Influence of genetics and the pre-vaccination blood transcriptome on the variability of antibody levels after vaccination against Mycoplasma hyopneumoniae in pigs.

BACKGROUND: The impact of individual genetic and genomic variations on immune responses is an emerging lever investigated in vaccination strategies. In our study, we used genetic and pre-vaccination blood transcriptomic data to study vaccine effectiveness in pigs. RESULTS: A cohort of 182 Large White pigs was vaccinated against Mycoplasma hyopneumoniae (M. hyo) at weaning (28 days of age), with a booster 21 days later. Vaccine response was assessed by measuring seric M. hyo antibodies (Ab) at 0 (vaccination day), 21 (booster day), 28, 35, and 118 days post-vaccination (dpv). Inter-individual variability of M. hyo Ab levels was observed at all time points and the corresponding heritabilities ranged from 0.46 to 0.57. Ab persistence was higher in females than in males. Genome-wide association studies with a 658 K SNP panel revealed two genomic regions associated with variations of M. hyo Ab levels at 21 dpv at positions where immunity-related genes have been mapped, DAB2IP on chromosome 1, and ASAP1, CYRIB and GSDMC on chromosome 4. We studied covariations of Ab responses with the pre-vaccination blood transcriptome obtained by RNA-Seq for a subset of 82 pigs. Weighted gene correlation network and differential expression analyses between pigs that differed in Ab responses highlighted biological functions that were enriched in heme biosynthesis and platelet activation for low response at 21 dpv, innate antiviral immunity and dendritic cells for high response at 28 and 35 dpv, and cell adhesion and extracellular matrix for high response at 118 dpv. Sparse partial least squares discriminant analysis identified 101 genes that efficiently predicted divergent responders at all time points. We found weak negative correlations of M. hyo Ab levels with body weight traits, which revealed a trade-off that needs to be further explored. CONCLUSIONS: We confirmed the influence of the host genetics on vaccine effectiveness to M. hyo and provided evidence that the pre-vaccination blood transcriptome co-varies with the Ab response. Our results highlight that both genetic markers and blood biomarkers could be used as potential predictors of vaccine response levels and more studies are required to assess whether they can be exploited in breeding programs.

Identification of strong candidate genes for backfat and intramuscular fatty acid composition in three crosses based on the Iberian pig.

Meat quality has an important genetic component and can be modified by the fatty acid (FA) composition and the amount of fat contained in adipose tissue and muscle. The present study aimed to find genomic regions associated with the FA composition in backfat and muscle (longissimus dorsi) in 439 pigs with three different genetic backgrounds but having the Iberian breed in common. Genome-wide association studies (GWAS) were performed between 38,424 single-nucleotide polymorphisms (SNPs) covering the pig genome and 60 phenotypic traits related to backfat and muscle FA composition. Nine significant associated regions were found in backfat on the Sus scrofa chromosomes (SSC): SSC1, SSC2, SSC4, SSC6, SSC8, SSC10, SSC12, and SSC16. For the intramuscular fat, six significant associated regions were identified on SSC4, SSC13, SSC14, and SSC17. A total of 52 candidate genes were proposed to explain the variation in backfat and muscle FA composition traits. GWAS were also reanalysed including SNPs on five candidate genes (ELOVL6, ELOVL7, FADS2, FASN, and SCD). Regions and molecular markers described in our study may be useful for meat quality selection of commercial pig breeds, although several polymorphisms were breed-specific, and further analysis would be needed to evaluate possible causal mutations.

Association between the pig genome and its gut microbiota composition.

