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The objectives of these experiments were to investigate (1) the relative abundance of transcripts for Cu-responsive genes in whole bovine liver vs. cultured hepatocytes and (2) the influence of Cu dose on the relative abundance of transcripts for Cu-responsive genes in cultured bovine hepatocytes. Experiment 1: Liver samples were obtained immediately post-mortem from one healthy Angus steer. Half of the tissue samples were placed in RNAlater solution; the remaining half was used to isolate hepatocytes. Experiment 2: A subset of cultured hepatocytes was incubated in media containing: 0 mg/L, 0.10 mg/L, 1.0 mg/L, 10.0 mg/L, and 100 mg/L Cu for 1 h. Transcripts analyzed were aldehyde dehydrogenase (ALDH2), apolipoprotein A-1 (APOA1), antioxidant 1 (ATOX1), ATPase copper transporting alpha (ATP7A), ATPase copper transporting beta (ATP7B), betaine homocysteine methyltransferase (BHMT), flavin reductase (BLVRB), carbonic anhydrase II (CA2), copper chaperone for superoxide dismutase (CCS), cytochrome c oxidase copper chaperone (COX17), Cu transporter 1 (CTR1), glutamate dehydrogenase (GLUD1), glutathione synthetase (GSS), protein disulfide isomerase A3 (PDIA3), and superoxide dismutase (Cu–Zn) (SOD1). Β-Actin (ACTB) was selected as the endogenous control in both experiments. Experiment 1: Whole liver had greater (P

As hindgut fermenters, horses are especially dependent on the microbiota residing in their cecum and large intestines. Interactions between these microbial populations and the horse are critical for maintaining gut homeostasis, which supports proper digestion. The current project was motivated to determine if any features of the fecal microbiota are informative of the microbial communities from the cecum, ventral colon, or dorsal colon. Digesta from the cecum, ventral colon, dorsal colon and feces were collected from 6 yearling miniature horses. Microbial DNA was isolated and the microbiota from each sample was characterized by profiling the V4 region of the 16S rRNA. Principal coordinate analysis of the beta diversity results revealed significant (p = 0.0001; F = 5.2393) similarities between the microbial populations from cecal and ventral colon and the dorsal colon and fecal samples, however, there was little overlap between the proximal and distal ends of the hindgut. These distinct population structures observed in our results coincide with the pelvic flexure, which itself separates intestinal compartments with distinct roles in digestive physiology. An indicator species analysis confirmed the population differences, supported by the identification of several microbial families characteristic of the compartments upstream of the pelvic flexure that were not represented following it. Our data suggest that the fecal microbiota is not informative of the proximal hindgut but can provide insight into communities of the distal compartments. Further, our results suggest that the pelvic flexure might be an important anatomical landmark relative to the microbial communities in the equine large intestine.

The objective of this experiment was to investigate the influence of copper (Cu) supplementation on Cu homeostatic genes in bovine liver. Liver samples were obtained from multiparous cows (n = 3 cows per treatment) from a previous experiment. Treatments consisted of: 1) no supplemental Cu (-Cu; total diet contained 7.0 mg Cu/kg DM) and 2) 3 mg Cu/kg DM (+Cu; total diet contained 10.0 mg Cu/kg DM). Cows were fed their respective diets for 420 d. Liver samples were harvested immediately upon slaughter, rinsed with phosphate-buffered saline solution, diced into small pieces, and placed into RNAlater and frozen at -80°C. Total RNA was extracted from all samples, and quantitative RT-PCR was used to determine the abundance of transcripts for proteins involved in Cu homeostasis in liver tissue. The target transcripts were: ALDH2, APOA1, ATOX1, ATP7A, ATP7B, BHMT, BLVRB, CA2, CCS, COX17, CTR1, ELN, GAPDH, GLUD1, GSS, LOXL1, PDIA3, SOD1, SOD3. The relative abundance of APOA1, ATOX1, ATP7B, BLVRB, CTR1, GLUD1, LOXL1, and SOD3 mRNA was greater (P < 0.05) in cows receiving supplemental Cu when compared to cattle not receiving supplemental Cu. These data indicate possible differences in Cu homeostasis in the liver based on Cu supplementation. Further investigation is warranted to determine if a difference in the relative abundance of identified transcripts are related to differences in associated protein production and function.

The sequencing and assembly of a reference genome for the horse has been revolutionary for investigation of horse health and performance. Next-generation sequencing (NGS) methods represent a second revolution in equine genomics. Researchers can align and compare DNA and RNA sequencing data to the reference genome to explore variation that may contribute or be attributed to disease. NGS has also facilitated the translation of research discovery to clinically relevant applications. This article discusses the history and development of NGS, details some of the available sequencing platforms, and describes currently available applications in the context of both discovery and clinical settings.

