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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.

BackgroundAlthough 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.ResultsOverall, 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.ConclusionsThe lack of conserved accessibility at distal elements is consistent with rapid evolution of enhancers, and further emphasizes the need to annotate regulatory elements in individual species, rather than inferring elements based on homology. This atlas of chromatin accessibility in cattle and pig constitutes a substantial step towards annotating livestock genomes and dissecting the regulatory link between genome and phenome.

The 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]

Genetic analysis of transcriptional profiling experiments is emerging as a promising approach for unraveling genes and pathways that underlie variation of complex biological traits. However, these genetical genomics approaches are currently limited by the high cost of microarrays. We studied five different strategies to optimally select subsets of individuals for transcriptional profiling, including (1) maximizing genetic dissimilarity between selected individuals, (2) maximizing the number of recombination events in selected individuals, (3) selecting phenotypic extremes within inferred genotypes of a previously identified quantitative trait locus (QTL), (4) purely random selection, and (5) profiling animals with the highest and lowest phenotypic values within each family--gender subclass. A simulation study was conducted on the basis of a linkage map and marker genotypes were derived from data on chromosome 6 for 510 [F.sub.2] animals from an existing pig resource population and on a simulated biallelic QTL with pleiotropic effects on performance and gene expression traits. Bivariate analyses were conducted for selected subset sample sizes of 80, 160, and 240 individuals under three different correlation scenarios between the two traits. The genetic dissimilarity and phenotypic extremes within genotype methods had the smallest mean square error on QTL effects and maximum sensitivity on QTL detection, thereby outperforming all other selection strategies, particularly at the smallest proportion of samples selected for gene expression profiling (80/510).

The swine industry feeds pharmacological zinc (Zn) to newly weaned pigs to improve health. Because most swine diets are plant-based with a high phytic acid content, we hypothesized that adding phytase to diets could reduce the amount of Zn required to obtain beneficial responses. The role of metallothionein (MT) in Zn homeostasis could be important in this positive response. Thus, the goal of this study was to investigate the effect of dietary Zn and phytase on relative MT mRNA abundance and protein concentration in newly weaned pigs. Diets containing adequate (150 mg Zn/kg) or pharmacological concentrations of Zn (1000 or 2000 mg Zn/kg), as zinc oxide, with or without phytase [0, 500 phytase units (FTU)/kg, Natuphos, BASF] were fed in a 3 X 2 factorial design. Plasma and tissue minerals were measured in pigs killed after 14 d of dietary intervention. Hepatic and renal relative MT mRNA abundance and protein were greater (P < 0.05) in pigs fed 1000 mg Zn/kg with phytase, or 2000 mg Zn/kg with or without phytase vs. the remaining treatments. Intestinal mucosa MT mRNA abundance and protein were greater (P < 0.05) in pigs fed 2000 mg Zn/kg with phytase than in pigs fed 2000 mg Zn/kg alone or 1000 mg Zn/kg with phytase. Pigs fed 1000 mg Zn/kg plus phytase or 2000 mg Zn/kg with or without phytase had higher plasma, hepatic, and renal Zn than those fed the adequate Zn diets or 1000 mg Zn/kg. We conclude that feeding 1000 mg Zn/kg with phytase enhances MT mRNA abundance and protein and Zn absorption to the same degree as 2000 mg Zn/kg with and without phytase. KEY WORDS: * metallothionein * pharmacological zinc * phytase * pig

Qualitative Behavior Assessments (QBA) are a useful way to assess animal welfare by capturing behavioral signals of emotionality not captured by quantitative measures. The objective of this study was to determine if QBA descriptors assigned to nursery age gilts during a social dyad test in a novel arena were associated with differences in quantitative behavioral measures recorded from the test such as latency to make social contact and amount of time spent in proximity. To investigate this, 30 weight-matched purebred Yorkshire gilt pairs underwent a social dyad test at 6 wk-of-age. Dyads were unfamiliar with each other, and one was assigned as the focal gilt. The test began as soon as both gilts were in the novel arena and lasted for 5 minutes. Four independent, untrained observers were asked to perform a QBA by circling all terms that they felt described the focal gilt throughout the test duration from a set list of 20 terms. Overhead video was recorded and used to collect quantitative measures including number of quadrant crosses, percentage of crosses entering an already occupied quadrant, percentage of crosses exiting an already occupied quadrant, latency of the focal pig to touch the other pig, and time spent in an occupied quadrant. For each focal gilt, only terms that were used by at least two observers were kept. To be included in the analysis a given term had to be assigned to at least 6 of the 30 focal gilts such that each group had sufficient sample size to make comparisons. This filtering resulted in 10 terms being kept for analysis including Active (22), Agitated (7), Aimless (10), Calm (15), Indifferent (8), Lively (13), Playful (9), Positively Occupied (16), Sociable (14), and Tense (8). Quantitative measures were analyzed using a t-test to determine if pigs described by each term differed from those that were not. Pigs described as Aimless, Calm, Positively Occupied, Sociable, or Tense did not significantly differ in any of the quantitative measures of behavior. Pigs described as Active, Indifferent, or Lively had significantly more quadrant crosses (P = 0.0003, 0.003, 0.022 respectively). Pigs described as Agitated had a greater proportion of crosses that were entering an occupied quadrant (P = 0.022), while those described as Playful had a significantly lower proportion of crosses that were exiting an occupied quadrant (P = 0.031). Understanding how qualitative descriptors associate with quantitative measures of behavior may assist in better interpretation of QBA results as well as confirming that a chosen metric is capturing the intended behavior. [ABSTRACT FROM AUTHOR]

