Pig fetal skeletal muscle development is associated with genome-wide DNA hypomethylation and corresponding alterations in transcript and microRNA expression.

Fetal myogenesis represents a critical period of porcine skeletal muscle development and requires coordinated expression of thousands of genes. Epigenetic mechanisms, including DNA methylation, drive transcriptional regulation during development; however, these processes are understudied in developing porcine tissues. We performed bisulfite sequencing to assess DNA methylation in pig longissimus dorsi muscle at 41- and 70-days gestation (dg), as well as RNA- and small RNA-sequencing to identify coordinated changes in methylation and expression between myogenic stages. We identified 45 739 differentially methylated regions (DMRs) between stages, and the majority (N = 34 232) were hypomethylated at 70 versus 41 dg. Integration of methylation and transcriptomic data revealed strong associations between differential gene methylation and expression. Differential miRNA methylation was significantly negatively correlated with abundance, and dynamic expression of assayed miRNAs persisted postnatally. Motif analysis revealed significant enrichment of myogenic regulatory factor motifs among hypomethylated regions, suggesting that DNA hypomethylation may function to increase accessibility of muscle-specific transcription factors. We show that developmental DMRs are enriched for GWAS SNPs for muscle- and meat-related traits, demonstrating the potential for epigenetic processes to influence phenotypic diversity. Our results enhance understanding of DNA methylation dynamics of porcine myogenesis and reveal putative cis-regulatory elements governed by epigenetic processes.

Genome-wide association analyses of lesion counts in group-housed pigs.

Aggression in group-housed pigs is a welfare concern and can negatively affect production. Skin lesions are reliable indicators of aggression and are moderately heritable, suggesting that selective breeding may reduce aggression. To further understand the genetic control of behavioral traits, such as the aggressive response to regrouping, associated single nucleotide polymorphisms (SNPs) can be identified within the genome, and the region in which these SNPs are located can be related to known genes. To investigate SNPs associated with aggression, 1093 purebred Yorkshire pigs were strategically remixed into new groups of familiar and unfamiliar animals at three life stages and lesion counts were recorded. Genomic best linear unbiased prediction (GBLUP) models were fitted for each trait. The genetic additive effect was obtained from a genetic relationship matrix constructed from the 50 924 SNPs. SNP effects and their variances were estimated from the GBLUP objects. SNPs that were associated with a significant portion of the trait variance were identified for lesions to the anterior (three SNPs, FDR <5%) and central (one SNP, FDR <5%) portions of the body in grow-finish pigs. These SNPs were located on chromosome 11, suggesting that chromosome 11 contains a region explaining variation in lesion counts that should be further explored to identify genes underlying biological control of aggression.

Estimates of the actual relationship between half-sibs in a pig population.

Genomic relationships based on markers capture the actual instead of the expected (based on pedigree) proportion of genome shared identical by descent (IBD). Several methods exist to estimate genomic relationships. In this research, we compare four such methods that were tested looking at the empirical distribution of the estimated relationships across 6704 pairs of half-sibs from a cross-bred pig population. The first method based on multiple marker linkage analysis displayed a mean and standard deviation (SD) in close agreement with the expected ones and was robust to changes in the minor allele frequencies (MAF). A single marker method that accounts for linkage disequilibrium (LD) and inbreeding came second, showing more sensitivity to changes in the MAF. Another single marker method that considers neither inbreeding nor LD showed the smallest empirical SD and was the most sensible to changes in MAF. A higher mean and SD were displayed by VanRaden's method, which was not sensitive to changes in MAF. Therefore, the method based on multiple marker linkage analysis and the single marker method that considers LD and inbreeding performed closer to theoretical values and were consistent with the estimates reported in literature for human half-sibs.

Evaluation of BTA1 and BTA5 QTL Regions for Growth and Carcass Traits in American and Korean Cattle.