At altitudes >1500 m, measurements of mean pulmonary arterial pressures (mPAP; mmHg) are a measure of pulmonary hypertension (PH) in cattle. Genotypes of a G/A single nucleotide polymorphism (SNP; rs208684340) in the endothelial PAS domain protein 1 (EPAS1) gene were determined for mPAP categories (low, moderate, high) on Angus cattle. The A allele of this SNP was postulated to be associated with altitude-induced PH. The first objective was to estimate allele and genotypic frequencies in Angus cattle at high- (elevation 1850 to 2800 m) and low-altitude (elevation 91 to 1192 m) ranches (n = 118; random sample of 1275). Percent of cattle at these low elevation ranches with genotypes G/G, G/A, and A/A were 64.4, 33.9, and 1.7%, respectively, and a minor allele frequency (MAF; A allele) of 18.6%. Bulls, steers, and heifers (n = 691) from 4 high-altitude Angus herds in Colorado and Wyoming were genotyped. Percent of cattle with genotypes G/G, G/A, and A/A were 48.6, 34.3, and 17.1%, respectively, with a MAF of 34.2%. Average mPAP was 40.5 ± 8.8 mmHg. Low, moderate, and high-mPAP category MAF were similar (χ2 = 3.03; P = 0.22), with values of 35.4, 31.0, and 37.8%, respectively. Additionally we sought to evaluate the genotype-to-phenotype association of this SNP with mPAP phenotypes (n = 532) from the Colorado State University Beef Improvement Center (elevation 2150 m) Angus herd. Linear mixed model analysis suggested genotype was not a predictor (P = 0.61) of mPAP. Results do not suggest the A allele of SNP in EPAS1 is associated with high altitude-induced PH in yearling Angus cattle.

The objective of the current experiment was to investigate the influence of Cu dose on the relative abundance of Cu trafficking genes in cultured bovine hepatocytes. A liver sample was obtained immediately post-mortem from one healthy Angus steer. Hepatocytes were isolated, counted, and seeded at equal density into 15 separate wells, and incubated for 1 hour in culture media containing: 0.0, 0.10, 1.0, 10.0, or 100 mg Cu/L (3 replicates per Cu dose). Following incubation, cells were collected and total RNA was isolated. Quantitative RT-PCR was used to determine the abundance of transcripts for proteins involved in Cu homeostasis. The identified targets were: ALDH2, APOA1, ATOX1, ATP7A, ATP7B, BHMT, BLVRB, CA2, CCS, COX17, CTR1, ELN, GAPDH, GLUD1, GSS, LOXL1, PDIA3, SOD1, SOD3. β-Actin (ACTB) served as the endogenous control. Significant linear responses existed for ALDH2 (P < 0.001), ATOX1 (P < 0.01), PDIA3 (P < 0.05). As Cu dose increased, the relative abundance of ALDH2 increased, and ATOX1 and PDIA3 decreased. Significant quadratic responses existed for ATP7B (P < 0.001), COX17 (P < 0.05), and SOD1 (P < 0.05). The relative abundance of COX17 was lesser at 0.1 and 1.0 mg Cu/L when compared to 0.0, 10, and 100 mg Cu/L. Transcript abundance for ATP7B and SOD1 was lower at 0, 1, and 100 mg Cu/L when compared to 0.1 and 10 mg Cu/L. These data indicate that certain transcripts are differentially expressed in cultured bovine hepatocytes in response to increasing Cu dose.

The objective of this study was to determine the effect of corn supplementation on muscle microRNA (miRNA) profiles. Twelve mares were blocked by weight and BCS and assigned to one of two treatments (6 hd/treatment): 1) control, (basal diet: 9 Kg/hd/d of chopped mixed grass hay and ad libitum mixed grass hay), 2) basal diet supplemented with 454 g/d steam flaked corn individually using feed bags. All mares were placed on the basal diet and in the same pen 14 days prior to the beginning of corn supplementation. Mares were weighed and BCS on d -7 and 28. Muscle biopsies of the gluteus medius were taken from all horses on d0 and d26. Muscle samples were analyzed using real time RT-qPCR for 277 endogenous miRNAs. Raw CT values were normalized with the geometric mean of three consistently appearing endogenous miRNAs. Sixteen miRNAs that consistently appeared in at least 8 horses including 3 relating to metabolic disorders appearing at d0, d26, or both were analyzed. Differential abundances of miRNAs using log transformed fold change and differences between BW and BCS of treatment groups were determined by ANOVA and LS means analysis. There was no difference between BW (P < .05) and BCS (P < .05) between the two treatment groups. Feeding corn lead to higher quantities (P < .05) of mir 133a, 1515p, 6155p, 770, and 99b within muscle compared to control group. Lower quantities of mir 382 and 433 were found within muscle of horses fed corn compared to the control group (P < .05). Based upon the results of this study corn supplementation appears to influence endogenous miRNA expression profiles within muscle of horses.

