Mapping splice QTLs reveals distinct transcriptional and post-transcriptional regulatory variation of gene expression and identifies putative alternative splicing variation mediating complex trait variation in pigs.

BACKGROUND: Alternative splicing is an important step in gene expression, generating multiple isoforms for the same genes and greatly expanding the diversity of proteomes. Genetic variation in alternative splicing contributes to phenotypic diversity in natural populations. However, the genetic basis of variation in alternative splicing in livestock including pigs remains poorly understood. RESULTS: In this study, using a Duroc x Pietrain F2 pig population, we performed genome-wide analysis of alternative splicing estimated from stranded RNA-Seq data in skeletal muscle. We characterized the genetic architecture of alternative splicing and compared its basic features with those of overall gene expression. We detected a large number of novel alternative splicing events that were not previously annotated. We found heritability of quantitative alternative splicing scores (percent spliced in or PSI) to be lower than that of overall gene expression. In addition, heritabilities showed little correlation between alternative splicing and overall gene expression. We mapped expression QTLs (eQTLs) and splice QTLs (sQTLs) and found them to be largely non-overlapping. Finally, we integrated sQTL mapping with phenotype QTL (pQTL mapping to identify potential mediator of pQTL effect by alternative splicing. CONCLUSIONS: Our results suggest that regulatory variation exists at multiple levels and that their genetic controls are distinct, offering opportunities for genetic improvement.

Transcriptome annotation of 17 porcine tissues using nanopore sequencing technology.

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.

Assessment of DNA methylation in porcine immune cells reveals novel regulatory elements associated with cell-specific gene expression and immune capacity traits.

BACKGROUND: Genetics studies in the porcine immune system have enhanced selection practices for disease resistance phenotypes and increased the efficacy of porcine models in biomedical research; however limited functional annotation of the porcine immunome has hindered progress on both fronts. Among epigenetic mechanisms that regulate gene expression, DNA methylation is the most ubiquitous modification made to the DNA molecule and influences transcription factor binding as well as gene and phenotype expression. Human and mouse DNA methylation studies have improved mapping of regulatory elements in these species, but comparable studies in the pig have been limited in scope. RESULTS: We performed whole-genome bisulfite sequencing to assess DNA methylation patterns in nine pig immune cell populations: CD21+ and CD21- B cells, four T cell fractions (CD4+, CD8+, CD8+CD4+, and SWC6γδ+), natural killer and myeloid cells, and neutrophils. We identified 54,391 cell differentially methylated regions (cDMRs), and clustering by cDMR methylation rate grouped samples by cell lineage. 32,737 cDMRs were classified as cell lowly methylated regions (cLMRs) in at least one cell type, and cLMRs were broadly enriched in genes and regions of intermediate CpG density. We observed strong correlations between differential methylation and expression across immune cell populations, with cell-specific low methylation disproportionately impacting genes exhibiting enriched gene expression in the same cell type. Motif analysis of cLMRs revealed cell type-specific enrichment of transcription factor binding motifs, indicating that cell-specific methylation patterns may influence accessibility by trans-acting factors. Lastly, cDMRs were enriched for immune capacity GWAS SNPs, and many such overlaps occurred within genes known to influence immune cell development and function (CD8B, NDRG1). CONCLUSION: Our DNA methylation data improve functional annotation of the porcine genome through characterization of epigenomic regulatory patterns that contribute to immune cell identity and function, and increase the potential for identifying mechanistic links between genotype and phenotype.

The Social Life of Pigs: Changes in Affiliative and Agonistic Behaviors following Mixing.