Previously identified QTL regions on BTA1 and BTA5 were investigated to validate the QTL regions and to identify candidate genes for growth and carcass traits in commercial cattle populations from the USA and Korea. Initially, a total of 8 polymorphic microsatellite (MS) markers in the BTA1 and 5 QTL regions were used for Chi-square tests to compare the frequencies of individual alleles between high and low phenotypic groups for the US (Michigan Cattleman's Association/Michigan State University; MCA/MSU) cattle. For a subsequent study, 24 candidate genes containing missense mutations and located within the QTL regions based on bovine genome sequence data were analyzed for genotyping in the two commercial cattle populations. Re-sequencing analyses confirmed 18 public missense SNPs and identified 9 new SNPs. Seventeen of these SNPs were used for genotyping of the MCA/MSU cattle (n = 98) and Korean native cattle (n = 323). On BTA1, UPK1B, HRG, and MAGEF1 polymorphisms residing between BM1312 and BMS4048 were significantly associated with growth and carcass traits in one or both of the MCA/MSU and Korean populations. On BTA5, ABCD2, IL22 and SNRPF polymorphisms residing between BL4 and BR2936 were associated with marbling and backfat traits in one or both of the MCA/MSU and Korean cattle populations. These results suggested that BTA 1 and 5 QTL regions may be segregating in both Korean Hanwoo and USA commercial cattle populations and DNA markers tested in this study may contribute to the identification of positional candidate genes for marker-assisted selection programs.

Transcriptional profiling during foetal skeletal muscle development of Piau and Yorkshire-Landrace cross-bred pigs.

Skeletal muscle development is a complex process involving the coordinated expression of thousands of genes. The aim of this study was to identify differentially expressed genes in longissimus dorsi (LD) muscle of pigs at 40 and 70 days (d) of gestation (developmental stages encompassing primary and secondary fibre formation) in Yorkshire-Landrace (YL) cross-bred pigs and Piau pigs (a naturalized Brazilian breed), which are two breed types that differ in muscularity. Foetuses were obtained from gilts at each gestational age (n = 3 YL; n = 4 Piau), and transcriptional profiling was performed using the Pigoligoarray microarray containing 20 400 oligonucleotides. A total of 486 oligonucleotides were differentially expressed (fold change (FC) ≥ 1.5; false discovery rate (FDR) ≤ 0.05) between 40 and 70 d gestation in either YL or Piau pigs, and a total of 1300 oligonucleotides were differentially expressed (FC ≥ 1.5; FDR ≤ 0.05) between YL and Piau pigs at either age. Gene ontology annotation and pathway analyses determined functional classifications for differentially expressed genes and revealed breed type-specific developmental expression patterns. Thirteen genes were selected for confirmation by qRT-PCR analyses, and expression patterns for most of these genes were confirmed, providing further insight into the roles of these genes in pig muscle development. This study revealed both developmental and breed type-specific patterns of gene expression in foetal pig skeletal muscle, including genes not previously associated with myogenesis. This information will contribute to future pig genetic improvement efforts.

Genome-wide association study in an F2 Duroc x Pietrain resource population for economically important meat quality and carcass traits.

