Combined GWAS and LDLA approaches to improve genome-wide quantitative trait loci detection affecting carcass and meat quality traits in pig.

Many QTL affecting meat quality and carcass traits have been reported. However, in most of the cases these QTL have been detected in non-commercial populations. Therefore, a family structured population of 457 F2 pigs issued from an inter-cross between 2 commercial sire lines was used to detect QTL affecting meat quality and carcass traits. All animals were genotyped using the Illumina PorcineSNP60 BeadChip platform. Genome-wide association studies were used in combination with linkage disequilibrium-linkage analysis to identify QTL. A total of 32 QTL were detected. Nine of these QTL exceeded the genome-wide 5% significance threshold. We detected 18 QTL affecting carcass composition traits and 16 QTL affecting meat quality traits. Using post-QTL bioinformatics analysis we highlighted 26 functional candidate genes related to fatness, muscle development, meat color and meat pH. Finally, our results shed light on the advantage of using different QTL detection methodologies to get a global overview of the QTL present in the studied population.

The Longissimus and Semimembranosus muscles display marked differences in their gene expression profiles in pig.

BACKGROUND: Meat quality depends on skeletal muscle structure and metabolic properties. While most studies carried on pigs focus on the Longissimus muscle (LM) for fresh meat consumption, Semimembranosus (SM) is also of interest because of its importance for cooked ham production. Even if both muscles are classified as glycolytic muscles, they exhibit dissimilar myofiber composition and metabolic characteristics. The comparison of LM and SM transcriptome profiles undertaken in this study may thus clarify the biological events underlying their phenotypic differences which might influence several meat quality traits. METHODOLOGY/PRINCIPAL FINDINGS: Muscular transcriptome analyses were performed using a custom pig muscle microarray: the 15 K Genmascqchip. A total of 3823 genes were differentially expressed between the two muscles (Benjamini-Hochberg adjusted P value ≤0.05), out of which 1690 and 2133 were overrepresented in LM and SM respectively. The microarray data were validated using the expression level of seven differentially expressed genes quantified by real-time RT-PCR. A set of 1047 differentially expressed genes with a muscle fold change ratio above 1.5 was used for functional characterization. Functional annotation emphasized five main clusters associated to transcriptome muscle differences. These five clusters were related to energy metabolism, cell cycle, gene expression, anatomical structure development and signal transduction/immune response. CONCLUSIONS/SIGNIFICANCE: This study revealed strong transcriptome differences between LM and SM. These results suggest that skeletal muscle discrepancies might arise essentially from different post-natal myogenic activities.

The structure of a gene co-expression network reveals biological functions underlying eQTLs.

What are the commonalities between genes, whose expression level is partially controlled by eQTL, especially with regard to biological functions? Moreover, how are these genes related to a phenotype of interest? These issues are particularly difficult to address when the genome annotation is incomplete, as is the case for mammalian species. Moreover, the direct link between gene expression and a phenotype of interest may be weak, and thus difficult to handle. In this framework, the use of a co-expression network has proven useful: it is a robust approach for modeling a complex system of genetic regulations, and to infer knowledge for yet unknown genes. In this article, a case study was conducted with a mammalian species. It showed that the use of a co-expression network based on partial correlation, combined with a relevant clustering of nodes, leads to an enrichment of biological functions of around 83%. Moreover, the use of a spatial statistics approach allowed us to superimpose additional information related to a phenotype; this lead to highlighting specific genes or gene clusters that are related to the network structure and the phenotype. Three main results are worth noting: first, key genes were highlighted as a potential focus for forthcoming biological experiments; second, a set of biological functions, which support a list of genes under partial eQTL control, was set up by an overview of the global structure of the gene expression network; third, pH was found correlated with gene clusters, and then with related biological functions, as a result of a spatial analysis of the network topology.

Genetic characterization and codon usage bias of full-length Hepatitis E virus sequences shed new lights on genotypic distribution, host restriction and genome evolution.

