Identification of candidate regulatory genes for intramuscular fatty acid composition in pigs by transcriptome analysis.

BACKGROUND: Intramuscular fat (IMF) content and its fatty acid (FA) composition are typically controlled by several genes, each with a small effect. In the current study, to pinpoint candidate genes and putative regulators involved in FA composition, we performed a multivariate integrative analysis between intramuscular FA and transcriptome profiles of porcine longissimus dorsi (LD) muscle. We also carried out a combination of network, regulatory impact factor (RIF), in silico prediction of putative target genes, and functional analyses to better support the biological relevance of our findings. RESULTS: For this purpose, we used LD RNA-Seq and intramuscular FA composition profiles of 129 Iberian × Duroc backcrossed pigs. We identified 378 correlated variables (13 FA and 365 genes), including six FA (C20:4n-6, C18:2n-6, C20:3n-6, C18:1n-9, C18:0, and C16:1n-7) that were among the most interconnected variables in the predicted network. The detected FA-correlated genes include genes involved in lipid and/or carbohydrate metabolism or in regulation of IMF deposition (e.g., ADIPOQ, CHUK, CYCS, CYP4B1, DLD, ELOVL6, FBP1, G0S2, GCLC, HMGCR, IDH3A, LEP, LGALS12, LPIN1, PLIN1, PNPLA8, PPP1R1B, SDR16C5, SFRP5, SOD3, SNW1, and TFRC), meat quality (GALNT15, GOT1, MDH1, NEU3, PDHA1, SDHD, and UNC93A), and transport (e.g., EXOC7 and SLC44A2). Functional analysis highlighted 54 over-represented gene ontology terms, including well-known biological processes and pathways that regulate lipid and carbohydrate metabolism. RIF analysis suggested a pivotal role for six transcription factors (CARHSP1, LBX1, MAFA, PAX7, SIX5, and TADA2A) as putative regulators of gene expression and intramuscular FA composition. Based on in silico prediction, we identified putative target genes for these six regulators. Among these, TADA2A and CARHSP1 had extreme RIF scores and present novel regulators in pigs. In addition, the expression of TADA2A correlated (either positively or negatively) with C20:4n-6, C18:2n-6, C20:3n-6, C18:1n-9, and that of CARHSP1 correlated (positively) with the C16:1n-7 lipokine. We also found that these two transcription factors share target genes that are involved in lipid metabolism (e.g., GOT1, PLIN1, and TFRC). CONCLUSIONS: This integrative analysis of muscle transcriptome and intramuscular FA profile revealed valuable information about key candidate genes and potential regulators for FA and lipid metabolism in pigs, among which some transcription factors are proposed to control gene expression and modulate FA composition differences.

Single and longitudinal genome-wide association studies for dairy traits available in goats with three recorded lactations.

Milk yield and composition phenotypes are systematically recorded across several lactations in goats, but the majority of genome-wide association studies (GWAS) performed so far have rather ignored the longitudinal nature of such data. Here, we have used two different GWAS approaches to analyse data from three lactations recorded in Murciano-Granadina goats. In Analysis 1, independent GWAS have been carried out for each trait and lactation, while a single longitudinal GWAS, jointly considering all data, has been performed in Analysis 2. In both analyses, genome-wide significant QTL for lactose percentage on chromosome 2 (129.77-131.01 Mb) and for milk protein percentage on the chromosome 6 (74.8-94.6 Mb) casein gene cluster region were detected. In Analysis 1, several QTL were not replicated in all three lactations, possibly due to the existence of lactation-specific genetic determinants. In Analysis 2, we identified several genome-wide significant QTL related to milk yield and protein content that were not uncovered in Analysis 1. The increased number of QTL identified in Analysis 2 suggests that the longitudinal GWAS is particularly well suited for the genetic analysis of dairy traits. Moreover, our data confirm that variability within or close to the casein complex is the main genetic determinant of milk protein percentage in Murciano-Granadina goats.

Variability in porcine microRNA genes and its association with mRNA expression and lipid phenotypes.