Equine maternal recognition of pregnancy (MRP) is a process whose signal remains unknown. During MRP the conceptus and endometrium communicate to attenuate prostaglandin F2&alpha, (PGF) secretion, sparing the corpus luteum and maintaining progesterone production. Recognition of a mobile conceptus by the endometrium is critical by days 14&ndash, 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 &le, 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.

Gastrointestinal (GI) homeostasis in a horse results from dynamic interactions between a horse's gut physiology and the microbes in the various compartments. MicroRNAs (miRNA), single-stranded 20–22 basepair RNA molecules involved in post-transcriptional control of gene expression, have been implicated as an essential mechanism for control of gastrointestinal physiology and communication between host and microbe. Researchers have described how host-derived miRNAs influence microbial communities' composition in the GI. We have recently described how the pelvic flexure separates distinct microbial populations in the equine hindgut and subsequently have wondered if equine miRNA transcripts expressed by GI tissues could have a role in maintaining this separation. Investigations to characterize miRNAs' expression profile and other non-coding RNAs in the equine GI tract are limited. To address this, it is critical that we first know something about the expression of non-coding transcripts in the equine GI. This study investigated miRNA expression in tissues of the hindgut surrounding the pelvic flexure. RNA was isolated from the intestinal epithelium of 3 4-year-old American Quarter Horses collected from the right and left ventral colon (VC), pelvic flexure (PF), and right and left dorsal colon (DC). The miRNA transcripts were reverse transcribed using the miScript II RT kit, and relative abundance was quantified using the miScript SYBR Green PCR kit and primer sets for 286 annotated mature equine miRNAs. Biological replicates were pooled, and differential expression was determined following normalization by the delta Ct = Ct(target) – Ct(sample) method. A total of 230 miRNA transcripts were expressed in at least one GI location, with 60 expressed in all 5 locations. Twenty-eight transcripts had expression restricted to a single site, 5 of which were only expressed in the pelvic flexure. Additionally, 42 transcripts have patterns of expression across a subset of anatomical locations. Notably, 10 transcripts were expressed in the VC and PF, 5 transcripts were expressed in the DC and PF, and 15 transcripts were expressed in the VC and DC but not PF. Pathway and target analysis for the miRNAs with compartment restricted expression was performed using DIANA TOOLS. Of note, miRNA transcripts expressed only in the PF were predicted to target genes in the Hippo signaling pathway, which is associated with the regulation of intestinal tissue proliferation and homeostasis. Further investigation of miRNA expression in the equine hindgut will improve our understanding of gastrointestinal homeostasis's regulatory processes.

All epididymal regions are lined with multiple epithelial cell types, each with different functions to provide the luminal environment for spermatozoal maturation. Epithelial cells also create apical blebs, which are released from the apical surface via apocrine secretion and disintegrate in the lumen, thereby releasing epididymosomes. Epididymosomes transport proteins to spermatozoa and contain microRNAs. We hypothesized that epididymosomes also transfer miRNA from epididymal epithelium to spermatozoa. Quantitative real-time polymerase chain reaction was used to determine miRNA profiles of epididymal tissue from caput and cauda, epididymal spermatozoa from caput and cauda, and epididymosomes and from caput, proximal corpus, distal corpus, and cauda. Pathway analysis was performed using DIANA tools on the miRNA unique to caudal spermatozoa. We found 66 newly acquired miRNAs in spermatozoa located in the caudal epididymis. Predicted pathways targeted by these miRNAs suggest a role in cell motility and viability and factors in oocyte and embryo maturation and development. These findings suggest that miRNAs are transported to spermatozoa from epididymal epithelium via epididymosomes.