Hepatitis E virus (HEV) is present in different species and ecological niches. It has been divided into 4 major mammalian genotypes. In this study, 3 new full-length genomes of swine HEV were sequenced and the results did not reveal any particular host determinant in comparison with human isolates belonging to the same genotype. Nucleotide composition and codon usage bias were determined to characterize HEV host restriction and genome evolution. Peculiar nucleotide bias was observed for A and C nucleotides in all HEV genotypes. Apart from the ORF1 hypervariable region and the ORF2/3 overlapping region, no nucleotide bias was observed between the 3 codon positions. CpG dinucleotides were also shown to be under-represented in HEV as in most RNA viruses. The effective number of codon used in HEV genome was high, indicating a lack of codon bias. Correspondence analysis of the relative synonymous codon usage was performed and demonstrated that evolution of HEV is not driven by geographical or host factors, but is representative of HEV phylogeny. These results confirm that HEV genome evolution is mainly based on mutational pressure. Natural selection, for instance involving fine-tuning translation kinetics and escape from the host immune system, may also play a role in shaping the HEV genome, particularly in the ORF1 hypervariable region and the ORF2/3 overlapping region. These regions might be involved in host restriction. Finally this study revealed the need to re-evaluate the possible subtyping classification.

Genetic variability of transcript abundance in pig peri-mortem skeletal muscle: eQTL localized genes involved in stress response, cell death, muscle disorders and metabolism.

BACKGROUND: The genetics of transcript-level variation is an exciting field that has recently given rise to many studies. Genetical genomics studies have mainly focused on cell lines, blood cells or adipose tissues, from human clinical samples or mice inbred lines. Few eQTL studies have focused on animal tissues sampled from outbred populations to reflect natural genetic variation of gene expression levels in animals. In this work, we analyzed gene expression in a whole tissue, pig skeletal muscle sampled from individuals from a half sib F2 family shortly after slaughtering. RESULTS: QTL detection on transcriptome measurements was performed on a family structured population. The analysis identified 335 eQTLs affecting the expression of 272 transcripts. The ontologic annotation of these eQTLs revealed an over-representation of genes encoding proteins involved in processes that are expected to be induced during muscle development and metabolism, cell morphology, assembly and organization and also in stress response and apoptosis. A gene functional network approach was used to evidence existing biological relationships between all the genes whose expression levels are influenced by eQTLs. eQTLs localization revealed a significant clustered organization of about half the genes located on segments of chromosome 1, 2, 10, 13, 16, and 18. Finally, the combined expression and genetic approaches pointed to putative cis-drivers of gene expression programs in skeletal muscle as COQ4 (SSC1), LOC100513192 (SSC18) where both the gene transcription unit and the eQTL affecting its expression level were shown to be localized in the same genomic region. This suggests cis-causing genetic polymorphims affecting gene expression levels, with (e.g. COQ4) or without (e.g. LOC100513192) potential pleiotropic effects that affect the expression of other genes (cluster of trans-eQTLs). CONCLUSION: Genetic analysis of transcription levels revealed dependence among molecular phenotypes as being affected by variation at the same loci. We observed the genetic variation of molecular phenotypes in a specific situation of cellular stress thus contributing to a better description of muscle physiologic response. In turn, this suggests that large amounts of genetic variation, mediated through transcriptional networks, can drive transient cell response phenotypes and contribute to organismal adaptative potential.

Joint analysis of quantitative trait loci and major-effect causative mutations affecting meat quality and carcass composition traits in pigs.

BACKGROUND: Detection of quantitative trait loci (QTLs) affecting meat quality traits in pigs is crucial for the design of efficient marker-assisted selection programs and to initiate efforts toward the identification of underlying polymorphisms. The RYR1 and PRKAG3 causative mutations, originally identified from major effects on meat characteristics, can be used both as controls for an overall QTL detection strategy for diversely affected traits and as a scale for detected QTL effects. We report on a microsatellite-based QTL detection scan including all autosomes for pig meat quality and carcass composition traits in an F2 population of 1,000 females and barrows resulting from an intercross between a Pietrain and a Large White-Hampshire-Duroc synthetic sire line. Our QTL detection design allowed side-by-side comparison of the RYR1 and PRKAG3 mutation effects seen as QTLs when segregating at low frequencies (0.03-0.08), with independent QTL effects detected from most of the same population, excluding any carrier of these mutations. RESULTS: Large QTL effects were detected in the absence of the RYR1 and PRKGA3 mutations, accounting for 12.7% of phenotypic variation in loin colour redness CIE-a* on SSC6 and 15% of phenotypic variation in glycolytic potential on SSC1. We detected 8 significant QTLs with effects on meat quality traits and 20 significant QTLs for carcass composition and growth traits under these conditions. In control analyses including mutation carriers, RYR1 and PRKAG3 mutations were detected as QTLs, from highly significant to suggestive, and explained 53% to 5% of the phenotypic variance according to the trait. CONCLUSIONS: Our results suggest that part of muscle development and backfat thickness effects commonly attributed to the RYR1 mutation may be a consequence of linkage with independent QTLs affecting those traits. The proportion of variation explained by the most significant QTLs detected in this work is close to the influence of major-effect mutations on the least affected traits, but is one order of magnitude lower than effect on variance of traits primarily affected by these causative mutations. This suggests that uncovering physiological traits directly affected by genetic polymorphisms would be an appropriate approach for further characterization of QTLs.

