Search

To identify and annotate transcript isoforms in the chicken genome, we generated Nanopore long-read sequencing data from 68 samples that encompassed 19 diverse tissues collected from experimental adult male and female White Leghorn chickens. More than 23.8 million reads with mean read length of 790 bases and average quality of 18.2 were generated. The annotation and subsequent filtering resulted in the identification of 55,382 transcripts at 40,547 loci with mean length of 1,700 bases. We predicted 30,967 coding transcripts at 19,461 loci, and 16,495 lncRNA transcripts at 15,512 loci. Compared to existing reference annotations, we found ∼52% of annotated transcripts could be partially or fully matched while ∼47% were novel. Seventy percent of novel transcripts were potentially transcribed from lncRNA loci. Based on our annotation, we quantified transcript expression across tissues and found two brain tissues (i.e., cerebellum and cortex) expressed the highest number of transcripts and loci. Furthermore, ∼22% of the transcripts displayed tissue specificity with the reproductive tissues (i.e., testis and ovary) exhibiting the most tissue-specific transcripts. Despite our wide sampling, ∼20% of Ensembl reference loci were not detected. This suggests that deeper sequencing and additional samples that include different breeds, cell types, developmental stages, and physiological conditions, are needed to fully annotate the chicken genome. The application of Nanopore sequencing in this study demonstrates the usefulness of long-read data in discovering additional novel loci (e.g., lncRNA loci) and resolving complex transcripts (e.g., the longest transcript for the TTN locus).

Characterizing transcription start sites is essential for understanding the regulatory mechanisms that control gene expression. Recently, a new bovine genome assembly (ARS-UCD1.2) with high continuity, accuracy, and completeness was released; however, the functional annotation of the bovine genome lacks precise transcription start sites and contains a low number of transcripts in comparison to human and mouse. By using the RAMPAGE approach, this study identified transcription start sites at high resolution in a large collection of bovine tissues. We found several known and novel transcription start sites attributed to promoters of protein-coding and lncRNA genes that were validated through experimental and in silico evidence. With these findings, the annotation of transcription start sites in cattle reached a level comparable to the mouse and human genome annotations. In addition, we identified and characterized transcription start sites for antisense transcripts derived from bidirectional promoters, potential lncRNAs, mRNAs, and pre-miRNAs. We also analyzed the quantitative aspects of RAMPAGE to produce a promoter activity atlas, reaching highly reproducible results comparable to traditional RNA-seq. Coexpression networks revealed considerable use of tissue-specific promoters, especially between brain and testicle, which expressed several genes in common from alternate loci. Furthermore, regions surrounding coexpressed modules were enriched in binding factor motifs representative of each tissue. The comprehensive annotation of promoters in such a large collection of tissues will substantially contribute to our understanding of gene expression in cattle and other mammalian species, shortening the gap between genotypes and phenotypes.

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.

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 F2α (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 (

Equine maternal recognition of pregnancy (MRP) is a process whose signal remains unknown. During MRP the conceptus and endometrium communicate to attenuate prostaglandin F2α (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.

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.

BackgroundThe 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.ResultsWe 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.ConclusionsWe 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.

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.

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.

SbIII and AsIII are known to exhibit similar chemical properties, but the degree of similarity in their effects on biological systems merits further exploration. Present work compares the responses of human epidermal keratinocytes, a known target cell type for arsenite-induced carcinogenicity, to these metalloids after treatment for a week at environmentally relevant concentrations. Previous work with these cells has shown that arsenite and antimonite have parallel effects in suppressing differentiation, altering levels of several critical enzymes and maintaining colony forming ability. More globally, protein profiling now reveals parallels in SbIII and AsIII effects. The more sensitive technique of transcriptional profiling also shows considerable parallels. Thus, gene expression changes were almost entirely in the same directions for the two treatments, although the degree of change was sometimes significantly different. Inspection of the changes revealed that RYR1 and LRIG1 were among the genes strongly suppressed, consistent with reduced calcium-dependent differentiation and maintenance of EGF-dependent proliferative potential. Moreover, levels of miRNAs in the cells were altered in parallel, with nearly 90% of the 198 most highly expressed ones being suppressed. Among these was miR-203, which is known to decrease proliferative potential. Finally, both SbIII and AsIII were seen to attenuate bone morphogenetic protein 6 induction of dual specificity phosphatases 2 and 14, consistent with maintaining epidermal growth factor receptor signaling. These findings raise the question whether SbIII, like AsIII, could act as a human skin carcinogen.