Beef cattle grazing patterns are influenced by topography in that cattle generally select grazing sites near water while avoiding steep, rugged terrain; therefore, forage is often left ungrazed in the mountainous uplands whereas lowland riparian zones can be heavily grazed. Improving uniformity in beef cattle terrain-use provides opportunities to increase forage harvest and sustain sensitive ecosystems. Studies suggest that terrain-use by cattle may be improved using genomic selection as combinations of single nucleotide polymorphisms (SNP) explained approximately 35% of the phenotypic variation in terrain-use indices that combined slope, elevation, and distance traveled from water. The objective of this study was to perform genome-wide association studies (GWAS) for six terrain-use traits (slope, elevation, vertical climb, distance traveled from water, rolling index, and rough index) in which we explored single-SNP associations, surveyed the genome in consecutive, one mega-base windows and examined quantitative trait loci (QTL) and underlying positional and functional candidate genes using bioinformatic tools to identify possible pleiotropic effects with other traits. Global positioning system (GPS) collars were used to track terrain-use patterns for 330 beef cows across 14 rangeland operations located in the western U.S. and Illumina BovineHD and BovineSNP50 SNP arrays were used to collect genomic data. Genotype quality control was conducted using PLINK software and association analyses were performed using BOLT, implementing a BayesC model. Quantitative trait loci detection was based upon the posterior inclusion probability (PIP) for individual loci and the proportion of total genetic variance explained by individual genomic windows. Analyses revealed 19 QTL that have been previously associated with health and production traits in dairy and beef cattle as well as 8 positional putative candidate genes that function in oxygen homeostasis, feed efficiency, and growth. These results suggest that beef cattle terrain-use traits are polygenic and may be pleiotropic with other production traits.

Pulmonary hypertension (PH) and subsequent right heart failure is an economic burden for high altitude beef production systems. An indicator of PH is pulmonary arterial pressure (PAP), which has been used to improve tolerance of cattle to high altitude due to its moderate heritability (0.20 to 0.40). A key mediator of myocardial contraction and relaxation is intracellular signaling of calcium. We hypothesized that genes involved in calcium signaling pathways have a role in susceptibility of cattle to PH at high altitude. Right ventricle tissues were collected from Angus steers (age 12 to 14 months; live weight 387 ± 2.5 kg) fed and harvested at high altitude (2,170 m). Experimental groups consisted of low PAP, healthy (average 39 ± 0.2 mm Hg; n = 7) and high PAP, sick/symptomatic of potential heart failure (average 82 ± 2.9 mm Hg; n = 3) steers. Gene expression counts were estimated from RNA-sequence data and then analyzed with DESeq, resulting in 622 differentially expressed genes between experimental groups (Fold change > 2.0; P < 0.05), after a multiple testing correction with the Benjamini-Hochberg procedure. Ingenuity Pathway Analysis revealed the top disease and biological function as cardiovascular malformation represented by 134 of the differentially expressed genes (P < 0.01). The principal intra-pathway genes identified were TGFβ1, an upstream regulator of ventricular chamber hypertrophy and systolic dysfunction leading to heart failure, and EDN1, a vasoconstrictor with downstream regulatory effects on intracellular calcium availability. Two additional genes, FBLN7, a calcium-binding glycoprotein; and ANKRD2, which interacts with calcium-dependent genes, represented the highest expression fold changes. These results support our hypothesis that calcium-dependent gene regulation appears to be influenced by PH (right ventricle malformation) in Angus steers fed at high altitude.

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.

While the phenomenon of polyadenylation has been well-studied, the dynamics of poly(A) tail size and its impact on transcript function and cell biology are less well-appreciated. The goal of this review is to encourage readers to view the poly(A) tail as a dynamic, changeable aspect of a transcript rather than a simple static entity that marks the 3' end of an mRNA. This could open up new angles of regulation in the post-transcriptional control of gene expression throughout development, differentiation and cancer.

As a result of climate change, salt lakes on Eyre Peninsula are predicted to have shorter hydroperiods. Already there are salinity increases due to land use changes, so major decreases in biodiversity are expected. The likely situation is different however for the few lakes receiving waters via marine springs, with Seagull Lake, 20 km south of Streaky Bay, the best example. It has been thoroughly surveyed as a base to assess future changes. This lake originated about 6000 years ago as a marine bay, since occluded by coastal dunes. Its waters are a balance between meteoric sources, groundwater and marine water via springs with seasonal fluctuations due to input variations and evaporation. A conceptual model closely matched field observations. Presently the main lake varied in salinity seasonally from 75 to 200 g/L, with the spring virtually constant at 37–40 g/L. Plants in the lake and its palustrine margins numbered 25 species arranged into 18 floristic associations. Of particular interest are the...