BACKGROUND: Mature microRNAs (miRNAs) play an important role in repressing the expression of a wide range of mRNAs. The presence of polymorphic sites in miRNA genes and their corresponding 3'UTR binding sites can disrupt canonical conserved miRNA-mRNA pairings, and thus modify gene expression patterns. However, to date such polymorphic sites in miRNA genes and their association with gene expression phenotypes and complex traits are poorly characterized in pigs. RESULTS: By analyzing whole-genome sequences from 120 pigs and wild boars from Europe and Asia, we identified 285 single nucleotide polymorphisms (SNPs) that map to miRNA loci, and 109,724 SNPs that are located in predicted 7mer-m8 miRNA binding sites within porcine 3'UTR. In porcine miRNA genes, SNP density is reduced compared with their flanking non-miRNA regions. By sequencing the genomes of five Duroc boars, we identified 12 miRNA SNPs that were subsequently genotyped in their offspring (N = 345, Lipgen population). Association analyses of miRNA SNPs with 38 lipid-related traits and hepatic and muscle microarray expression phenotypes recorded in the Lipgen population were performed. The most relevant detected association was between the genotype of the rs319154814 (G/A) SNP located in the apical loop of the ssc-miR-326 hairpin precursor and PPP1CC mRNA levels in the liver (q-value = 0.058). This result was subsequently confirmed by qPCR (P-value = 0.027). The rs319154814 (G/A) genotype was also associated with several fatty acid composition traits. CONCLUSIONS: Our findings show a reduced variability of porcine miRNA genes, which is consistent with strong purifying selection, particularly in the seed region that plays a critical role in miRNA binding. Although it is generally assumed that SNPs mapping to the seed region are those with the most pronounced consequences on mRNA expression, we show that a SNP mapping to the apical region of ssc-miR-326 is significantly associated with hepatic mRNA levels of the PPP1CC gene, one of its predicted targets. Although experimental confirmation of such an interaction is reported in humans but not in pigs, this result highlights the need to further investigate the functional effects of miRNA polymorphisms that are located outside the seed region on gene expression in pigs.

A systems biology framework integrating GWAS and RNA-seq to shed light on the molecular basis of sperm quality in swine.

BACKGROUND: Genetic pressure in animal breeding is sparking the interest of breeders for selecting elite boars with higher sperm quality to optimize ejaculate doses and fertility rates. However, the molecular basis of sperm quality is not yet fully understood. Our aim was to identify candidate genes, pathways and DNA variants associated to sperm quality in swine by analysing 25 sperm-related phenotypes and integrating genome-wide association studies (GWAS) and RNA-seq under a systems biology framework. RESULTS: By GWAS, we identified 12 quantitative trait loci (QTL) associated to the percentage of head and neck abnormalities, abnormal acrosomes and motile spermatozoa. Candidate genes included CHD2, KATNAL2, SLC14A2 and ABCA1. By RNA-seq, we identified a wide repertoire of mRNAs (e.g. PRM1, OAZ3, DNAJB8, TPPP2 and TNP1) and miRNAs (e.g. ssc-miR-30d, ssc-miR-34c, ssc-miR-30c-5p, ssc-miR-191, members of the let-7 family and ssc-miR-425-5p) with functions related to sperm biology. We detected 6128 significant correlations (P-value ≤ 0.05) between sperm traits and mRNA abundances. By expression (e)GWAS, we identified three trans-expression QTL involving the genes IQCJ, ACTR2 and HARS. Using the GWAS and RNA-seq data, we built a gene interaction network. We considered that the genes and interactions that were present in both the GWAS and RNA-seq networks had a higher probability of being actually involved in sperm quality and used them to build a robust gene interaction network. In addition, in the final network we included genes with RNA abundances correlated with more than four semen traits and miRNAs interacting with the genes on the network. The final network was enriched for genes involved in gamete generation and development, meiotic cell cycle, DNA repair or embryo implantation. Finally, we designed a panel of 73 SNPs based on the GWAS, eGWAS and final network data, that explains between 5% (for sperm cell concentration) and 36% (for percentage of neck abnormalities) of the phenotypic variance of the sperm traits. CONCLUSIONS: By applying a systems biology approach, we identified genes that potentially affect sperm quality and constructed a SNP panel that explains a substantial part of the phenotypic variance for semen quality in our study and that should be tested in other swine populations to evaluate its relevance for the pig breeding sector.

A genome-wide analysis of copy number variation in Murciano-Granadina goats.