Differences in pig muscle proteome according to HAL genotype: implications for meat quality defects.

Bidimensional electrophoresis was used to compare sarcoplasmic protein profiles of early post-mortem pig semimembranosus muscles, sampled from pigs of different HAL genotypes (RYR1 mutation 1841T/C): 6 NN, 6 Nn, 6 nn. ANOVA showed that 55 (18%) of the total of 300 matched protein spots were influenced by genotype, and hierarchical clustering analysis identified 31 (10% of the matched proteins) additional proteins coregulated with these proteins. Fold-changes of differentially expressed proteins were between 1.3 and 21.8. Peptide mass fingerprinting identification of 78 of these 86 proteins indicates that faster pH decline of nn pigs was not explained by higher abundance of glycolytic enzymes. Results indicate further that nn muscles contained fewer proteins of the oxidative metabolic pathway, fewer antioxidants, and more protein fragments. Lower abundance of small heat shock proteins and myofibrillar proteins in nn muscles may at least partly be explained by the effect of pH on their extractability. Possible consequences of lower levels of antioxidants and repair capacities, increased protein fragmentation, and lower extractability of certain proteins in nn muscles on meat quality are discussed.

Differentially-expressed genes in pig Longissimus muscles with contrasting levels of fat, as identified by combined transcriptomic, reverse transcription PCR, and proteomic analyses.

Intramuscular fat content is important for many meat quality parameters. This work is aimed at identifying functional categories of genes associated with natural variation among individuals in intramuscular fat content to help the design of genetic schemes for high marbling potential. Taking advantage of the global nature of transcriptomic and proteomic technologies, 40 genes were identified as differently expressed between high fat and low fat pig Longissimus muscles at slaughter weight. They are involved in metabolic processes, cell communication, binding, and response to stimulus. Using real-time PCR in muscle biopsies taken earlier in the fattening period, the group with a high intramuscular fat content was also characterized by the down-expression of genes playing a negative role in adipogenesis, such as architectural transcription factor high-motility hook A1, mitogen activated protein-kinase14, and cyclin D1. These results suggest that interindividual variability in intramuscular fat content might arise essentially from differences in early adipogenesis.

Comparison of the antiviral potentials among the pseudorabies-resistant transgenes encoding different soluble forms of porcine nectin-1 in transgenic mice.

Nectin-1 is an alphaherpesvirus receptor that binds to virion glycoprotein D by the first immunoglobulin (Ig)-like domain. The possibility of making animals resistant to pseudorabies virus (PRV) infection has been investigated by generating transgenic mice expressing soluble forms of porcine nectin-1. Previously, transgenic mice were generated that expressed a fusion protein made of the entire ectodomain of nectin-1 fused to the Fc portion of human IgG, or the first Ig-like domain fused to the Fc portion of porcine IgG. Here, the contribution of the second and third Ig-like domains of nectin-1 was analysed by generating transgenic mice expressing the entire ectodomain of nectin-1 fused to the porcine Fc portion. Transgenic mice expressing each of three different fusion proteins were challenged with PRV for comparison of their resistance. Altogether, mice transgenic for a chimera that carried the entire ectodomain were more resistant than those transgenic for a chimera that carried the first Ig-like domain.

Comparison of sarcoplasmic proteomes between two groups of pig muscles selected for shear force of cooked meat.