BackgroundMouse 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.ResultsPhenotypic 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.ConclusionsThe 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.

A variety of proteases release hundreds of endogenous peptide fragments from intact bovine milk proteins. Mass spectrometry-based peptidomics allows for high throughput sequence assignment of a large number of these peptides. Mastitis is known to result in increased protease activity in the mammary gland. Therefore, we hypothesized that subclinically mastitic milks would contain higher concentrations of released peptides. In this work, milks were sampled from three cows and, for each, one healthy and one subclinically mastitic teat were sampled for milk. Peptides were analyzed by nano-liquid chromatography quadrupole time of flight tandem mass spectrometry and identified with database searching. In total, 682 peptides were identified. The total number of released peptides increased 146% from healthy to subclinically mastitic milks (p < 0.05), and the total abundance of released peptides also increased significantly (p < 0.05). Bioinformatic analysis of enzyme cleavage revealed increases in activity of cathepsin D and elastase (p < 0.05) with subclinical mastitis.

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.

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.

Abstract 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.

Abstract Background Cow milk is a complex bioactive fluid consumed by humans beyond infancy. Even though the chemical and physical properties of cow milk are well characterized, very limited research has been done on characterizing the milk transcriptome. This study performs a comprehensive expression profiling of genes expressed in milk somatic cells of transition (day 15), peak (day 90) and late (day 250) lactation Holstein cows by RNA sequencing. Milk samples were collected from Holstein cows at 15, 90 and 250 days of lactation, and RNA was extracted from the pelleted milk cells. Gene expression analysis was conducted by Illumina RNA sequencing. Sequence reads were assembled and analyzed in CLC Genomics Workbench. Gene Ontology (GO) and pathway analysis were performed using the Blast2GO program and GeneGo application of MetaCore program. Results A total of 16,892 genes were expressed in transition lactation, 19,094 genes were expressed in peak lactation and 18,070 genes were expressed in late lactation. Regardless of the lactation stage approximately 9,000 genes showed ubiquitous expression. Genes encoding caseins, whey proteins and enzymes in lactose synthesis pathway showed higher expression in early lactation. The majority of genes in the fat metabolism pathway had high expression in transition and peak lactation milk. Most of the genes encoding for endogenous proteases and enzymes in ubiquitin-proteasome pathway showed higher expression along the course of lactation. Conclusions This is the first study to describe the comprehensive bovine milk transcriptome in Holstein cows. The results revealed that 69% of NCBI Btau 4.0 annotated genes are expressed in bovine milk somatic cells. Most of the genes were ubiquitously expressed in all three stages of lactation. However, a fraction of the milk transcriptome has genes devoted to specific functions unique to the lactation stage. This indicates the ability of milk somatic cells to adapt to different molecular functions according to the biological need of the animal. This study provides a valuable insight into the biology of lactation in the cow, as well as many avenues for future research on the bovine lactome.

This study examines the genes coding for enzymes involved in bovine milk oligosaccharide metabolism by comparing the oligosaccharide profiles with the expressions of glycosylation-related genes. Fresh milk samples (n = 32) were collected from four Holstein and Jersey cows at days 1, 15, 90 and 250 of lactation and free milk oligosaccharide profiles were analyzed. RNA was extracted from milk somatic cells at days 15 and 250 of lactation (n = 12) and gene expression analysis was conducted by RNA-Sequencing. A list was created of 121 glycosylation-related genes involved in oligosaccharide metabolism pathways in bovine by analyzing the oligosaccharide profiles and performing an extensive literature search. No significant differences were observed in either oligosaccharide profiles or expressions of glycosylation-related genes between Holstein and Jersey cows. The highest concentrations of free oligosaccharides were observed in the colostrum samples and a sharp decrease was observed in the concentration of free oligosaccharides on day 15, followed by progressive decrease on days 90 and 250. Ninety-two glycosylation-related genes were expressed in milk somatic cells. Most of these genes exhibited higher expression in day 250 samples indicating increases in net glycosylation-related metabolism in spite of decreases in free milk oligosaccharides in late lactation milk. Even though fucosylated free oligosaccharides were not identified, gene expression indicated the likely presence of fucosylated oligosaccharides in bovine milk. Fucosidase genes were expressed in milk and a possible explanation for not detecting fucosylated free oligosaccharides is the degradation of large fucosylated free oligosaccharides by the fucosidases. Detailed characterization of enzymes encoded by the 92 glycosylation-related genes identified in this study will provide the basic knowledge for metabolic network analysis of oligosaccharides in mammalian milk. These candidate genes will guide the design of a targeted breeding strategy to optimize the content of beneficial oligosaccharides in bovine milk.