This study investigated potentially affiliative behaviors in grow-finish pigs, how these behaviors changed over time and their relationship to agonistic behaviors. A total of 257 Yorkshire barrows were observed for agonistic (reciprocal fights, attacks) and affiliative (nosing, play, non-agonistic contact) behaviors after mixing (at 10 weeks of age), and weeks 3, 6, and 9 after mix. The least square means of affiliative behaviors were compared across time points. Relationships among affiliative and agonistic behaviors were assessed using generalized linear mixed models. Non-agonistic contact with conspecifics increased until week 6 then remained stable between weeks 6 and 9. Nosing was highest at mix, then decreased in the following weeks. Play was lowest at mix and highest at week 3. Affiliative behaviors were negatively related with aggression at mix (p < 0.001). Pigs who engaged in play and nosing behaviors were more likely to be involved in agonistic interactions in the weeks after mixing (p < 0.05), while pigs engaging in non-agonistic contact were less likely to be involved in agonistic interactions (p < 0.001). There appear to be relationships between affiliative and agonistic behaviors in pigs, with contact being the most predictive of less aggression. Future studies could focus on promoting positive non-agonistic contact in unfamiliar pigs as a way to mitigate aggressive interactions.

Pig genome functional annotation enhances the biological interpretation of complex traits and human disease.

The functional annotation of livestock genomes is crucial for understanding the molecular mechanisms that underpin complex traits of economic importance, adaptive evolution and comparative genomics. Here, we provide the most comprehensive catalogue to date of regulatory elements in the pig (Sus scrofa) by integrating 223 epigenomic and transcriptomic data sets, representing 14 biologically important tissues. We systematically describe the dynamic epigenetic landscape across tissues by functionally annotating 15 different chromatin states and defining their tissue-specific regulatory activities. We demonstrate that genomic variants associated with complex traits and adaptive evolution in pig are significantly enriched in active promoters and enhancers. Furthermore, we reveal distinct tissue-specific regulatory selection between Asian and European pig domestication processes. Compared with human and mouse epigenomes, we show that porcine regulatory elements are more conserved in DNA sequence, under both rapid and slow evolution, than those under neutral evolution across pig, mouse, and human. Finally, we provide biological insights on tissue-specific regulatory conservation, and by integrating 47 human genome-wide association studies, we demonstrate that, depending on the traits, mouse or pig might be more appropriate biomedical models for different complex traits and diseases.

Estimation of direct and social effects of feeding duration in growing pigs using records from automatic feeding stations.

Automatic feeding systems in pig production allow for the recording of individual feeding behavior traits, which might be influenced by the social interactions among individuals. This study fitted mixed models to estimate the direct and social effects on visit duration at the feeder of group-housed pigs. The dataset included 74,413 records of each visit duration time (min) event at the automatic feeder from 135 pigs housed in 14 pens. The sequence of visits at the feeder was employed as a proxy for the social interaction between individuals. To estimate animal effects, the direct effect was apportioned to the animal feeding (feeding pig), and the social effect was apportioned to the animal that entered the feeder immediately after the feeding pig left the feeding station (follower). The data were divided into two subsets: "non-immediate replacement" time (NIRT, N = 6,256), where the follower pig occupied the feeder at least 600 s after the feeding pig left the feeder, and "immediate replacement" time (IRT, N = 58,255), where the elapsed time between replacements was less than or equal to 60 s. The marginal posterior distribution of the parameters was obtained by Bayesian method. Using the IRT subset, the posterior mean of the proportion of variance explained by the direct effect (PrpσTemefós) was 18% for all models. The proportion of variance explained by the follower social effect (Prpσ^f2) was 2%, and the residual variance (σ^e2) decreased, suggesting an improved model fit by including the follower effect. Fitting the models with the NIRT subset, the estimate of PrpσTemefós was 20% but the Prpσ^f2 was almost zero and σ^e2 was identical for all models. For the IRT subset, the predicted best linear unbiased predictor (BLUP) of direct (Direct BLUP) and social (Follower BLUP) random effects on visit duration at the feeder of an animal was calculated. Feeder visit duration time was not correlated with traits, such as weight gain or average feed intake (P > 0.05), whereas for the daily feeder occupation time, the estimated correlation was positive with the Direct BLUP (r^ = 0.51, P < 0.05) and negative with the Follower BLUP (r^= -0.26, P < 0.05). The results suggest that the visit duration of an animal at the single-space feeder was influenced by both direct and social effects when the replacement time between visits was less than 1 min. Finally, animals that spent a longer time per day at the feeder seemed to do so by shortening the meal length of the preceding individual at the feeder.