BACKGROUND: In this work, our aim was to generate a map of the copy number variations (CNV) segregating in a population of Murciano-Granadina goats, the most important dairy breed in Spain, and to ascertain the main biological functions of the genes that map to copy number variable regions. RESULTS: Using a dataset that comprised 1036 Murciano-Granadina goats genotyped with the Goat SNP50 BeadChip, we were able to detect 4617 and 7750 autosomal CNV with the PennCNV and QuantiSNP software, respectively. By applying the EnsembleCNV algorithm, these CNV were assembled into 1461 CNV regions (CNVR), of which 486 (33.3% of the total CNVR count) were consistently called by PennCNV and QuantiSNP and used in subsequent analyses. In this set of 486 CNVR, we identified 78 gain, 353 loss and 55 gain/loss events. The total length of all the CNVR (95.69 Mb) represented 3.9% of the goat autosomal genome (2466.19 Mb), whereas their size ranged from 2.0 kb to 11.1 Mb, with an average size of 196.89 kb. Functional annotation of the genes that overlapped with the CNVR revealed an enrichment of pathways related with olfactory transduction (fold-enrichment = 2.33, q-value = 1.61 × 10-10), ABC transporters (fold-enrichment = 5.27, q-value = 4.27 × 10-04) and bile secretion (fold-enrichment = 3.90, q-value = 5.70 × 10-03). CONCLUSIONS: A previous study reported that the average number of CNVR per goat breed was ~ 20 (978 CNVR/50 breeds), which is much smaller than the number we found here (486 CNVR). We attribute this difference to the fact that the previous study included multiple caprine breeds that were represented by small to moderate numbers of individuals. Given the low frequencies of CNV (in our study, the average frequency of CNV is 1.44%), such a design would probably underestimate the levels of the diversity of CNV at the within-breed level. We also observed that functions related with sensory perception, metabolism and embryo development are overrepresented in the set of genes that overlapped with CNV, and that these loci often belong to large multigene families with tens, hundreds or thousands of paralogous members, a feature that could favor the occurrence of duplications or deletions by non-allelic homologous recombination.

A genome-wide association analysis for body, udder, and leg conformation traits recorded in Murciano-Granadina goats.

Morphological traits are of great importance to dairy goat production given their effect on phenotypes of economic interest. However, their underlying genomic architecture has not yet been extensively characterized. Herein, we aimed to identify genomic regions associated with body, udder, and leg conformation traits recorded in 825 Murciano-Granadina goats. We genotyped this resource population using the GoatSNP50 BeadChip (Illumina Inc., San Diego, CA) and performed genome-wide association analyses using the GEMMA software. We found 2 genome-wide significant associations between markers rs268273468 [Capra hircus (CHI) 16:69617700] and rs268249346 (CHI 28:18321523) and medial suspensory ligament. In contrast, we did not detect any genome-wide significant associations for body and leg traits. Moreover, we found 12, 19, and 7 chromosome-wide significant associations for udder, body, and leg traits, respectively. Comparison of our data with previous studies revealed a low level of positional concordance between regions associated with morphological traits. In addition to technical factors, this lack of concordance could be due to a substantial level of genetic heterogeneity among breeds or to the strong polygenic background of morphological traits, which makes it difficult to detect genetic factors that have small phenotypic effects.

About the existence of common determinants of gene expression in the porcine liver and skeletal muscle.

BACKGROUND: The comparison of expression QTL (eQTL) maps obtained in different tissues is an essential step to understand how gene expression is genetically regulated in a context-dependent manner. In the current work, we have compared the transcriptomic and eQTL profiles of two porcine tissues (skeletal muscle and liver) which typically show highly divergent expression profiles, in 103 Duroc pigs genotyped with the Porcine SNP60 BeadChip (Illumina) and with available microarray-based measurements of hepatic and muscle mRNA levels. Since structural variation could have effects on gene expression, we have also investigated the co-localization of cis-eQTLs with copy number variant regions (CNVR) segregating in this Duroc population. RESULTS: The analysis of differential expresssion revealed the existence of 1204 and 1490 probes that were overexpressed and underexpressed in the gluteus medius muscle when compared to liver, respectively (|fold-change| > 1.5, q-value < 0.05). By performing genome scans in 103 Duroc pigs with available expression and genotypic data, we identified 76 and 28 genome-wide significant cis-eQTLs regulating gene expression in the gluteus medius muscle and liver, respectively. Twelve of these cis-eQTLs were shared by both tissues (i.e. 42.8% of the cis-eQTLs identified in the liver were replicated in the gluteus medius muscle). These results are consistent with previous studies performed in humans, where 50% of eQTLs were shared across tissues. Moreover, we have identified 41 CNVRs in a set of 350 pigs from the same Duroc population, which had been genotyped with the Porcine SNP60 BeadChip by using the PennCNV and GADA softwares, but only a small proportion of these CNVRs co-localized with the cis-eQTL signals. CONCLUSION: Despite the fact that there are considerable differences in the gene expression patterns of the porcine liver and skeletal muscle, we have identified a substantial proportion of common cis-eQTLs regulating gene expression in both tissues. Several of these cis-eQTLs influence the mRNA levels of genes with important roles in meat (CTSF) and carcass quality (TAPT1), lipid metabolism (TMEM97) and obesity (MARC2), thus evidencing the practical importance of dissecting the genetic mechanisms involved in their expression.