Previously, we have shown that horses could be divided into susceptible and resistant groups based on an in vitro assay using dual-color flow cytometric analysis of CD3 + T cells infected with equine arteritis virus (EAV). Here, we demonstrate that the differences in in vitro susceptibility of equine CD3 + T lymphocytes to EAV infection have a genetic basis. To investigate the possible hereditary basis for this trait, we conducted a genome-wide association study (GWAS) to compare susceptible and resistant phenotypes. Testing of 267 DNA samples from four horse breeds that had a susceptible or a resistant CD3 + T lymphocyte phenotype using both Illumina Equine SNP50 BeadChip and Sequenom's MassARRAY system identified a common, genetically dominant haplotype associated with the susceptible phenotype in a region of equine chromosome 11 (ECA11), positions 49572804 to 49643932. The presence of a common haplotype indicates that the trait occurred in a common ancestor of all four breeds, suggesting that it may be segregated among other modern horse breeds. Biological pathway analysis revealed several cellular genes within this region of ECA11 encoding proteins associated with virus attachment and entry, cytoskeletal organization, and NF-κB pathways that may be associated with the trait responsible for the in vitro susceptibility/resistance of CD3 + T lymphocytes to EAV infection. The data presented in this study demonstrated a strong association of genetic markers with the trait, representing de facto proof that the trait is under genetic control. To our knowledge, this is the first GWAS of an equine infectious disease and the first GWAS of equine viral arteritis.

Concentrated grazing may destroy wildlife habitat, reduce forage harvest, and degrade riparian areas; therefore, it is necessary to develop tools to improve grazing distribution for extensive pastures in beef production systems. A previous genome-wide association study identified 5 QTL on BTA 4, 12, 17, and 29 that were associated with grazing distribution indices (rolling and rough). The objective of this study was to validate these QTL using additional association analyses with a Bayesian approach. Global positioning system (GPS) technology was used to collect grazing distribution phenotypes from 80 beef cows on 5 ranches in New Mexico, Montana, and Arizona. Percent slope, elevation, and distance from water were calculated using GPS coordinates and incorporated into two terrain-use indices (rough and rolling). Cows were genotyped with 777,962 SNP and after applying standard SNP quality filters, 733,713 SNP from 75 cows were available for analysis. A single chromosome (BTA 4, 12, 17, 29) association analysis was performed for both the rough and rolling indices using Bayes C (within the BOLT software package. The posterior inclusion probability (PIP) for each SNP was estimated. The association between rs109619368 on BTA 17 and the rough index was confirmed (PIP = 2.1%); however, the QTL on BTA 4, 12, and 29 were not verified. To further investigate this, 50 SNP spanning 0.2 MB on BTA 29 were analyzed with the rolling index. Two SNP were confirmed in this analysis: rs42161939 (PIP = 54.9%) and rs43744222 (PIP = 21.6%). The Bayesian approach applied in this study, in which markers are simultaneously fit in the model, revealed 3 QTL concordant with previous analyses which used a single-marker regression approach. These results suggest that grazing distribution traits are under genetic control; however, a greater number of observations are needed to confirm the QTL associated with grazing distribution (SW15-015).

Pulmonary hypertension (PH) can cause heart failure in cattle and is measured with pulmonary arterial pressure (PAP). Although historically associated with high elevation (> 1,500 m), PH and heart failure are increasing in moderate altitude feedlots. High elevation production systems wean and stocker calves before placing them in feedlot finishing systems of moderate elevations on the Great Plains of North America. The objective was to evaluate PAP and growth of steers finished at moderate or high elevation with varied lengths of post-weaning grazing. Forty steers from Colorado’s Beef Improvement Center (2,150 m) with PAP averaging 41.5 ± 0.5 mmHg were randomly assigned to one of four management groups: 1) traditional stocker and finishing system (i.e., grown and finished at 1,420 m beginning at 10 months of age), 2) extended stocker grazing at high elevation (2,150 m), but finishing at 1,420 m at 18 months of age, 3) stocker and finishing at high elevation (2,150 m) at 24 months of age, or 4) grass finishing (2,150 m) > 24 months of age. Weight and PAP data were analyzed with a repeated measures model. Effects of group, PAP-date, and their interaction (P < 0.01) were observed. Pre-harvest, steers in the stocker-finishing group at high elevation had higher PAP than the moderate elevation finishing groups (52.9 > 46.5 ± 1.7 mmHg), but PAP (41.1 ± 1.6) was unchanged across dates in the grass finishing group and lower than the grain-fed groups. Pre-harvest live weight averaged 621.6 ± 11.8 kg in all three grain finishing groups, whereas grass finishing steers weighed 436.6 ± 11.5 kg on these dates. In summary, grain-fattening appeared to cause PH independent of elevation. Steers grazing in a high elevation grass finishing system did not experience increased PAP, but required several more months of grazing to reach harvest criteria.