Two-dimensional electrophoresis was used to compare Longissimus sarcoplasmic protein abundance between two groups (tough meat and tender meat), defined on the basis of extreme Warner-Bratzler shear force values measured on cooked pork. Fourteen protein spots differed in quantity (P<0.05) between the two groups and were identified. Adypocyte fatty acid binding protein and acyl-CoA binding protein involved in lipid traffic and in the control of gene expression regulating cell proliferation and differentiation, and Enoyl-CoA hydratase, aldose reductase and triosephosphate isomerase indirectly related to lipid metabolism were overrepresented in the tender group. The tender group was further characterized by increased levels of proteins involved in protein folding and polymerization (initiation factor elf-3beta, chaperonin subunit 2, profilin II). The results suggest that the lower post-cooking shear force could at least in part be related to muscle adipogenetic and/or myogenetic status of which the possible underlying mechanisms are discussed.

Proteome analysis of the sarcoplasmic fraction of pig semimembranosus muscle: implications on meat color development.

Two-dimensional electrophoresis was used to investigate sarcoplasmic protein expression in pig Semimembranosus muscles sampled 20 min after slaughter. Two groups (light and dark) of 12 animals were selected from 1000 pigs, based on meat L values measured 36 h postmortem. Twenty-two proteins or fragments (p < 0.05) were differentially expressed. Muscles leading to darker meat had a more oxidative metabolism, indicated by more abundant mitochondrial enzymes of the respiratory chain, hemoglobin, and chaperone or regulator proteins (HSP27, alphaB-crystallin, and glucose-regulated protein 58 kDa). Conversely, enzymes of glycolysis were overexpressed in the lighter group. Such samples were also characterized by higher levels of glutathione S-transferase omega, which can activate the RyR calcium channels, and higher levels of cyclophilin D. This protein pattern is likely to have severe implications on postmortem metabolism, namely, acceleration of ATP depletion and pH fall and subsequent enhanced protein denaturation, well-known to induce discoloration.

Comparison of protection levels against pseudorabies virus infection of transgenic mice expressing a soluble form of porcine nectin-1/HveC and vaccinated mice.

We recently generated transgenic mice expressing a soluble form of porcine nectin-1 (PHveCIg) showing remarkable resistance to pseudorabies virus (PRV) infection. Nectin-1, also known as herpesvirus entry mediator C (HveC), is an alphaherpesvirus receptor that binds to virion glycoprotein D (gD). In order to evaluate the level of resistance to PRV infection induced by the expression of PHveCIg in the transgenic mice, the protective effects of vaccinated and transgenic mice were directly compared. Mice were immunized with a live vaccine, through intraperitoneal injection of PRV strain Begonia (an attenuated vaccine strain deleted for gE and thymidine kinase genes) at 4 weeks before challenge. The vaccinated and transgenic mice were challenged with 10LD(50), 20LD(50) or 50LD(50) of PRV strainYS-81 via intranasal route. In the vaccinated mice, no protection was observed in the challenges with 20LD(50) and 50LD(50). Only two out of six vaccinated mice survived in the challenge with 10LD(50). In contrast, four transgenic mouse lines showed significant resistance to PRV infection, although the survival rates varied in the challenge with each viral dose. These results demonstrate clearly the high potential of transgenic strategy in control of pseudorabies.

Transgenic mice expressing a soluble form of porcine nectin-1/herpesvirus entry mediator C as a model for pseudorabies-resistant livestock.

An approach to genetically engineered resistance to pseudorabies virus (PRV) infection was examined by using a transgene encoding a soluble form of nectin-1, also known as herpesvirus entry mediator C. Nectin-1 is an alpha-herpesvirus receptor that binds to virion glycoprotein D. Nectin-1 mediates entry of PRV, herpes simplex virus types 1 and 2, and bovine herpesvirus type 1. To assess the antiviral potential of an ectopic expression of the nectin-1 ectodomain in vivo, six transgenic mouse lines expressing a soluble form of nectin-1, consisting of an extracellular domain of porcine nectin-1 and the Fc portion of human IgG1, were generated. All of the transgenic mouse lines showed nearly complete resistance to PRV infection by means of both i.p. and intranasal routes. These results suggest that the introduction into farm animals of a transgene encoding a soluble form of nectin-1 would offer a potent biological approach to generating alpha-herpesvirus-resistant livestock.