The gut microbiota has been evolving with its host along the time creating a symbiotic relationship. In this study, we assess the role of the host genome in the modulation of the microbiota composition in pigs. Gut microbiota compositions were estimated through sequencing the V3-V4 region of the 16S rRNA gene from rectal contents of 285 pigs. A total of 1,261 operational taxonomic units were obtained and grouped in 18 phyla and 101 genera. Firmicutes (45.36%) and Bacteroidetes (37.47%) were the two major phyla obtained, whereas at genus level Prevotella (7.03%) and Treponema (6.29%) were the most abundant. Pigs were also genotyped with a high-throughput method for 45,508 single nucleotide polymorphisms that covered the entire pig genome. Subsequently, genome-wide association studies were made among the genotypes of these pigs and their gut microbiota composition. A total of 52 single-nucleotide polymorphisms distributed in 17 regions along the pig genome were associated with the relative abundance of six genera; Akkermansia, CF231, Phascolarctobacterium, Prevotella, SMB53, and Streptococcus. Our results suggest 39 candidate genes that may be modulating the microbiota composition and manifest the association between host genome and gut microbiota in pigs.

Indel detection from Whole Genome Sequencing data and association with lipid metabolism in pigs.

The selection in commercial swine breeds for meat-production efficiency has been increasing among the past decades, reducing the intramuscular fat content, which has changed the sensorial and technological properties of pork. Through processes of natural adaptation and selective breeding, the accumulation of mutations has driven the genetic divergence between pig breeds. The most common and well-studied mutations are single-nucleotide polymorphisms (SNPs). However, insertions and deletions (indels) usually represents a fifth part of the detected mutations and should also be considered for animal breeding. In the present study, three different programs (Dindel, SAMtools mpileup, and GATK) were used to detect indels from Whole Genome Sequencing data of Iberian boars and Landrace sows. A total of 1,928,746 indels were found in common with the three programs. The VEP tool predicted that 1,289 indels may have a high impact on protein sequence and function. Ten indels inside genes related with lipid metabolism were genotyped in pigs from three different backcrosses with Iberian origin, obtaining different allelic frequencies on each backcross. Genome-Wide Association Studies performed in the Longissimus dorsi muscle found an association between an indel located in the C1q and TNF related 12 (C1QTNF12) gene and the amount of eicosadienoic acid (C20:2(n-6)).

Characterization of bacterial microbiota compositions along the intestinal tract in pigs and their interactions and functions.

In addition to its value in meat production, the pig is an interesting animal model for human digestive tract studies due to its physiological similarities. The aim of this study was to describe the microbiome composition, distribution and interaction along the Iberian pig intestinal tract and its role in whole-body energy homeostasis. The V3-V4 region of the 16S rRNA gene was amplified and sequenced from the microbiomes of five gut sections (duodenum, jejunum, ileum, and proximal and distal colon) in thirteen castrated male pigs. A total of 1,669 operational taxonomic units distributed in 179 genera were found among all samples. The two most abundant genera in the small intestine were Lactobacillus and Clostridium, while Prevotella was predominant in the colon. The colon samples were more similar among the pigs and richer in species than the small intestine samples were. In the small intestine, the metagenome prediction pointed to rapid internalization and conversion of the available simple carbohydrates for microbial proliferation and maintenance. In the colon, a competition among anaerobic bacteria for plant polysaccharide degradation to produce short chain fatty acids was found. This study confirms that the energy pathways of the gut microbiome differ along its sections and provides a description of the correlations between genera.

Expression analysis of candidate genes for fatty acid composition in adipose tissue and identification of regulatory regions.

The aim of this work was to study the genetic basis of the backfat expression of lipid-related genes associated with meat quality traits in pigs. We performed a genome-wide association study with the backfat gene expression measured in 44 genes by qPCR and the PorcineSNP60 BeadChip genotypes in 115 Iberian x Landrace backcross animals. A total of 193 expression-associated SNPs located in 19 chromosomal regions were associated with expression levels of ACSM5, ELOVL6, FABP4, FADS2, and SLC27A4 genes. Three expression quantitative trail loci (eQTLs) corresponding to ACSM5, FABP4, and FADS2 were classified as cis-acting eQTLs, whereas the remaining 16 eQTLs have trans-regulatory effects. Remarkably, a SNP in the ACSM5 promoter region and a SNP in the 3'UTR region of FABP4 were the most associated polymorphisms with the ACSM5 and FABP4 expression levels, respectively. Moreover, relevant lipid-related genes mapped in the trans-eQTLs regions associated with the ACSM5, FABP4, FADS2, and SLC27A4 genes. Interestingly, a trans-eQTL hotspot on SSC13 regulating the gene expression of ELOVL6, ELOLV5, and SCD, three important genes implicated in the elongation and desaturation of fatty acids, was identified. These findings provide new data to further understand the functional regulatory mechanisms implicated in the variation of fatty acid composition in pigs.