Integrated Genome-Wide Analysis of MicroRNA Expression Quantitative Trait Loci in Pig Longissimus Dorsi Muscle.

Determining mechanisms regulating complex traits in pigs is essential to improve the production efficiency of this globally important protein source. MicroRNAs (miRNAs) are a class of non-coding RNAs known to post-transcriptionally regulate gene expression affecting numerous phenotypes, including those important to the pig industry. To facilitate a more comprehensive understanding of the regulatory mechanisms controlling growth, carcass composition, and meat quality phenotypes in pigs, we integrated miRNA and gene expression data from longissimus dorsi muscle samples with genotypic and phenotypic data from the same animals. We identified 23 miRNA expression Quantitative Trait Loci (miR-eQTL) at the genome-wide level and examined their potential effects on these important production phenotypes through miRNA target prediction, correlation, and colocalization analyses. One miR-eQTL miRNA, miR-874, has target genes that colocalize with phenotypic QTL for 12 production traits across the genome including backfat thickness, dressing percentage, muscle pH at 24 h post-mortem, and cook yield. The results of our study reveal genomic regions underlying variation in miRNA expression and identify miRNAs and genes for future validation of their regulatory effects on traits of economic importance to the global pig industry.

Time budgets of group-housed pigs in relation to social aggression and production.

Commercial producers house growing pigs by sex and weight to allow for efficient use of resources and provide pigs the welfare benefits of interacting with their conspecifics and more freedom of movement. However, the introduction of unfamiliar pigs can cause increased aggression for 24 to 48 h as pigs establish social relationships. To address this issue, a better understanding of pig behavior is needed. The objectives of this study were to quantify time budgets of pigs following introduction into a new social group and how these changed over time and to investigate how social aggression influences the overall time budgets and production parameters. A total of 257 grow-finish Yorkshire barrows across 20 pens were introduced into new social groups at 10 wk of age (~23 kg) and observed for aggression and time budgets of behavior at four periods: immediately after introduction and 3, 6, and 9 wk later. Pigs were observed for the duration of total aggression and initiated aggression (s) for 9 h after introduction and for 4 h at 3, 6, and 9 wk later. Time budgets were created by scan sampling inactive, movement, ingestion, social, and exploration behaviors every 2 min for 4 h in the afternoon and summarizing the proportion of time each behavior was performed by period. The least square means of each behavior were compared across time points. Pigs spent most of their time inactive. In general, the greatest change in pig behavior was observed between introduction and week 3 (P < 0.003), with gradual changes throughout the study period as pigs became more inactive (week 3 vs. week 6: P = 0.209; week 6 vs. week 9: P = 0.007) and spent less time on other behaviors. Pigs' nonaggressive behavior and production parameters were compared with aggression using generalized linear mixed models. The time pigs spent on nonaggressive behaviors was negatively related to aggression (P < 0.045) with few exceptions. Initiated aggression after introduction was negatively related to loin muscle area (P = 0.003). These results show how finishing pigs spend their time in commercial facilities and indicate that behavior continues to change for up to 9 wk after introduction into a new social group. Efforts to reduce chronic levels of aggression should focus on promoting nonaggressive behaviors, such as exploration and movement, after the initial fighting that occurs immediately after introduction has waned, and should be implemented for up to 9 wk after introduction into new social groups.

Weaning Induces Stress-Dependent DNA Methylation and Transcriptional Changes in Piglet PBMCs.