The horse, like the majority of animal species, has a limited amount of species-specific expressed sequence data available in public databases. As a result, structural models for the majority of genes defined in the equine genome are predictions based on ab initio sequence analysis or the projection of gene structures from other mammalian species. The current study used Illumina-based sequencing of messenger RNA (RNA-seq) to help refine structural annotation of equine protein-coding genes and for a preliminary assessment of gene expression patterns. Sequencing of mRNA from eight equine tissues generated 293,758105 sequence tags of 35 bases each, equalling 10.28 gbp of total sequence data. The tag alignments represent approximately 207 × coverage of the equine mRNA transcriptome and confirmed transcriptional activity for roughly 90% of the protein-coding gene structures predicted by Ensembl and NCBI. Tag coverage was sufficient to refine the structural annotation for 11,356 of these predicted genes, while also identifying an additional 456 transcripts with exon/intron features that are not listed by either Ensembl or NCBI. Genomic locus data and intervals for the protein-coding genes predicted by the Ensembl and NCBI annotation pipelines were combined with 75,116 RNA-seq-derived transcriptional units to generate a consensus equine protein-coding gene set of 20,302 defined loci. Gene ontology annotation was used to compare the functional and structural categories of genes expressed in either a tissue-restricted pattern or broadly across all tissue samples.

Toll-like receptors 3, 7, and 8 (TLR3, TLR7, and TLR8) were studied in the genomes of the domestic horse and several other mammals. The messenger RNA sequences and exon/intron structures of these TLR genes were determined. An equine bacterial artificial chromosome clone containing the TLR3 gene was assigned by fluorescent in situ hybridization to the horse chromosomal location ECA27q16-q17 and this map location was confirmed using an equine radiation hybrid panel. Direct sequencing revealed 13 single-nucleotide polymorphisms in the coding regions of the equine TLR 3, 7, and 8 genes. Of these polymorphisms, 12 were not previously reported. The allelic frequency was estimated for each single-nucleotide polymorphism from genotyping data obtained for 154 animals from five horse breeds. Some of these frequencies varied significantly among different horse breeds. Domain architecture predictions for the three equine TLR protein sequences revealed several conserved regions within the variable leucine-rich repeats between the corresponding horse and cattle TLR proteins. A phylogenetic analysis did not indicate that any significant exchanges had occurred between paralogous TLR7 and TLR8 genes in 20 vertebrate species analyzed.

Australia’s only federally listed endangered samphire, the fan or bead samphire Tecticornia flabelliformis (Paul G.Wilson) K.A.Sheph. & Paul G.Wilson, is a small deciduous forb up to twenty centimetres high that is generally found growing in monospecific patches on clay pans or sabkhas directly behind coastal barrier dunes or on salt lakes further inland. The habitat requirements that control the distribution of fan samphires are poorly understood.During September 2004, thirty-six soil samples were collected from within and surrounding three stands of fan samphires near Gulf St Vincent, South Australia. Analysis of the samples showed that pH had the strongest association with the presence of fan samphires, with field moist chlorinity also strongly associated. Oven dried chlorinity and percentage moisture were not significantly different between sites with fan samphires and those sites without, although the fan samphires occurred on soils with a wider range for these parameters than the surrounding...

Cellular mechanisms that achieve protein diversity in eukaryotes are multifaceted, including transcriptional components such as RNA splicing. Through alternative splicing, a single protein-coding gene can generate multiple mRNA transcripts and protein isoforms, some of which are tissue-specific. We have conducted qualitative and quantitative analyses of the Bodymap 2.0 messenger RNA-sequencing data from 16 human tissue samples and identified 209,363 splice junctions. Of these, 22,231 (10.6%) were not previously annotated and 21,650 (10.3%) were expressed in a tissue-restricted pattern. Tissue-restricted alternative splicing was found to be widespread, with approximately 65% of expressed multi-exon genes containing at least one tissue-specific splice junction. Interestingly, we observed many tissue-specific splice junctions not only in genes expressed in one or a few tissues, but also from gene loci with a broad pattern of expression.

Current gene annotation of the horse genome is largely derived from in silico predictions and cross-species alignments. Only a small number of genes are annotated based on equine EST and mRNA sequences. To expand the number of equine genes annotated from equine experimental evidence, we sequenced mRNA from a pool of forty-three different tissues. From these, we derived the structures of 68,594 transcripts. In addition, we identified 301,829 positions with SNPs or small indels within these transcripts relative to EquCab2. Interestingly, 780 variants extend the open reading frame of the transcript and appear to be small errors in the equine reference genome, since they are also identified as homozygous variants by genomic DNA resequencing of the reference horse. Taken together, we provide a resource of equine mRNA structures and protein coding variants that will enhance equine and cross-species transcriptional and genomic comparisons.

