed assignment and population structure analysis of commercial and Creole breeds.

We assessed the relationships of polar bears (Ursus maritimus), brown bears (U. arctos), and black bears (U. americanus) with high throughput genomic sequencing data with an average coverage of 25× for each species. A total of 1.4 billion 100-bp paired-end reads were assembled using the polar bear and annotated giant panda (Ailuropoda melanoleuca) genome sequences as references. We identified 13.8 million single nucleotide polymorphisms (SNP) in the 3 species aligned to the polar bear genome. These data indicate that polar bears and brown bears share more SNP with each other than either does with black bears. Concatenation and coalescence-based analysis of consensus sequences of approximately 1 million base pairs of ultraconserved elements in the nuclear genome resulted in a phylogeny with black bears as the sister group to brown and polar bears, and all brown bears are in a separate clade from polar bears. Genotypes for 162 SNP loci of 336 bears from Alaska and Montana showed that the species are genetically differentiated and there is geographic population structure of brown and black bears but not polar bears.

The genomic architecture and expression of the Igf-1 gene are complex yielding multiple IGF-I transcript isoforms with putative functional contributions to growth and metabolism. Using RNA-seq on different tissues, physiological states, and species, the breadth of transcripts expressed was determined. Tissues from pre- and post-pubertal heifers and mature mice were collected and the transcript isoforms characterized. Three different IGF-I isoforms were detected in heifers with Class 1 transcripts most abundantly expressed. The pituitary reduced IGF-I expression post-pubertally whereas the uterus increased expression. Murine IGF-I transcript expression was more diverse utilizing multiple exons, start sites, and 3'UTRs. The RNA-seq methodology to characterize expression profiles permits assessment of the transcript isoforms yielding insight into functional roles of each transcript., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background: Studies of normal human mammary gland development and function have mostly relied on cell culture, limited surgical specimens, and rodent models. Although RNA extracted from human milk has been used to assay the mammary transcriptome non-invasively, this assay has not been adequately validated in primates. Thus, the objectives of the current study were to assess the suitability of lactating rhesus macaques as a model for lactating humans and to determine whether RNA extracted from milk fractions is representative of RNA extracted from mammary tissue for the purpose of studying the transcriptome of milk-producing cells., Results: We confirmed that macaque milk contains cytoplasmic crescents and that ample high-quality RNA can be obtained for sequencing. Using RNA sequencing, RNA extracted from macaque milk fat and milk cell fractions more accurately represented RNA from mammary epithelial cells (cells that produce milk) than did RNA from whole mammary tissue. Mammary epithelium-specific transcripts were more abundant in macaque milk fat, whereas adipose or stroma-specific transcripts were more abundant in mammary tissue. Functional analyses confirmed the validity of milk as a source of RNA from milk-producing mammary epithelial cells., Conclusions: RNA extracted from the milk fat during lactation accurately portrayed the RNA profile of milk-producing mammary epithelial cells in a non-human primate. However, this sample type clearly requires protocols that minimize RNA degradation. Overall, we validated the use of RNA extracted from human and macaque milk and provided evidence to support the use of lactating macaques as a model for human lactation.

Background: Use of RNA-Seq presents unique benefits in terms of gene expression analysis because of its wide dynamic range and ability to identify functional sequence variants. This technology provides the opportunity to assay the developing embryo, but the paucity of biological material available from individual embryos has made this a challenging prospect., Results: We report here the first application of RNA-Seq for the analysis of individual blastocyst gene expression, SNP detection, and characterization of allele specific expression (ASE). RNA was extracted from single bovine blastocysts (n = 5), amplified, and analyzed using high-throughput sequencing. Approximately 38 million sequencing reads were generated per embryo and 9,489 known bovine genes were found to be expressed, with a high correlation of expression levels between samples (r > 0.97). Transcriptomic data was analyzed to identify SNP in expressed genes, and individual SNP were examined to characterize allele specific expression. Expressed biallelic SNP variants with allelic imbalances were observed in 473 SNP, where one allele represented between 65-95% of a variant's transcripts., Conclusions: This study represents the first application of RNA-seq technology in single bovine embryos allowing a representation of the embryonic transcriptome and the analysis of transcript sequence variation to describe specific allele expression.