Changes to the epigenome, including those to DNA methylation, have been proposed as mechanisms by which stress can induce long-term physiological changes in livestock species. Pig weaning is associated with dietary and social stress, both of which elicit an immune response and changes to the hypothalamic-pituitary-adrenal (HPA) axis. While differential methylation following stress has been assessed in model organisms, it remains poorly understood how the pig methylome is altered by stressors in production settings. We quantified changes in CpG methylation and transcript abundance in piglet peripheral blood mononuclear cells (PBMCs) following weaning and also assessed differential patterns in pigs exhibiting high and low stress response as measured by cortisol concentration and lesion scores. Blood was collected from nine gilt piglets 24 h before and after weaning, and whole-genome bisulfite sequencing (WGBS) and RNA-sequencing were performed on six and nine animals, respectively, at both time points. We identified 2,674 differentially methylated regions (DMRs) that were enriched within promoters of genes associated with lymphocyte stimulation and transcriptional regulation. Stress groups displayed unique differential methylation and expression patterns associated with activation and suppression of T cell immunity in low and high stress animals, respectively. Differential methylation was strongly associated with differential expression; specifically, upregulated genes were enriched among hypomethylated genes. We observed post-weaning hypermethylation of the glucocorticoid receptor (NR3C1) promoter and a significant decrease in NR3C1 expression (n = 9, p = 6.1 × 10-3). Our results indicate that weaning-associated stress elicits genome-wide methylation changes associated with differential gene expression, reduced T cell activation, and an altered HPA axis response.

Genome-Wide Assessment of DNA Methylation in Chicken Cardiac Tissue Exposed to Different Incubation Temperatures and CO2 Levels.

Temperature and CO2 concentration during incubation have profound effects on broiler chick development, and numerous studies have identified significant effects on hatch heart weight (HW) as a result of differences in these parameters. Early life environment has also been shown to affect broiler performance later in life; it has thus been suggested that epigenetic mechanisms may mediate long-term physiological changes induced by environmental stimuli. DNA methylation is an epigenetic modification that can confer heritable changes in gene expression. Using reduced-representation bisulfite sequencing (RRBS), we assessed DNA methylation patterns in cardiac tissue of 84 broiler hatchlings incubated at two egg shell temperatures (EST; 37.8°C and 38.9°C) and three CO2 concentrations (0.1%, 0.4%, and 0.8%) from day 8 of incubation onward. We assessed differential methylation between EST treatments and identified 2,175 differentially methylated (DM) CpGs (1,121 hypermethylated, 1,054 hypomethylated at 38.9° vs. 37.8°) in 269 gene promoters and 949 intragenic regions. DM genes (DMGs) were associated with heart developmental processes, including cardiomyocyte proliferation and differentiation. We identified enriched binding motifs among DM loci, including those for transcription factors associated with cell proliferation and heart development among hypomethylated CpGs that suggest increased binding ability at higher EST. We identified 9,823 DM CpGs between at least two CO2 treatments, with the greatest difference observed between 0.8 and 0.1% CO2 that disproportionately impacted genes involved in cardiac muscle development and response to low oxygen levels. Using HW measurements from the same chicks, we performed an epigenome-wide association study (EWAS) for HW, and identified 23 significantly associated CpGs, nine of which were also DM between ESTs. We found corresponding differences in transcript abundance between ESTs in three DMGs (ABLIM2, PITX2, and THRSP). Hypomethylation of an exonic CpG in PITX2 at 38.9°C was associated with increased expression, and suggests increased cell proliferation in broiler hatchlings incubated at higher temperatures. Overall, these results identified numerous epigenetic associations between chick incubation factors and heart development that may manifest in long-term differences in animal performance.

A comparative analysis of chromatin accessibility in cattle, pig, and mouse tissues.

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.

Deciphering Sex-Specific Genetic Architectures Using Local Bayesian Regressions.

Many complex human traits exhibit differences between sexes. While numerous factors likely contribute to this phenomenon, growing evidence from genome-wide studies suggest a partial explanation: that males and females from the same population possess differing genetic architectures. Despite this, mapping gene-by-sex (G×S) interactions remains a challenge likely because the magnitude of such an interaction is typically and exceedingly small; traditional genome-wide association techniques may be underpowered to detect such events, due partly to the burden of multiple test correction. Here, we developed a local Bayesian regression (LBR) method to estimate sex-specific SNP marker effects after fully accounting for local linkage-disequilibrium (LD) patterns. This enabled us to infer sex-specific effects and G×S interactions either at the single SNP level, or by aggregating the effects of multiple SNPs to make inferences at the level of small LD-based regions. Using simulations in which there was imperfect LD between SNPs and causal variants, we showed that aggregating sex-specific marker effects with LBR provides improved power and resolution to detect G×S interactions over traditional single-SNP-based tests. When using LBR to analyze traits from the UK Biobank, we detected a relatively large G×S interaction impacting bone mineral density within ABO, and replicated many previously detected large-magnitude G×S interactions impacting waist-to-hip ratio. We also discovered many new G×S interactions impacting such traits as height and body mass index (BMI) within regions of the genome where both male- and female-specific effects explain a small proportion of phenotypic variance (R2 < 1 × 10-4), but are enriched in known expression quantitative trait loci.