Integrative approach using liver and duodenum RNA-Seq data identifies candidate genes and pathways associated with feed efficiency in pigs.

This study aims identifying candidate genes and pathways associated with feed efficiency (FE) in pigs. Liver and duodenum transcriptomes of 37 gilts showing high and low residual feed intake (RFI) were analysed by RNA-Seq. Gene expression data was explored through differential expression (DE) and weighted gene co-expression network analyses. DE analysis revealed 55 and 112 differentially regulated genes in liver and duodenum tissues, respectively. Clustering genes according to their connectivity resulted in 23 (liver) and 25 (duodenum) modules of genes with a co-expression pattern. Four modules, one in liver (with 444 co-expressed genes) and three in duodenum (gathering 37, 126 and 41 co-expressed genes), were significantly associated with FE indicators. Intra-module analyses revealed tissue-specific candidate genes; 12 of these genes were also identified as DE between individuals with high and low RFI. Pathways enriched by the list of genes showing DE and/or belonging to FE co-expressed modules included response to oxidative stress, inflammation, immune response, lipid metabolism and thermoregulation. Low overlapping between genes identified in duodenum and liver tissues was observed but heat shock proteins were associated to FE in both tissues. Our results suggest tissue-specific rather than common transcriptome regulatory processes associated with FE in pigs.

A global analysis of CNVs in swine using whole genome sequence data and association analysis with fatty acid composition and growth traits.

Copy number variations (CNVs) are important genetic variants complementary to SNPs, and can be considered as biomarkers for some economically important traits in domestic animals. In the present study, a genomic analysis of porcine CNVs based on next-generation sequencing data was carried out to identify CNVs segregating in an Iberian x Landrace backcross population and study their association with fatty acid composition and growth-related traits. A total of 1,279 CNVs, including duplications and deletions, were detected, ranging from 106 to 235 CNVs across samples, with an average of 183 CNVs per sample. Moreover, we detected 540 CNV regions (CNVRs) containing 245 genes. Functional annotation suggested that these genes possess a great variety of molecular functions and may play a role in production traits in commercial breeds. Some of the identified CNVRs contained relevant functional genes (e.g., CLCA4, CYP4X1, GPAT2, MOGAT2, PLA2G2A and PRKG1, among others). The variation in copy number of four of them (CLCA4, GPAT2, MOGAT2 and PRKG1) was validated in 150 BC1_LD (25% Iberian and 75% Landrace) animals by qPCR. Additionally, their contribution regarding backfat and intramuscular fatty acid composition and growth-related traits was analyzed. Statistically significant associations were obtained for CNVR112 (GPAT2) for the C18:2(n-6)/C18:3(n-3) ratio in backfat and carcass length, among others. Notably, GPATs are enzymes that catalyze the first step in the biosynthesis of both triglycerides and glycerophospholipids, suggesting that this CNVR may contribute to genetic variation in fatty acid composition and growth traits. These findings provide useful genomic information to facilitate the further identification of trait-related CNVRs affecting economically important traits in pigs.

Integration of liver gene co-expression networks and eGWAs analyses highlighted candidate regulators implicated in lipid metabolism in pigs.

