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.

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.

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.

Differences in muscle transcriptome among pigs phenotypically extreme for fatty acid composition.

BACKGROUND: Besides having an impact on human health, the porcine muscle fatty acid profile determines meat quality and taste. The RNA-Seq technologies allowed us to explore the pig muscle transcriptome with an unprecedented detail. The aim of this study was to identify differentially-expressed genes between two groups of 6 sows belonging to an Iberian × Landrace backcross with extreme phenotypes according to FA profile. RESULTS: We sequenced the muscle transcriptome acquiring 787.5 M of 75 bp paired-end reads. About 85.1% of reads were mapped to the reference genome. Of the total reads, 79.1% were located in exons, 6.0% in introns and 14.9% in intergenic regions, indicating expressed regions not annotated in the reference genome. We identified a 34.5% of the intergenic regions as interspersed repetitive regions. We predicted a total of 2,372 putative proteins. Pathway analysis with 131 differentially-expressed genes revealed that the most statistically-significant metabolic pathways were related with lipid metabolism. Moreover, 18 of the differentially-expressed genes were located in genomic regions associated with IMF composition in an independent GWAS study in the same genetic background. Thus, our results indicate that the lipid metabolism of FAs is differently modulated when the FA composition in muscle differs. For instance, a high content of PUFA may reduce FA and glucose uptake resulting in an inhibition of the lipogenesis. These results are consistent with previous studies of our group analysing the liver and the adipose tissue transcriptomes providing a view of each of the main organs involved in lipid metabolism. CONCLUSIONS: The results obtained in the muscle transcriptome analysis increase the knowledge of the gene regulation of IMF deposition, FA profile and meat quality, in terms of taste and nutritional value. Besides, our results may be important in terms of human health.

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.

Analysis of porcine adipose tissue transcriptome reveals differences in de novo fatty acid synthesis in pigs with divergent muscle fatty acid composition.

BACKGROUND: In pigs, adipose tissue is one of the principal organs involved in the regulation of lipid metabolism. It is particularly involved in the overall fatty acid synthesis with consequences in other lipid-target organs such as muscles and the liver. With this in mind, we have used massive, parallel high-throughput sequencing technologies to characterize the porcine adipose tissue transcriptome architecture in six Iberian x Landrace crossbred pigs showing extreme phenotypes for intramuscular fatty acid composition (three per group). RESULTS: High-throughput RNA sequencing was used to generate a whole characterization of adipose tissue (backfat) transcriptome. A total of 4,130 putative unannotated protein-coding sequences were identified in the 20% of reads which mapped in intergenic regions. Furthermore, 36% of the unmapped reads were represented by interspersed repeats, SINEs being the most abundant elements. Differential expression analyses identified 396 candidate genes among divergent animals for intramuscular fatty acid composition. Sixty-two percent of these genes (247/396) presented higher expression in the group of pigs with higher content of intramuscular SFA and MUFA, while the remaining 149 showed higher expression in the group with higher content of PUFA. Pathway analysis related these genes to biological functions and canonical pathways controlling lipid and fatty acid metabolisms. In concordance with the phenotypic classification of animals, the major metabolic pathway differentially modulated between groups was de novo lipogenesis, the group with more PUFA being the one that showed lower expression of lipogenic genes. CONCLUSIONS: These results will help in the identification of genetic variants at loci that affect fatty acid composition traits. The implications of these results range from the improvement of porcine meat quality traits to the application of the pig as an animal model of human metabolic diseases.

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

BACKGROUND: The ELOVL fatty acid elongase 6 (ELOVL6), the only elongase related to de novo lipogenesis, catalyzes the rate-limiting step in the elongation cycle by controlling the fatty acid balance in mammals. It is located on pig chromosome 8 (SSC8) in a region where a QTL affecting palmitic, and palmitoleic acid composition was previously detected, using an Iberian x Landrace intercross. The main goal of this work was to fine-map the QTL and to evaluate the ELOVL6 gene as a positional candidate gene affecting the percentages of palmitic and palmitoleic fatty acids in pigs. METHODOLOGY AND PRINCIPAL FINDINGS: The combination of a haplotype-based approach and single-marker analysis allowed us to identify the main, associated interval for the QTL, in which the ELOVL6 gene was identified and selected as a positional candidate gene. A polymorphism in the promoter region of ELOVL6, ELOVL6:c.-533C>T, was highly associated with the percentage of palmitic and palmitoleic acids in muscle and backfat. Significant differences in ELOVL6 gene expression were observed in backfat when animals were classified by the ELOVL6:c.-533C>T genotype. Accordingly, animals carrying the allele associated with a decrease in ELOVL6 gene expression presented an increase in C16:0 and C16:1(n-7) fatty acid content and a decrease of elongation activity ratios in muscle and backfat. Furthermore, a SNP genome-wide association study with ELOVL6 relative expression levels in backfat showed the strongest effect on the SSC8 region in which the ELOVL6 gene is located. Finally, different potential genomic regions associated with ELOVL6 gene expression were also identified by GWAS in liver and muscle, suggesting a differential tissue regulation of the ELOVL6 gene. CONCLUSIONS AND SIGNIFICANCE: Our results suggest ELOVL6 as a potential causal gene for the QTL analyzed and, subsequently, for controlling the overall balance of fatty acid composition in pigs.