Estimation of indirect social genetic effects for skin lesion count in group-housed pigs by quantifying behavioral interactions1.

Mixing of pigs into new social groups commonly induces aggressive interactions that result in skin lesions on the body of the animals. The relationship between skin lesions and aggressive behavioral interactions in group-housed pigs can be analyzed within the framework of social genetic effects (SGE). This study incorporates the quantification of aggressive interactions between pairs of animals in the modeling of SGE for skin lesions in different regions of the body in growing pigs. The dataset included 792 pigs housed in 59 pens. Skin lesions in the anterior, central, and caudal regions of the body were counted 24 h after pig mixing. Animals were video-recorded for 9 h postmixing and trained observers recorded the type and duration of aggressive interactions between pairs of animals. The number of seconds that pairs of pigs spent engaged in reciprocal fights and unilateral attack behaviors were used to parametrize the intensity of social interactions (ISI). Three types of models were fitted: direct genetic additive model (DGE), traditional social genetic effect model (TSGE) assuming uniform interactions between dyads, and an intensity-based social genetic effect model (ISGE) that used ISI to parameterize SGE. All models included fixed effects of sex, replicate, lesion scorer, weight at mixing, premixing lesion count, and the total time that the animal spent engaged in aggressive interactions (reciprocal fights and unilateral attack behaviors) as a covariate; a random effect of pen; and a random direct genetic effect. The ISGE models recovered more direct genetic variance than DGE and TSGE, and the estimated heritabilities (h^D2) were highest for all traits (P < 0.01) for the ISGE with ISI parametrized with unilateral attack behavior. The TSGE produced estimates that did not differ significantly from DGE (P > 0.5). Incorporating the ISI into ISGE, even in a small dataset, allowed separate estimation of the genetic parameters for direct and SGE, as well as the genetic correlation between direct and SGE (rs), which was positive for all lesion traits. The estimates from ISGE suggest that if behavioral observations are available, selection incorporating SGE may reduce the consequences of aggressive behaviors after mixing pigs.

Genetic control of longissimus dorsi muscle gene expression variation and joint analysis with phenotypic quantitative trait loci in pigs.

BACKGROUND: Economically important growth and meat quality traits in pigs are controlled by cascading molecular events occurring during development and continuing throughout the conversion of muscle to meat. However, little is known about the genes and molecular mechanisms involved in this process. Evaluating transcriptomic profiles of skeletal muscle during the initial steps leading to the conversion of muscle to meat can identify key regulators of polygenic phenotypes. In addition, mapping transcript abundance through genome-wide association analysis using high-density marker genotypes allows identification of genomic regions that control gene expression, referred to as expression quantitative trait loci (eQTL). In this study, we perform eQTL analyses to identify potential candidate genes and molecular markers regulating growth and meat quality traits in pigs. RESULTS: Messenger RNA transcripts obtained with RNA-seq of longissimus dorsi muscle from 168 F2 animals from a Duroc x Pietrain pig resource population were used to estimate gene expression variation subject to genetic control by mapping eQTL. A total of 339 eQTL were mapped (FDR ≤ 0.01) with 191 exhibiting local-acting regulation. Joint analysis of eQTL with phenotypic QTL (pQTL) segregating in our population revealed 16 genes significantly associated with 21 pQTL for meat quality, carcass composition and growth traits. Ten of these pQTL were for meat quality phenotypes that co-localized with one eQTL on SSC2 (8.8-Mb region) and 11 eQTL on SSC15 (121-Mb region). Biological processes identified for co-localized eQTL genes include calcium signaling (FERM, MRLN, PKP2 and CHRNA9), energy metabolism (SUCLG2 and PFKFB3) and redox hemostasis (NQO1 and CEP128), and results support an important role for activation of the PI3K-Akt-mTOR signaling pathway during the initial conversion of muscle to meat. CONCLUSION: Co-localization of eQTL with pQTL identified molecular markers significantly associated with both economically important phenotypes and gene transcript abundance. This study reveals candidate genes contributing to variation in pig production traits, and provides new knowledge regarding the genetic architecture of meat quality phenotypes.