Comparing the mRNA expression profile and the genetic determinism of intramuscular fat traits in the porcine gluteus medius and longissimus dorsi muscles.

BACKGROUND: Intramuscular fat (IMF) content and composition have a strong impact on the nutritional and organoleptic properties of porcine meat. The goal of the current work was to compare the patterns of gene expression and the genetic determinism of IMF traits in the porcine gluteus medius (GM) and longissimus dorsi (LD) muscles. RESULTS: A comparative analysis of the mRNA expression profiles of the pig GM and LD muscles in 16 Duroc pigs with available microarray mRNA expression measurements revealed the existence of 106 differentially expressed probes (fold-change > 1.5 and q-value < 0.05). Amongst the genes displaying the most significant differential expression, several loci belonging to the Hox transcription factor family were either upregulated (HOXA9, HOXA10, HOXB6, HOXB7 and TBX1) or downregulated (ARX) in the GM muscle. Differences in the expression of genes with key roles in carbohydrate and lipid metabolism (e.g. FABP3, ORMDL1 and SLC37A1) were also detected. By performing a GWAS for IMF content and composition traits recorded in the LD and GM muscles of 350 Duroc pigs, we identified the existence of one region on SSC14 (110-114 Mb) displaying significant associations with C18:0, C18:1(n-7), saturated and unsaturated fatty acid contents in both GM and LD muscles. Moreover, we detected several genome-wide significant associations that were not consistently found in both muscles. Further studies should be performed to confirm whether these associations are muscle-specific. Finally, the performance of an eQTL scan for 74 genes, located within GM QTL regions and with available microarray measurements of gene expression, made possible to identify 14 cis-eQTL regulating the expression of 14 loci, and six of them were confirmed by RNA-Seq. CONCLUSIONS: We have detected significant differences in the mRNA expression patterns of the porcine LD and GM muscles, evidencing that the transcriptomic profile of the skeletal muscle tissue is affected by anatomical, metabolic and functional factors. A highly significant association with IMF composition on SSC14 was replicated in both muscles, highlighting the existence of a common genetic determinism, but we also observed the existence of a few associations whose magnitude and significance varied between LD and GM muscles.

Differential expression of mRNA isoforms in the skeletal muscle of pigs with distinct growth and fatness profiles.

BACKGROUND: The identification of genes differentially expressed in the skeletal muscle of pigs displaying distinct growth and fatness profiles might contribute to identify the genetic factors that influence the phenotypic variation of such traits. So far, the majority of porcine transcriptomic studies have investigated differences in gene expression at a global scale rather than at the mRNA isoform level. In the current work, we have investigated the differential expression of mRNA isoforms in the gluteus medius (GM) muscle of 52 Duroc HIGH (increased backfat thickness, intramuscular fat and saturated and monounsaturated fatty acids contents) and LOW pigs (opposite phenotype, with an increased polyunsaturated fatty acids content). RESULTS: Our analysis revealed that 10.9% of genes expressed in the GM muscle generate alternative mRNA isoforms, with an average of 2.9 transcripts per gene. By using two different pipelines, one based on the CLC Genomics Workbench and another one on the STAR, RSEM and DESeq2 softwares, we have identified 10 mRNA isoforms that both pipelines categorize as differentially expressed in HIGH vs LOW pigs (P-value < 0.01 and ±0.6 log2fold-change). Only five mRNA isoforms, produced by the ITGA5, SEMA4D, LITAF, TIMP1 and ANXA2 genes, remain significant after correction for multiple testing (q-value < 0.05 and ±0.6 log2fold-change), being upregulated in HIGH pigs. CONCLUSIONS: The increased levels of specific ITGA5, LITAF, TIMP1 and ANXA2 mRNA isoforms in HIGH pigs is consistent with reports indicating that the overexpression of these four genes is associated with obesity and metabolic disorders in humans. A broader knowledge about the functional attributes of these mRNA variants would be fundamental to elucidate the consequences of transcript diversity on the determinism of porcine phenotypes of economic interest.