Meat quality is essential for consumer acceptance, it ultimately impacts pork production profitability and it is subject to genetic control. The objective of this study was to map genomic regions associated with economically important meat quality and carcass traits. We performed a genome-wide association (GWA) analysis to map regions associated with 38 meat quality and carcass traits recorded for 948 F2 pigs from the Michigan State University Duroc × Pietrain resource population. The F0, F1, and 336 F2 pigs were genotyped with the Illumina Porcine SNP60 BeadChip, while the remaining F2 pigs were genotyped with the GeneSeek Genomic Profiler for Porcine Low Desnisty (LD) chip, and imputed with high accuracy ( = 0.97). Altogether the genomic dataset comprised 1,019 animals and 44,911 SNP. A Gaussian linear mixed model was fitted to estimate the breeding values and the variance components. A linear transformation was performed to estimate the marker effects and variances. Type I error rate was controlled at a False Discovery Rate of 5%. Seven putative QTL found in this study were previously reported in other studies. Two novel QTL associated with tenderness (TEN) were located on SSC3 [135.6:137.5Mb; False Discovery rate (FDR) < 0.03] and SSC5 (67.3:69.1Mb; FDR < 0.02). The QTL region identified on SSC15 includes Protein Kinase AMP-activated É£ 3-subunit gene (), which has been associated with 24-h pH (pH24), drip loss (DL) and cook yield (CY). Also, novel candidate genes were identified for TEN in the region on SSC5 [A Kinase (PRKA) Anchor Protein 3 (], and for tenth rib backfat thickness (BF10) [Carnitine O-Acetyltransferase ()] on SSC1. The association of gene polymorphisms with pork quality traits has been reported for several pig populations. However, there are no SNP for this gene on the chip used, thus we genotyped the animals for 2 non-synonymous variants ( and ). We then performed a GWA conditioning on the genotype of both SNP and was associated with pH24, DL, protein content (PRO) and CY ( < 0.004) and T30N with Juiciness, TEN, shear force, pH24, PRO, and CY < 0.04). Finally, we performed a GWA conditioning on the genotype of the SNP peak detected in this study, and T30N remained associated only with PRO ( < 0.02). Therefore, in this study we identified 2 novel QTL regions, suggest 2 novel candidate genes, and conclude that other SNP in PRKAG3 or nearby gene(s) explain the observed associations on SSC15 in this population.

Refining genomewide association for growth and fat deposition traits in an F pig population.

The identification of genomic regions that affect additive genetic variation and contain genes involved in controlling growth and fat deposition has enormous impact in the farm animal industry (e.g., carcass merit and meat quality). Therefore, a genomewide association study was implemented in an F pig population using a 60,000 SNP marker panel for traits related to growth and fat deposition. Estimated genomic EBV were linearly transformed to calculate SNP effects and to identify genomic positions possibly associated with the genetic variability of each trait. Genomic segments were then defined considering the markers included in a region 1 Mb up- and downstream from the SNP with the smallest -value and a false discovery rate < 0.05 for each trait. The significance for each 2-Mb segment was tested using the Bonferroni correction. Significant SNP were detected on SSC2, SSC3, SSC5, and SSC6, but 2-Mb segment significant effects were observed on SSC3 for weight at birth (wt_birth) and on SSC6 for 10th-rib backfat and last-rib backfat measured by ultrasound at different ages. Furthermore, a 6-Mb segment on SSC6 was also considered because the 2-Mb segments for 10 different fat deposition traits were overlapped. Although the segment effects for each trait remain significant, the proportion of additive variance explained by this larger segment was slightly smaller in some traits. In general, the results confirm the presence of genetic variability for wt_birth on SSC3 (18.0-20.2 Mb) and for fat deposition traits on SSC6 (133.8-136.0 Mb). Within these regions, fibrosin () and myosin light chain, phosphorylatable, fast skeletal muscle () genes could be considered as candidates for the wt_birth signal on SSC3, and the SERPINE1 mRNAbinding protein 1 gene () may be a candidate for the fat deposition trait signals on SSC6.

Comparative mapping of genes flanking the human chromosome 12 evolutionary breakpoint in the pig.

Genes located on human chromosome 12 (HSA12) are conserved on pig chromosomes 5 and 14 (SSC5 and SSC14), with HSA12q23.3-->q24.11 harboring the evolutionary breakpoint between these chromosomes. For this study, pig sequence-tagged sites (STS) were developed for nine HSA12 genes flanking this breakpoint. Radiation hybrid (RH) mapping using the IMpRH panel revealed that COL2A1, DUSP6, KITLG, PAH and STAB2 map to SSC5, while PXN, PLA2G1B, SART3 and TCF1 map to SSC14. Polymorphisms identified in COL2A1, DUSP6, PAH, PLA2G1B and TCF1 were used for genetic linkage mapping and confirmed the map locations for these genes. Our results indicate that the HSA12 evolutionary breakpoint occurs between STAB2 and SART3 in a region spanning less than five million basepairs. These results refine the comparative map of the HSA12 evolutionary breakpoint region and help to further elucidate the extensive gene order rearrangements between HSA12 and SSC5 and 14.