Evidence of evolutionary history and selective sweeps in the genome of Meishan pig reveals its genetic and phenotypic characterization.

Background: Meishan is a pig breed indigenous to China and famous for its high fecundity. The traits of Meishan are strongly associated with its distinct evolutionary history and domestication. However, the genomic evidence linking the domestication of Meishan pigs with its unique features is still poorly understood. The goal of this study is to investigate the genomic signatures and evolutionary evidence related to the phenotypic traits of Meishan via large-scale sequencing. Results: We found that the unique domestication of Meishan pigs occurred in the Taihu Basin area between the Majiabang and Liangzhu Cultures, during which 300 protein-coding genes have underwent positive selection. Notably, enrichment of the FoxO signaling pathway with significant enrichment signal and the harbored gene IGF1R were likely associated with the high fertility of Meishan pigs. Moreover, NFKB1 exhibited strong selective sweep signals and positively participated in hyaluronan biosynthesis as the key gene of NF-kB signaling, which may have resulted in the wrinkled skin and face of Meishan pigs. Particularly, three population-specific synonymous single-nucleotide variants occurred in PYROXD1, MC1R, and FAM83G genes; the T305C substitution in the MCIR gene explained the black coat of the Meishan pigs well. In addition, the shared haplotypes between Meishan and Duroc breeds confirmed the previous Asian-derived introgression and demonstrated the specific contribution of Meishan pigs. Conclusions: These findings will help us explain the unique genetic and phenotypic characteristics of Meishan pigs and offer a plausible method for their utilization of Meishan pigs as valuable genetic resources in pig breeding and as an animal model for human wrinkled skin disease research.

Profiling and characterization of a longissimus dorsi muscle microRNA dataset from an F2 Duroc × Pietrain pig resource population.

To elucidate the effects of microRNA (miRNA) regulation in skeletal muscle of adult pigs, miRNA expression profiling was performed with RNA extracted from longissimus dorsi (LD) muscle samples from 174 F2 pigs (~ 5.5 months of age) from a Duroc × Pietrain resource population. Total RNA was extracted from LD samples, and libraries were sequenced on an Illumina HiSeq 2500 platform in 1 × 50 bp format. After processing, 232,826,977 total reads were aligned to the Sus scrofa reference genome (v10.2.79), with 74.8% of total reads mapping successfully. The miRDeep2 software package was utilized to quantify annotated Sus scrofa mature miRNAs from miRBase (Release 21) and to predict candidate novel miRNA precursors. Among the retained 295 normalized mature miRNA expression profiles ssc-miR-1, ssc-miR-133a-3p, ssc-miR-378, ssc-miR-206, and ssc-miR-10b were the most abundant, all of which have previously been shown to be expressed in pig skeletal muscle. Additionally, 27 unique candidate novel miRNA precursors were identified exhibiting homologous sequence to annotated human miRNAs. The composition of classes of small RNA present in this dataset was also characterized; while the majority of unique expressed sequence tags were not annotated in any of the queried databases, the most abundantly expressed class of small RNA in this dataset was miRNAs. This data provides a resource to evaluate miRNA regulation of gene expression and effects on complex trait phenotypes in adult pig skeletal muscle. The raw sequencing data were deposited in the Sequence Read Archive, BioProject PRJNA363073.

Evidence for transcriptome-wide RNA editing among Sus scrofa PRE-1 SINE elements.