Background: In a marker-trait association study we estimated the statistical significance of 65 single nucleotide polymorphisms (SNP) in 23 candidate genes on HDL levels of two independent Caucasian populations. Each population consisted of men and women and their HDL levels were adjusted for gender and body weight. We used a linear regression model. Selected genes corresponded to folate metabolism, vitamins B-12, A, and E, and cholesterol pathways or lipid metabolism., Methods: Extracted DNA from both the Sacramento and Beltsville populations was analyzed using an allele discrimination assay with a MALDI-TOF mass spectrometry platform. The adjusted phenotype, y, was HDL levels adjusted for gender and body weight only statistical analyses were performed using the genotype association and regression modules from the SNP Variation Suite v7., Results: Statistically significant SNP (where P values were adjusted for false discovery rate) included: CETP (rs7499892 and rs5882); SLC46A1 (rs37514694; rs739439); SLC19A1 (rs3788199); CD36 (rs3211956); BCMO1 (rs6564851), APOA5 (rs662799), and ABCA1 (rs4149267). Many prior association trends of the SNP with HDL were replicated in our cross-validation study. Significantly, the association of SNP in folate transporters (SLC46A1 rs37514694 and rs739439; SLC19A1 rs3788199) with HDL was identified in our study., Conclusions: Given recent literature on the role of niacin in the biogenesis of HDL, focus on status and metabolism of B-vitamins and metabolites of eccentric cleavage of β-carotene with lipid metabolism is exciting for future study.

The technological properties of milk have significant importance for the dairy industry. Citrate, a normal constituent of milk, forms one of the main buffer systems that regulate the equilibrium between Ca(2+) and H(+) ions. Higher-than-normal citrate content is associated with poor coagulation properties of milk. To identify the genes responsible for the variation of citrate content in milk in dairy cattle, the metabolic steps involved in citrate and fatty acid synthesis pathways in ruminant mammary tissue using RNA sequencing were studied. Genetic markers that could influence milk citrate content in Holstein cows were used in a marker-trait association study to establish the relationship between 74 single nucleotide polymorphisms (SNP) in 20 candidate genes and citrate content in 250 Holstein cows. This analysis revealed 6 SNP in key metabolic pathway genes [isocitrate dehydrogenase 1 (NADP+), soluble (IDH1); pyruvate dehydrogenase (lipoamide) β (PDHB); pyruvate kinase (PKM2); and solute carrier family 25 (mitochondrial carrier; citrate transporter), member 1 (SLC25A1)] significantly associated with increased milk citrate content. The amount of the phenotypic variation explained by the 6 SNP ranged from 10.1 to 13.7%. Also, genotype-combination analysis revealed the highest phenotypic variation was explained combining IDH1&#95;23211, PDHB&#95;5562, and SLC25A1&#95;4446 genotypes. This specific genotype combination explained 21.3% of the phenotypic variation. The largest citrate associated effect was in the 3' untranslated region of the SLC25A1 gene, which is responsible for the transport of citrate across the mitochondrial inner membrane. This study provides an approach using RNA sequencing, metabolic pathway analysis, and association studies to identify genetic variation in functional target genes determining complex trait phenotypes., (Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Linkage mapping in a backcross of {Brown Norway [BN/Crl (BN)] × ZUC-Lepr (faSte) (ZUC)} × ZUC identified a male-specific quantitative trait locus (QTL) for urinary albumin excretion (UAE) on rat chromosome 1. A homozygous ZUC.BN-(D1Rat42-D1Rat90)/Ste congenic was produced containing BN donor alleles from 135 to 276 Mb from chromosome 1 on the ZUC background. We observed threefold higher urinary albumin-to-creatinine ratios (ACR) in 15-wk-old Zucker background strain males than in same sex and age congenic animals when both strains are also homozygous for the ZUC leptin receptor fatty mutation (Lepr (faSte)) (P < 0.0001). We then linkage mapped within the donor region without confounded effects from other chromosomes. Phenotypes were collected in 248 F2 male rats in a population made by crossing parents heterozygous for both the BN donor region and ZUC Lepr (faSte). Significant interactions were observed between the Lepr genotype and chromosome 1 QTL for six renal traits: urine volume, UAE at 10 and 15 wk, ACR, right kidney weight, and plasma urea nitrogen. A few traits, such as UAE and ACR, exhibit a second peak at the distal end of the chromosome. Hydronephrosis exhibited one or two QTLs contingent on adjustment for body weight. The results now demonstrate at least two sets of coincident traits with different correlations to kidney function.