Evaluation of serum insulin-like growth factor binding proteins (IGFBP) in Angus cattle divergently selected for serum IGF-I concentration.

Postweaning serum insulin-like growth factor-I (IGF-I) concentrations and serum IGF binding proteins (IGFBP) were investigated in 68 (1992 Fall-born) and 84 (1999 Fall-born) Angus cattle selected for either high or low serum IGF-I concentrations since 1989. Relative serum levels of IGFBP were determined by [125I]IGF-I Western ligand blotting. IGFBP species of 38-42, 34, 30, and 24 kDa were identified. The 34 kDa species was identified as IGFBP-2 by immunoblot analysis. No significant line effects were observed for any of the IGFBP. In both 1992 and 1999, heifers had higher IGFBP-2 levels than bulls (P<0.0005). In 1992 calves, relative levels of the 38-42 and 24 kDa species were significantly correlated with serum IGF-I concentration. In 1999 calves, none of the IGFBP were correlated with serum IGF-I, although IGFBP-2 was negatively correlated with several measures of body weight. No significant line effects were observed for growth or serum IGF-I traits in 1992 calves. However, 1999 high line calves had higher serum IGF-I concentrations and body weights than low line calves (P<0.05). In both 1992 and 1999 calves, bulls had higher serum IGF-I concentrations and body weights than heifers (P<0.05). Thus, while selection for high versus low serum IGF-I concentrations has resulted in divergence between the selection lines and also in changes in body weights, it has not resulted in changes in serum IGFBP levels. Furthermore, circulating IGFBP-2 appears to be higher in heifers than in bulls, and also appears to be negatively correlated with body weights.

Complementary DNA macroarray analyses of differential gene expression in porcine fetal and postnatal muscle.

To study differential gene expression in porcine skeletal muscle, a porcine complementary DNA (cDNA) macroarray was produced that contained 327 expressed sequence tags (EST) derived from whole embryo and adult skeletal muscle, and differential display PCR products from fetal and postnatal muscle. Total RNA from four muscle samples, 75- and 105-d fetal hind limb muscles, and 1- and 7-wk postnatal semitendinosus muscle was used to make radiolabeled targets for duplicate hybridization to the macroarray membranes in an initial screen for expression. All EST that gave clear signals (n = 238) were then re-arrayed, and hybridization was conducted with additional biological replication of samples in the 75-d and 1-wk ages. Signal intensity for each gene was normalized to signal intensity measured at control spots on each membrane, which consisted of total cDNA from liver, lung, spleen, and skeletal muscle. Both normalized ratio levels and a mixed linear model analyses were used to identify genes differentially expressed among the muscle samples. Results showed 28 genes had differences in expression level greater than twofold between the 75-d fetal and 1-wk muscle RNA samples. All 28 genes were also identified as genes with significantly different (P < 0.01) expression using a mixed linear model analysis. Nineteen of these 28 genes had significant matches (basic local alignment search tool [BLAST] score > 100; P < 0.01) to known genes, two matched genes encoding human hypothetical proteins, and seven had no significant matches to Genbank nonredundant and dbEST (database of expressed sequence tags) entries. These results were confirmed for representative genes with RNA blot analysis of seven developmental time points, including RNA from the same muscle samples tested previously in the macroarray. The RNA blot results confirmed the macroarray results for all selected genes, demonstrating that the macroarray technique used in this study is accurate and reproducible. An unknown muscle clone (M218) with a slightly less than twofold increase in expression from the 75-d to the 1-wk age (1 wk/75 d = 1.94; P = 0.0114) was also shown to differ between these two ages using RNA blot analysis, demonstrating the methods used to identify differentially expressed genes may be conservative. The association between expression patterns of vimentin and desmin was also investigated. Results indicate the switch in intermediate filament protein from vimentin to desmin occurs primarily at the level of transcription and/or RNA processing.