BACKGROUND: RNA editing by ADAR (adenosine deaminase acting on RNA) proteins is a form of transcriptional regulation that is widespread among humans and other primates. Based on high-throughput scans used to identify putative RNA editing sites, ADAR appears to catalyze a substantial number of adenosine to inosine transitions within repetitive regions of the primate transcriptome, thereby dramatically enhancing genetic variation beyond what is encoded in the genome. RESULTS: Here, we demonstrate the editing potential of the pig transcriptome by utilizing DNA and RNA sequence data from the same pig. We identified a total of 8550 mismatches between DNA and RNA sequences across three tissues, with 75% of these exhibiting an A-to-G (DNA to RNA) discrepancy, indicative of a canonical ADAR-catalyzed RNA editing event. When we consider only mismatches within repetitive regions of the genome, the A-to-G percentage increases to 94%, with the majority of these located within the swine specific SINE retrotransposon PRE-1. We also observe evidence of A-to-G editing within coding regions that were previously verified in primates. CONCLUSIONS: Thus, our high-throughput evidence suggests that pervasive RNA editing by ADAR can exist outside of the primate lineage to dramatically enhance genetic variation in pigs.

Estimation of genome-wide and locus-specific breed composition in pigs.

Advances in pig genomic technologies enable implementation of new methods to estimate breed composition, allowing innovative and efficient ways to evaluate and ensure breed and line background. Existing methods to test for homozygosity at key loci involve test mating the animal in question and observing phenotypic patterns among offspring, requiring extensive resources. In this study, whole-genome pig DNA microarray data from over 8,000 SNP was used to profile the composition of U.S. registered purebred pigs using a refined linear regression method that enhances the interpretation of coefficients. In a simulation analysis, a strong correlation between true and estimated breed composition was observed (R2 = 0.94). Applying these methods to 930 Yorkshire animals registered with the National Swine Registry, 95% were estimated to have a "genome-wide" Yorkshire breed composition of at least 0.825 or 82.5%, with similar performance for evaluating datasets of registered Duroc (n = 88) Landrace (n = 129), and Hampshire (n = 17) breeds. We also developed new methods to evaluate locus-based breed probabilities. Such methods have been applied to multi-locus SNP genotypes flanking the KIT gene known to predominantly control coat color, thereby inferring the probability that an animal has haplotypes in the KIT region that are predominant in white breeds. These methods have been adopted by the National Swine Registry as a means to identify purebred Yorkshire animals.

Genomic Prediction Accounting for Residual Heteroskedasticity.

Whole-genome prediction (WGP) models that use single-nucleotide polymorphism marker information to predict genetic merit of animals and plants typically assume homogeneous residual variance. However, variability is often heterogeneous across agricultural production systems and may subsequently bias WGP-based inferences. This study extends classical WGP models based on normality, heavy-tailed specifications and variable selection to explicitly account for environmentally-driven residual heteroskedasticity under a hierarchical Bayesian mixed-models framework. WGP models assuming homogeneous or heterogeneous residual variances were fitted to training data generated under simulation scenarios reflecting a gradient of increasing heteroskedasticity. Model fit was based on pseudo-Bayes factors and also on prediction accuracy of genomic breeding values computed on a validation data subset one generation removed from the simulated training dataset. Homogeneous vs. heterogeneous residual variance WGP models were also fitted to two quantitative traits, namely 45-min postmortem carcass temperature and loin muscle pH, recorded in a swine resource population dataset prescreened for high and mild residual heteroskedasticity, respectively. Fit of competing WGP models was compared using pseudo-Bayes factors. Predictive ability, defined as the correlation between predicted and observed phenotypes in validation sets of a five-fold cross-validation was also computed. Heteroskedastic error WGP models showed improved model fit and enhanced prediction accuracy compared to homoskedastic error WGP models although the magnitude of the improvement was small (less than two percentage points net gain in prediction accuracy). Nevertheless, accounting for residual heteroskedasticity did improve accuracy of selection, especially on individuals of extreme genetic merit.