High-throughput sequencing of RNA (RNA-Seq) was developed primarily to analyze global gene expression in different tissues. However, it also is an efficient way to discover coding SNPs. The objective of this study was to perform a SNP discovery analysis in the milk transcriptome using RNA-Seq. Seven milk samples from Holstein cows were analyzed by sequencing cDNAs using the Illumina Genome Analyzer system. We detected 19,175 genes expressed in milk samples corresponding to approximately 70% of the total number of genes analyzed. The SNP detection analysis revealed 100,734 SNPs in Holstein samples, and a large number of those corresponded to differences between the Holstein breed and the Hereford bovine genome assembly Btau4.0. The number of polymorphic SNPs within Holstein cows was 33,045. The accuracy of RNA-Seq SNP discovery was tested by comparing SNPs detected in a set of 42 candidate genes expressed in milk that had been resequenced earlier using Sanger sequencing technology. Seventy of 86 SNPs were detected using both RNA-Seq and Sanger sequencing technologies. The KASPar Genotyping System was used to validate unique SNPs found by RNA-Seq but not observed by Sanger technology. Our results confirm that analyzing the transcriptome using RNA-Seq technology is an efficient and cost-effective method to identify SNPs in transcribed regions. This study creates guidelines to maximize the accuracy of SNP discovery and prevention of false-positive SNP detection, and provides more than 33,000 SNPs located in coding regions of genes expressed during lactation that can be used to develop genotyping platforms to perform marker-trait association studies in Holstein cattle.

BackgroundThe identification of novel genes is critical to understanding the molecular basis of body weight. Towards this goal, we have identified secretogranin V (Scg5; also referred to as Sgne1), as a candidate gene for growth traits.ResultsThrough a combination of DNA microarray analysis and quantitative PCR we identified a strong expression quantitative trait locus (eQTL) regulating Scg5 expression in two mouse chromosome 2 congenic strains and three additional F2 intercrosses. More importantly, the eQTL was coincident with a body weight QTL in congenic mice and Scg5 expression was negatively correlated with body weight in two of the F2 intercrosses. Analysis of haplotype blocks and genomic sequencing of Scg5 in high (C3H/HeJ, DBA/2J, BALB/cByJ, CAST/EiJ) and low (C57BL/6J) expressing strains revealed mutations unique to C57BL/6J and possibly responsible for the difference in mRNA abundance. To evaluate the functional consequence of Scg5 overexpression we measured the pituitary levels of 7B2 protein and PCSK2 activity and found both to be increased. In spite of this increase, the level of pituitary alpha-MSH, a PCSK2 processing product, was unaltered.ConclusionTogether, these data support a role for Scg5 in the modulation of body weight.

The annotation of animal genomes plays an important role in elucidating molecular mechanisms behind the genetic control of economically important traits. Here, we employed long‐read sequencing technology, Oxford Nanopore Technology, to annotate the pig transcriptome across 17 tissues from two Yorkshire littermate pigs. More than 9.8 million reads were obtained from a single flow cell, and 69 781 unique transcripts at 50 108 loci were identified. Of these transcripts, 16 255 were found to be novel isoforms, and 22 344 were found at loci that were novel and unannotated in the Ensembl (release 102) and NCBI (release 106) annotations. Novel transcripts were mostly expressed in cerebellum, followed by lung, liver, spleen, and hypothalamus. By comparing the unannotated transcripts to existing databases, there were 21 285 (95.3%) transcripts matched to the NT database (v5) and 13 676 (61.2%) matched to the NR database (v5). Moreover, there were 4324 (19.4%) transcripts matched to the SwissProt database (v5), corresponding to 11 356 proteins. Tissue‐specific gene expression analyses showed that 9749 transcripts were highly tissue‐specific, and cerebellum contained the most tissue‐specific transcripts. As the same samples were used for the annotation of cis‐regulatory elements in the pig genome, the transcriptome annotation generated by this study provides an additional and complementary annotation resource for the Functional Annotation of Animal Genomes effort to comprehensively annotate the pig genome. [ABSTRACT FROM AUTHOR]