In the present study, liver co-expression networks and expression Genome Wide Association Study (eGWAS) were performed to identify DNA variants and molecular pathways implicated in the functional regulatory mechanisms of meat quality traits in pigs. With this purpose, the liver mRNA expression of 44 candidates genes related with lipid metabolism was analysed in 111 Iberian x Landrace backcross animals. The eGWAS identified 92 eSNPs located in seven chromosomal regions and associated with eight genes: CROT, CYP2U1, DGAT1, EGF, FABP1, FABP5, PLA2G12A, and PPARA. Remarkably, cis-eSNPs associated with FABP1 gene expression which may be determining the C18:2(n-6)/C18:3(n-3) ratio in backfat through the multiple interaction of DNA variants and genes were identified. Furthermore, a hotspot on SSC8 associated with the gene expression of eight genes was identified and the TBCK gene was pointed out as candidate gene regulating it. Our results also suggested that the PI3K-Akt-mTOR pathway plays an important role in the control of the analysed genes highlighting nuclear receptors as the NR3C1 or PPARA. Finally, sex-dimorphism associated with hepatic lipid metabolism was identified with over-representation of female-biased genes. These results increase our knowledge of the genetic architecture underlying fat composition traits.

Expression-based GWAS identifies variants, gene interactions and key regulators affecting intramuscular fatty acid content and composition in porcine meat.

The aim of this work is to better understand the genetic mechanisms determining two complex traits affecting porcine meat quality: intramuscular fat (IMF) content and its fatty acid (FA) composition. With this purpose, expression Genome-Wide Association Study (eGWAS) of 45 lipid-related genes associated with meat quality traits in swine muscle (Longissimus dorsi) of 114 Iberian × Landrace backcross animals was performed. The eGWAS identified 241 SNPs associated with 11 genes: ACSM5, CROT, FABP3, FOS, HIF1AN, IGF2, MGLL, NCOA1, PIK3R1, PLA2G12A and PPARA. Three expression Quantitative Trait Loci (eQTLs) for IGF2, ACSM5 and MGLL were identified, showing cis-acting effects, whereas 16 eQTLs had trans regulatory effects. A polymorphism in the ACSM5 promoter region associated with its expression was identified. In addition, strong candidate genes regulating ACSM5, FOS, PPARA, PIK3R1, PLA2G12A and HIF1AN gene expression were also seen. Notably, the analysis highlighted the NR3C1 transcription factor as a strong candidate gene involved in the regulation of the 45 genes analysed. Finally, the IGF2, MGLL, MC2R, ARHGAP6, and NR3C1 genes were identified as potential regulators co-localizing within QTLs for fatness and growth traits in the IBMAP population. The results obtained increase our knowledge in the functional regulatory mechanisms involved in these complex traits.

Epigenetic regulation of the ELOVL6 gene is associated with a major QTL effect on fatty acid composition in pigs.

BACKGROUND: In previous studies on an Iberian x Landrace cross, we have provided evidence that supported the porcine ELOVL6 gene as the major causative gene of the QTL on pig chromosome 8 for palmitic and palmitoleic acid contents in muscle and backfat. The single nucleotide polymorphism (SNP) ELOVL6:c.-533C > T located in the promoter region of ELOVL6 was found to be highly associated with ELOVL6 expression and, accordingly, with the percentages of palmitic and palmitoleic acids in longissimus dorsi and adipose tissue. The main goal of the current work was to further study the role of ELOVL6 on these traits by analyzing the regulation of the expression of ELOVL6 and the implication of ELOVL6 polymorphisms on meat quality traits in pigs. RESULTS: High-throughput sequencing of BAC clones that contain the porcine ELOVL6 gene coupled to RNAseq data re-analysis showed that two isoforms of this gene are expressed in liver and adipose tissue and that they differ in number of exons and 3'UTR length. Although several SNPs in the 3'UTR of ELOVL6 were associated with palmitic and palmitoleic acid contents, this association was lower than that previously observed with SNP ELOVL6:c.-533C > T. This SNP is in full linkage disequilibrium with SNP ELOVL6:c.-394G > A that was identified in the binding site for estrogen receptor alpha (ERα). Interestingly, the ELOVL6:c.-394G allele is associated with an increase in methylation levels of the ELOVL6 promoter and with a decrease of ELOVL6 expression. Therefore, ERα is clearly a good candidate to explain the regulation of ELOVL6 expression through dynamic epigenetic changes in the binding site of known regulators of ELOVL6 gene, such as SREBF1 and SP1. CONCLUSIONS: Our results strongly suggest the ELOVL6:c.-394G > A polymorphism as the causal mutation for the QTL on pig chromosome 8 that affects fatty acid composition in pigs.