Identification of protein-damaging mutations in 10 swine taste receptors and 191 appetite-reward genes.

BACKGROUND: Taste receptors (TASRs) are essential for the body's recognition of chemical compounds. In the tongue, TASRs sense the sweet and umami and the toxin-related bitter taste thus promoting a particular eating behaviour. Moreover, their relevance in other organs is now becoming evident. In the intestine, they regulate nutrient absorption and gut motility. Upon ligand binding, TASRs activate the appetite-reward circuitry to signal the nervous system and keep body homeostasis. With the aim to identify genetic variation in the swine TASRs and in the genes from the appetite and the reward pathways, we have sequenced the exons of 201 TASRs and appetite-reward genes from 304 pigs belonging to ten breeds, wild boars and to two phenotypically extreme groups from a F2 resource with data on growth and fat deposition. RESULTS: We identified 2,766 coding variants 395 of which were predicted to have a strong impact on protein sequence and function. 334 variants were present in only one breed and at predicted alternative allele frequency (pAAF) ≥ 0.1. The Asian pigs and the wild boars showed the largest proportion of breed specific variants. We also compared the pAAF of the two F2 groups and found that variants in TAS2R39 and CD36 display significant differences suggesting that these genes could influence growth and fat deposition. We developed a 128-variant genotyping assay and confirmed 57 of these variants. CONCLUSIONS: We have identified thousands of variants affecting TASRs as well as genes involved in the appetite and the reward mechanisms. Some of these genes have been already associated to taste preferences, appetite or behaviour in humans and mouse. We have also detected indications of a potential relationship of some of these genes with growth and fat deposition, which could have been caused by changes in taste preferences, appetite or reward and ultimately impact on food intake. A genotyping array with 57 variants in 31 of these genes is now available for genotyping and start elucidating the impact of genetic variation in these genes on pig biology and breeding.

A genome-wide association analysis for porcine serum lipid traits reveals the existence of age-specific genetic determinants.

BACKGROUND: The genetic determinism of blood lipid concentrations, the main risk factor for atherosclerosis, is practically unknown in species other than human and mouse. Even in model organisms, little is known about how the genetic determinants of lipid traits are modulated by age-specific factors. To gain new insights into this issue, we have carried out a genome-wide association study (GWAS) for cholesterol (CHOL), triglyceride (TRIG) and low (LDL) and high (HDL) density lipoprotein concentrations measured in Duroc pigs at two time points (45 and 190 days). RESULTS: Analysis of data with mixed-model methods (EMMAX, GEMMA, GenABEL) and PLINK showed a low positional concordance between trait-associated regions (TARs) for serum lipids at 45 and 190 days. Besides, the proportion of phenotypic variance explained by SNPs at these two time points was also substantially different. The four analyses consistently detected two regions on SSC3 (124 Mb, CHOL and LDL at 190 days) and SSC6 (135 Mb, CHOL and TRIG at 190 days) with highly significant effects on the porcine blood lipid profile. Moreover, we have found that SNP variation within SSC3, SSC6, SSC10, SSC13 and SSC16 TARs is associated with the expression of several genes mapping to other chromosomes and related to lipid metabolism. CONCLUSIONS: Our data demonstrate that the effects of genomic determinants influencing lipid concentrations in pigs, as well as the amount of phenotypic variance they explain, are influenced by age-related factors.

From SNP co-association to RNA co-expression: novel insights into gene networks for intramuscular fatty acid composition in porcine.