Abstract Background The success of marker assisted selection depends on the amount of linkage disequilibrium (LD) across the genome. To implement marker assisted selection in the swine breeding industry, information about extent and degree of LD is essential. The objective of this study is to estimate LD in four US breeds of pigs (Duroc, Hampshire, Landrace, and Yorkshire) and subsequently calculate persistence of phase among them using a 60 k SNP panel. In addition, we report LD when using only a fraction of the available markers, to estimate persistence of LD over distance. Results Average r2 between adjacent SNP across all chromosomes was 0.36 for Landrace, 0.39 for Yorkshire, 0.44 for Hampshire and 0.46 for Duroc. For markers 1 Mb apart, r2 ranged from 0.15 for Landrace to 0.20 for Hampshire. Reducing the marker panel to 10% of its original density, average r2 ranged between 0.20 for Landrace to 0.25 for Duroc. We also estimated persistence of phase as a measure of prediction reliability of markers in one breed by those in another and found that markers less than 10 kb apart could be predicted with a maximal accuracy of 0.92 for Landrace with Yorkshire. Conclusions Our estimates of LD, although in good agreement with previous reports, are more comprehensive and based on a larger panel of markers. Our estimates also confirmed earlier findings reporting higher LD in pigs than in American Holstein cattle, especially at increasing marker distances (> 1 Mb). High average LD (r2 > 0.4) between adjacent SNP found in this study is an important precursor for the implementation of marker assisted selection within a livestock species. Results of this study are relevant to the US purebred pig industry and critical for the design of programs of whole genome marker assisted evaluation and selection. In addition, results indicate that a more cost efficient implementation of marker assisted selection using low density panels with genotype imputation, would be feasible for these breeds.

Abstract Background Skeletal muscle growth and development from embryo to adult consists of a series of carefully regulated changes in gene expression. Understanding these developmental changes in agriculturally important species is essential to the production of high quality meat products. For example, consumer demand for lean, inexpensive meat products has driven the turkey industry to unprecedented production through intensive genetic selection. However, achievements of increased body weight and muscle mass have been countered by an increased incidence of myopathies and meat quality defects. In a previous study, we developed and validated a turkey skeletal muscle-specific microarray as a tool for functional genomics studies. The goals of the current study were to utilize this microarray to elucidate functional pathways of genes responsible for key events in turkey skeletal muscle development and to compare differences in gene expression between two genetic lines of turkeys. To achieve these goals, skeletal muscle samples were collected at three critical stages in muscle development: 18d embryo (hyperplasia), 1d post-hatch (shift from myoblast-mediated growth to satellite cell-modulated growth by hypertrophy), and 16wk (market age) from two genetic lines: a randombred control line (RBC2) maintained without selection pressure, and a line (F) selected from the RBC2 line for increased 16wk body weight. Array hybridizations were performed in two experiments: Experiment 1 directly compared the developmental stages within genetic line, while Experiment 2 directly compared the two lines within each developmental stage. Results A total of 3474 genes were differentially expressed (false discovery rate; FDR < 0.001) by overall effect of development, while 16 genes were differentially expressed (FDR < 0.10) by overall effect of genetic line. Ingenuity Pathways Analysis was used to group annotated genes into networks, functions, and canonical pathways. The expression of 28 genes involved in extracellular matrix regulation, cell death/apoptosis, and calcium signaling/muscle function, as well as genes with miscellaneous function was confirmed by qPCR. Conclusions The current study identified gene pathways and uncovered novel genes important in turkey muscle growth and development. Future experiments will focus further on several of these candidate genes and the expression and mechanism of action of their protein products.

Abstract Background A variety of analysis approaches have been applied to detect quantitative trait loci (QTL) in experimental populations. The initial genome scan of our Duroc x Pietrain F2 resource population included 510 F2 animals genotyped with 124 microsatellite markers and analyzed using a line-cross model. For the second scan, 20 additional markers on 9 chromosomes were genotyped for 954 F2 animals and 20 markers used in the first scan were genotyped for 444 additional F2 animals. Three least-squares Mendelian models for QTL analysis were applied for the second scan: a line-cross model, a half-sib model, and a combined line-cross and half-sib model. Results In total, 26 QTL using the line-cross model, 12 QTL using the half-sib model and 3 additional QTL using the combined line-cross and half-sib model were detected for growth traits with a 5% false discovery rate (FDR) significance level. In the line-cross analysis, highly significant QTL for fat deposition at 10-, 13-, 16-, 19-, and 22-wk of age were detected on SSC6. In the half-sib analysis, a QTL for loin muscle area at 19-wk of age was detected on SSC7 and QTL for 10th-rib backfat at 19- and 22-wk of age were detected on SSC15. Conclusions Additional markers and animals contributed to reduce the confidence intervals and increase the test statistics for QTL detection. Different models allowed detection of new QTL which indicated differing frequencies for alternative alleles in parental breeds.