Abstract Background The identification of novel genes is critical to understanding the molecular basis of body weight. Towards this goal, we have identified secretogranin V (Scg5; also referred to as Sgne1), as a candidate gene for growth traits. Results Through a combination of DNA microarray analysis and quantitative PCR we identified a strong expression quantitative trait locus (eQTL) regulating Scg5 expression in two mouse chromosome 2 congenic strains and three additional F2 intercrosses. More importantly, the eQTL was coincident with a body weight QTL in congenic mice and Scg5 expression was negatively correlated with body weight in two of the F2 intercrosses. Analysis of haplotype blocks and genomic sequencing of Scg5 in high (C3H/HeJ, DBA/2J, BALB/cByJ, CAST/EiJ) and low (C57BL/6J) expressing strains revealed mutations unique to C57BL/6J and possibly responsible for the difference in mRNA abundance. To evaluate the functional consequence of Scg5 overexpression we measured the pituitary levels of 7B2 protein and PCSK2 activity and found both to be increased. In spite of this increase, the level of pituitary α-MSH, a PCSK2 processing product, was unaltered. Conclusion Together, these data support a role for Scg5 in the modulation of body weight.

Progenitor cells of the tendon proper and peritenon have unique properties that could impact their utilization in tendon repair strategies. While a few markers have been found to aid in distinguishing progenitors cells from each region, there is great value in identifying more markers. In this study, we hypothesized that RNAseq could be used to improve our understanding of those markers that define these cell types. Transcriptome profiles were generated for pools of mouse Achilles tendon progenitor cells from both regions and catalogues of potential markers were generated. Moreover, common (e.g., glycoprotein, signaling, and proteinaceous extracellular matrix) and unique (e.g., cartilage development versus angiogenesis and muscle contraction) biological processes and molecular functions were described for progenitors from each region. Real‐time quantitative PCR of a subset of genes was used to gain insight into the heterogeneity amongst individual progenitor colonies from each region. Markers like Scx, Mkx, Thbs4, and Wnt10a were consistently able to distinguish tendon proper progenitors from peritenon progenitors; expression variability for other genes suggested greater cell type complexity for potential peritenon progenitor markers. This is the first effort to define Achilles tendon progenitor markers by region. Further efforts to investigate the value of these cataloged markers are required by screening more individual colonies of progenitors for more markers. Clinical Significance: Findings from this study advance efforts in the discernment of cell type specific markers for tendon proper and peritenon progenitor cells; insight into marker sets could improve tracking and sorting strategies for these cells for future therapeutic strategies. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1409–1418, 2019. [ABSTRACT FROM AUTHOR]

SbsupIII and AssupIII are known to exhibit similar chemical properties, but the degree of similarity in their effects on biological systems merits further exploration. The present work compares the responses of human epidermal keratinocytes, a known target cell type for arsenite-induced carcinogenicity, to these metalloids after treatment for 1 week at environmentally relevant concentrations. Previous work with these cells has shown that arsenite and antimonite have parallel effects in suppressing differentiation, altering levels of several critical enzymes and maintaining colony-forming ability. More globally, protein profiling now reveals parallels in SbsupIII and AssupIII effects. The more sensitive technique of transcriptional profiling also shows considerable parallels. Thus, gene expression changes were almost entirely in the same directions for the two treatments, although the degree of change was sometimes significantly different. Inspection of the changes revealed that RYR1 and LRIG1 were among the genes strongly suppressed, consistent with reduced calciumdependent differentiation and maintenance of epidermal growth factor-dependent proliferative potential. Moreover, levels of microRNAs in the cells were altered in parallel, with nearly 90% of the 198 most highly expressed ones being suppressed. Among these was miR-203, which is known to decrease proliferative potential. Finally, both SbsupIII and AssupIII were seen to attenuate bone morphogenetic protein 6 induction of dual-specificity phosphatases 2 and 14, consistent with maintaining epidermal growth factor receptor signalling. These findings raise the question of whether SbsupIII, like AssupIII, could act as a human skin carcinogen. [ABSTRACT FROM AUTHOR]