Association of halothane sensitivity with growth and meat quality in pigs.

Previous reports have indicated that a proportion of pigs, homozygous normal for the skeletal muscle ryanodine receptor gene (RYR1), was halothane sensitive, and this was associated with poor meat quality when pigs were handled aggressively. This study was conducted to evaluate halothane sensitivity in RYR1-normal pigs, managed under simulated commercial conditions, to ascertain the association of halothane sensitivity with growth rate and meat quality. A total of 363 pigs across four farrowing groups, from seven Landrace sires and 38 Yorkshire-Landrace F1 dams, were tested at 8 weeks of age for halothane sensitivity using a closed system that delivered 5% halothane at 2 l/min for 3 (group 1) or 2 (groups 2 to 4) min. After 1 min, limb rigidity, limb tremors and abdominal discoloration were evaluated on a binomial scale with 0 indicating no reaction and 1 indicating reaction. Testing was repeated 2 days later. At 10 weeks of age, pigs were moved to finishing pens and not moved again until marketing. Within farrowing group, pigs were harvested in one of two groups, and at marketing were moved a distance of 91 m, weighed, tattooed, loaded and transported a distance of 550 km to a commercial harvest plant. After overnight rest, pigs were harvested and the pH of the loin muscle was measured at 45 min (pH45) after stunning. After an 18-h chill, loin muscle pH (pHu), International Commission on Illumination (CIE) L*, a*, b*, color (1 to 6) and marbling (1 to 10) scores and fluid loss percent were collected. Generalized linear mixed models were used to estimate repeatabilities for response to halothane challenge. Repeatabilities for limb rigidity for the front right and left legs were 0.24 and 0.31, respectively, whereas rear right and left leg repeatabilities were 0.19 and 0.17, respectively. Repeatabilities for front right and left leg tremors were 0.16 and 0.20, respectively. Growth rate was not influenced by any measure of halothane sensitivity. Carcasses from pigs exhibiting limb rigidity tended to have lower pH45 (5.88 v. 5.97; P = 0.06), similar pHu (5.47 v. 5.49; P = 0.32), less pH decline from 45 min to 18 h (-0.40 v. -0.50; P = 0.04) and a tendency for greater fluid loss percent (5.01 v. 4.55; P = 0.08) than carcasses from pigs that did not exhibit limb rigidity during halothane challenge. A proportion of pigs normal for RYR1 did exhibit limb rigidity during halothane gas challenge, and subsequently tended to have lower 45 min pH and greater longissimus muscle fluid loss post harvest.

Quantitative trait locus mapping in an F2 Duroc x Pietrain resource population: II. Carcass and meat quality traits.

Pigs from the F(2) generation of a Duroc x Pietrain resource population were evaluated to discover QTL affecting carcass composition and meat quality traits. Carcass composition phenotypes included primal cut weights, skeletal characteristics, backfat thickness, and LM area. Meat quality data included LM pH, temperature, objective and subjective color information, marbling and firmness scores, and drip loss. Additionally, chops were analyzed for moisture, protein, and fat composition as well as cook yield and Warner-Bratzler shear force measurements. Palatability of chops was determined by a trained sensory panel. A total of 510 F(2) animals were genotyped for 124 microsatellite markers evenly spaced across the genome. Data were analyzed with line cross, least squares regression interval, mapping methods using sex and litter as fixed effects and carcass weight or slaughter age as covariates. Significance thresholds of the F-statistic for single QTL with additive, dominance, or imprinted effects were determined on chromosome- and genome-wise levels by permutation tests. A total of 94 QTL for 35 of the 38 traits analyzed were found to be significant at the 5% chromosome-wise level. Of these 94 QTL, 44 were significant at the 1% chromosome-wise, 28 of these 44 were also significant at the 5% genome-wise, and 14 of these 28 were also significant at the 1% genome-wise significance thresholds. Putative QTL were discovered for 45-min pH and pH decline from 45 min to 24 h on SSC 3, marbling score and carcass backfat on SSC 6, carcass length and number of ribs on SSC 7, marbling score on SSC 12, and color measurements and tenderness score on SSC 15. These results will facilitate fine mapping efforts to identify genes controlling carcass composition and meat quality traits that can be incorporated into marker-assisted selection programs to accelerate genetic improvement in pig populations.