A co-association network analysis of the genetic determination of pig conformation, growth and fatness.

BACKGROUND: Several QTLs have been identified for major economically relevant traits in livestock, such as growth and meat quality, revealing the complex genetic architecture of these traits. The use of network approaches considering the interactions of multiple molecules and traits provides useful insights into the molecular underpinnings of complex traits. Here, a network based methodology, named Association Weight Matrix, was applied to study gene interactions and pathways affecting pig conformation, growth and fatness traits. RESULTS: The co-association network analysis underpinned three transcription factors, PPARγ, ELF1, and PRDM16 involved in mesoderm tissue differentiation. Fifty-four genes in the network belonged to growth-related ontologies and 46 of them were common with a similar study for growth in cattle supporting our results. The functional analysis uncovered the lipid metabolism and the corticotrophin and gonadotrophin release hormone pathways among the most important pathways influencing these traits. Our results suggest that the genes and pathways here identified are important determining either the total body weight of the animal and the fat content. For instance, a switch in the mesoderm tissue differentiation may determinate the age-related preferred pathways being in the puberty stage those related with the miogenic and osteogenic lineages; on the contrary, in the maturity stage cells may be more prone to the adipocyte fate. Hence, our results demonstrate that an integrative genomic co-association analysis is a powerful approach for identifying new connections and interactions among genes. CONCLUSIONS: This work provides insights about pathways and key regulators which may be important determining the animal growth, conformation and body proportions and fatness traits. Molecular information concerning genes and pathways here described may be crucial for the improvement of genetic breeding programs applied to pork meat production.

New insight into the SSC8 genetic determination of fatty acid composition in pigs.

BACKGROUND: Fat content and fatty acid composition in swine are becoming increasingly studied because of their effect on sensory and nutritional quality of meat. A QTL (quantitative trait locus) for fatty acid composition in backfat was previously detected on porcine chromosome 8 (SSC8) in an Iberian x Landrace F2 intercross. More recently, a genome-wide association study detected the same genomic region for muscle fatty acid composition in an Iberian x Landrace backcross population. ELOVL6, a strong positional candidate gene for this QTL, contains a polymorphism in its promoter region (ELOVL6:c.-533C < T), which is associated with percentage of palmitic and palmitoleic acids in muscle and adipose tissues. Here, a combination of single-marker association and the haplotype-based approach was used to analyze backfat fatty acid composition in 470 animals of an Iberian x Landrace F2 intercross genotyped with 144 SNPs (single nucleotide polymorphisms) distributed along SSC8. RESULTS: Two trait-associated SNP regions were identified at 93 Mb and 119 Mb on SSC8. The strongest statistical signals of both regions were observed for palmitoleic acid (C16:1(n-7)) content and C18:0/C16:0 and C18:1(n-7)/C16:1(n-7) elongation ratios. MAML3 and SETD7 are positional candidate genes in the 93 Mb region and two novel microsatellites in MAML3 and nine SNPs in SETD7 were identified. No significant association for the MAML3 microsatellite genotypes was detected. The SETD7:c.700G > T SNP, although statistically significant, was not the strongest signal in this region. In addition, the expression of MAML3 and SETD7 in liver and adipose tissue varied among animals, but no association was detected with the polymorphisms in these genes. In the 119 Mb region, the ELOVL6:c.-533C > T polymorphism showed a strong association with percentage of palmitic and palmitoleic fatty acids and elongation ratios in backfat. CONCLUSIONS: Our results suggest that the polymorphisms studied in MAML3 and SETD7 are not the causal mutations for the QTL in the 93 Mb region. However, the results for ELOVL6 support the hypothesis that the ELOVL6:c.-533C > T polymorphism has a pleiotropic effect on backfat and intramuscular fatty acid composition and that it has a role in the determination of the QTL in the 119 Mb region.