The reference assembly for the domestic horse, EquCab2, published in 2009, was built using approximately 30 million Sanger reads from a Thoroughbred mare named Twilight. Contiguity in the assembly was facilitated using nearly 315 thousand BAC end sequences from Twilight’s half brother Bravo. Since then, it has served as the foundation for many genome-wide analyses that include not only the modern horse, but ancient horses and other equid species as well. As data mapped to this reference has accumulated, consistent variation between mapped datasets and the reference, in terms of regions with no read coverage, single nucleotide variants, and small insertions/deletions have become apparent. In many cases, it is not clear whether these differences are the result of true sequence variation between the research subjects’ and Twilight’s genome or due to errors in the reference. EquCab2 is regarded as “The Twilight Assembly.” The objective of this study was to identify inconsistencies between the EquCab2 assembly and the source Twilight Sanger data used to build it. To that end, the original Sanger and BAC end reads have been mapped back to this equine reference and assessed with the addition of approximately 40X coverage of new Illumina Paired-End sequence data. The resulting mapped datasets identify those regions with low Sanger read coverage, as well as variation in genomic content that is not consistent with either the original Twilight Sanger data or the new genomic sequence data generated from Twilight on the Illumina platform. As the haploid EquCab2 reference assembly was created using Sanger reads derived largely from a single individual, the vast majority of variation detected in a mapped dataset comprised of those same Sanger reads should be heterozygous. In contrast, homozygous variations would represent either errors in the reference or contributions from Bravo's BAC end sequences. Our analysis identifies 720,843 homozygous discrepancies between new, high throughput genomic sequence data generated for Twilight and the EquCab2 reference assembly. Most of these represent errors in the assembly, while approximately 10,000 are demonstrated to be contributions from another horse. Other results are presented that include the binary alignment map file of the mapped Sanger reads, a list of variants identified as discrepancies between the source data and resulting reference, and a BED annotation file that lists the regions of the genome whose consensus was likely derived from low coverage alignments.

Trials conducted at Dry Creek, a solar saltfield north of Adelaide, Australia, indicate that manganese, as manganous sulphate, can increase both the purity and strength of salt crystals grown at an ambient or neutral-slightly acid pH range resulting in a salt suitable for high purity uses. The improved load bearing strength of the crystals indicates that manganous sulphate could also be useful in salt floor maintenance. However, the impact of an apparently slight rise in pH to 8.5 was significant. Crystals produced at the higher pH from the manganese enriched brine were opaque with many tiny, almost colloidal crystals. The salt did not drain well, had low load bearing strength and was notably impure.

Sequencing of equine mRNA (RNA-seq) identified 428 putative transcripts which do not map to any previously annotated or predicted horse genes. Most of these encode the equine homologs of known protein-coding genes described in other species, yet the potential exists to identify novel and perhaps equine-specific gene structures. A set of 36 transcripts were prioritized for further study by filtering for levels of expression (depth of RNA-seq read coverage), distance from annotated features in the equine genome, the number of putative exons, and patterns of gene expression between tissues. From these, four were selected for further investigation based on predicted open reading frames of greater than or equal to 50 amino acids and lack of detectable homology to known genes across species. Sanger sequencing of RT-PCR amplicons from additional equine samples confirmed expression and structural annotation of each transcript. Functional predictions were made by conserved domain searches. A single transcript, expressed in the cerebellum, contains a putative kruppel-associated box (KRAB) domain, suggesting a potential function associated with zinc finger proteins and transcriptional regulation. Overall levels of conserved synteny and sequence conservation across a 1MB region surrounding each transcript were approximately 73% compared to the human, canine, and bovine genomes; however, the four loci display some areas of low conservation and sequence inversion in regions that immediately flank these previously unannotated equine transcripts. Taken together, the evidence suggests that these four transcripts are likely to be equine-specific.