Quantitative trait loci mapping in an F2 Duroc x Pietrain resource population: I. Growth traits.

Pigs from the F(2) generation of a Duroc x Pietrain resource population were evaluated to discover QTL affecting growth and composition traits. Body weight and ultrasound estimates of 10th-rib backfat, last-rib backfat, and LM area were serially measured throughout development. Estimates of fat-free total lean, total body fat, empty body protein, empty body lipid, and ADG from 10 to 22 wk of age were calculated, and random regression analyses were performed to estimate individual animal phenotypes representing intercept and linear rates of increase in these serial traits. A total of 510 F(2) animals were genotyped for 124 micro-satellite markers evenly spaced across the genome. Data were analyzed with line cross, least squares regression, interval mapping methods using sex and litter as fixed effects. Significance thresholds of the F-statistic for single QTL with additive, dominance, or imprinted effects were determined at the chromosome- and genome-wise levels by permutation tests. A total of 43 QTL for 22 of the 29 measured traits were found to be significant at the 5% chromosome-wise level. Of these 43 QTL, 20 were significant at the 1% chromosome-wise significance threshold, 14 of these 20 were also significant at the 5% genome-wise significance threshold, and 10 of these 14 were also significant at the 1% genome-wise significance threshold. A total of 22 QTL for the animal random regression terms were found to be significant at the 5% chromosome-wise level. Of these 22 QTL, 6 were significant at the 1% chromosome-wise significance threshold, 4 of these 6 were also significant at the 5% genome-wise significance threshold, and 3 of these 4 were also significant at the 1% genome-wise significance threshold. Putative QTL were discovered for 10th-rib and last-rib backfat on SSC 6, body composition traits on SSC 9, backfat and lipid composition traits on SSC 11, 10th-rib backfat and total body fat tissue on SSC 12, and linear regression of last-rib backfat and total body fat tissue on SSC 8. These results will facilitate fine-mapping efforts to identify genes controlling growth and body composition of pigs that can be incorporated into marker-assisted selection programs to accelerate genetic improvement in pig populations.

Mapping of porcine genetic markers generated by representational difference analysis.

Representational difference analysis (RDA) was performed using pig genomic DNA from a Landrace non-selected control population and a Landrace population selected for increased loin muscle area (LMA) for five generations. Pigs used for the analysis differed phenotypically for various carcass traits and were divergent in genotype at the skeletal muscle ryanodine receptor 1 locus. Two RDA experiments were performed using BamHI and BglII. Fourteen BamHI and 37 BglII difference products were cloned and sequenced. Oligonucleotide primers were designed to amplify RDA difference products and sequence-tagged sites (STS) were developed for 16 RDA fragments (two BamHI and 14 BglII). These 16 STS were mapped using the INRA-Minnesota porcine Radiation Hybrid panel. Polymorphisms identified in nine of the STS were used to place these markers on the PiGMaP genetic linkage map. Sequence-tagged sites were localized to 11 different chromosomes including three markers on chromosome 11 and four markers on chromosome 14. Development of RDA markers increases the resolution of the pig genome maps and markers located within putative quantitative trait locus (QTL) regions can be used to refine QTL positions.