A funding organization and a preferred acronym for one of the funding organizations already listed were incorrectly omitted from the Funding Statement. The Funding Statement should read: "This work was funded by the University of Kentucky Gluck Equine Research Foundation, the Morris Animal Foundation, the Lourie Foundation, the National Science Foundation (EF-0850237), and the Kentucky Biomedical Research Infrastructure Network (KBRIN) supported in part by National Institutes of Health grant KY-INBRE P20 RR16481. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

Summary Brother of CDO (BOC) is a cell surface receptor that derives its name from the structurally related protein, cell adhesion molecule-related/down-regulated by oncogenes (CDO, sometimes CDON). High levels of BOC mRNA and protein expression have been described in embryonic tissues with active cell proliferation and ongoing cellular differentiation 1,2 . A microarray-based screen of RNA isolated from 11 different adult equine tissues unexpectedly identified BOC as having an expression pattern restricted to articular cartilage. The objective of this study was to further investigate BOC expression in adult articular cartilage relative to other tissues. Both RT-qPCR and mRNA sequencing confirmed the microarray data. Steady state BOC mRNA levels in articular cartilage were substantially higher than in the other adult tissues tested, neonatal tendon, placenta, and whole embryo. The expression of BOC displayed a pattern of tissue specificity comparable to well established cartilage matrix protein biomarkers. BOC mRNA levels in articular cartilage increased with age, but were rapidly down-regulated when chondrocytes were enzymatically isolated from the cartilage matrix and expanded in monolayer culture. Relative expression patterns of CDO were broadly similar, but displayed lower fold change differences. A functional role in articular cartilage that involves Hedgehog signaling is suggested by the known binding affinity of BOC for all three Hedgehog ligands. These data also extend BOC and CDO biology to a post-mitotic and highly differentiated cell type within a mature tissue.

The accurate mapping of reads that span splice junctions is a critical component of all analytic techniques that work with RNA-seq data. We introduce a second generation splice detection algorithm, MapSplice, whose focus is high sensitivity and specificity in the detection of splices as well as CPU and memory efficiency. MapSplice can be applied to both short (75 bp). MapSplice is not dependent on splice site features or intron length, consequently it can detect novel canonical as well as non-canonical splices. MapSplice leverages the quality and diversity of read alignments of a given splice to increase accuracy. We demonstrate that MapSplice achieves higher sensitivity and specificity than TopHat and SpliceMap on a set of simulated RNA-seq data. Experimental studies also support the accuracy of the algorithm. Splice junctions derived from eight breast cancer RNA-seq datasets recapitulated the extensiveness of alternative splicing on a global level as well as the differences between molecular subtypes of breast cancer. These combined results indicate that MapSplice is a highly accurate algorithm for the alignment of RNA-seq reads to splice junctions. Software download URL: http://www.netlab.uky .edu/p/bioinfo/MapSplice.

Background RNA-sequencing (RNA-seq) data from eight equine tissue samples (34-day whole embryo, full term placental villous, adult testes, adult cerebellum, adult articular cartilage, adult LPS-stimulated articular cartilage, adult synovial membrane, and adult LPS-stimulated synovial membrane) were used to refine the structural annotation of protein-coding genes in the horse and for a preliminary assessment of tissue-specific expression patterns.

A Horse Is a Horse, of Course The history of horse domestication is closely tied to the history of the human society. Wade et al. (p. 865 ) report on the sequencing and provide a single nucleotide polymorphism map of the horse ( Equus caballus ) genome. Horses are a member of the order perissodactyla (odd-toed animals with hooves). The analysis reveals an evolutionarily new centromere on equine chromosome 11 that displays properties of an immature but fully functioning centromere and is devoid of centromeric satellite sequence. The findings clarify the nature of genetic diversity within and across horse breeds and suggest that the horse was domesticated from a relatively large number of females, but few males.

Loci for 9322 equine expressed sequence tags (ESTs) were predicted using the Comparative Mapping by Annotation and Sequence Similarity (Compass) strategy in order to evaluate the programme's ability to make accurate locus predictions in species with comparative gene maps. Using human genome sequence information from Build 35 (May 2004) and published marker information from the radiation hybrid (RH) maps for equine chromosomes (ECA) 17 and X, 162 ESTs were predicted to locations on ECA17 and 328 ESTs to locations on ECAX by selection of the 'top blast hit'. The locations of 30 ESTs were assessed experimentally by RH mapping analysis to evaluate the accuracy of the Compass predictions. The data revealed that 53% (16 of 30) of the ESTs predicted on ECA17 and ECAX mapped to those chromosomes. Analysis of the results suggested the need to identify expressed orthologous sequences in order to generate more accurate predictions for ESTs. Locus predictions were reassessed with three modifications to the Compass strategy's orthologue selection parameters. Selection of the 'top gene hit' improved accuracy to 72% (21 of 29), while selection of the 'top expressed gene hit' improved accuracy to 86% (24 of 28). Using the default Compass parameters with the UniGene database improved prediction accuracy to 96% (22 of 23); however, this level of accuracy came with a substantial decrease in the total number of predictions. When used with optimized prediction parameters, the Compass strategy can be a practical in silico map location prediction tool for large EST sample sets from unsequenced animal genomes.