BACKGROUND: Fatty acids (FA) play a critical role in energy homeostasis and metabolic diseases; in the context of livestock species, their profile also impacts on meat quality for healthy human consumption. Molecular pathways controlling lipid metabolism are highly interconnected and are not fully understood. Elucidating these molecular processes will aid technological development towards improvement of pork meat quality and increased knowledge of FA metabolism, underpinning metabolic diseases in humans. RESULTS: The results from genome-wide association studies (GWAS) across 15 phenotypes were subjected to an Association Weight Matrix (AWM) approach to predict a network of 1,096 genes related to intramuscular FA composition in pigs. To identify the key regulators of FA metabolism, we focused on the minimal set of transcription factors (TF) that the explored the majority of the network topology. Pathway and network analyses pointed towards a trio of TF as key regulators of FA metabolism: NCOA2, FHL2 and EP300. Promoter sequence analyses confirmed that these TF have binding sites for some well-know regulators of lipid and carbohydrate metabolism. For the first time in a non-model species, some of the co-associations observed at the genetic level were validated through co-expression at the transcriptomic level based on real-time PCR of 40 genes in adipose tissue, and a further 55 genes in liver. In particular, liver expression of NCOA2 and EP300 differed between pig breeds (Iberian and Landrace) extreme in terms of fat deposition. Highly clustered co-expression networks in both liver and adipose tissues were observed. EP300 and NCOA2 showed centrality parameters above average in the both networks. Over all genes, co-expression analyses confirmed 28.9% of the AWM predicted gene-gene interactions in liver and 33.0% in adipose tissue. The magnitude of this validation varied across genes, with up to 60.8% of the connections of NCOA2 in adipose tissue being validated via co-expression. CONCLUSIONS: Our results recapitulate the known transcriptional regulation of FA metabolism, predict gene interactions that can be experimentally validated, and suggest that genetic variants mapped to EP300, FHL2, and NCOA2 modulate lipid metabolism and control energy homeostasis in pigs.

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.

Interrelation between gut microbiota, SCFA, and fatty acid composition in pigs.

The gut microbiota is a key player in the host metabolism. Some bacteria are able to ferment non-digestible compounds and produce short-chain fatty acids that the host can later transform and accumulate in tissue. In this study, we aimed to better understand the relationships between the microorganisms and the short-chain fatty acid composition of the rectal content, including the possible linkage with the fatty acid composition in backfat and muscle of the pig. We studied a Duroc × Iberian crossbred population, and we found significant correlations between different bacterial and archaeal genera and the fatty acid profile. The abundance of n-butyric acid in the rectal content was positively associated with Prevotella spp. and negatively associated with Akkermansia spp., while conversely, the abundance of acetic acid was negatively and positively associated with the levels of Prevotella spp. and Akkermansia spp., respectively. The most abundant genus, Rikenellaceae RC9 gut group, had a positive correlation with palmitic acid in muscle and negative correlations with stearic acid in backfat and oleic acid in muscle. These results suggest the possible role of Prevotella spp. and Akkermansia spp. as biomarkers for acetic and n-butyric acids, and the relationship of Rikenellaceae RC9 gut group with the lipid metabolism, building up the potential, although indirect, role of the microbiota in the modification of the backfat and muscle fatty acid composition of the host.IMPORTANCEThe vital role of the gut microbiota on its host metabolism makes it essential to know how its modulation is mirrored on the fatty acid composition of the host. Our findings suggest Prevotella spp. and Akkermansia spp. as potential biomarkers for the levels of beneficial short-chain fatty acids and the possible influence of Rikenellaceae RC9 gut group in the backfat and muscle fatty acid composition of the pig.

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.

Identifying miRNA-mRNA regulatory networks on extreme n-6/n-3 polyunsaturated fatty acid ratio expression profiles in porcine skeletal muscle.

Omega-3 (n-3) and omega-6 (n-6) polyunsaturated fatty acids (PUFAs) are essential fatty acids with antagonistic inflammatory functions that play vital roles in metabolic health and immune response. Current commercial swine diets tend to over-supplement with n-6 PUFAs, which may increase the likelihood of developing inflammatory diseases and affect the overall well-being of the animals. However, it is still poorly understood how n-6/n-3 PUFA ratios affect the porcine transcriptome expression and how messenger RNAs (mRNAs) and microRNAs (miRNAs) might regulate biological processes related to PUFA metabolism. On account of this, we selected a total of 20 Iberian × Duroc crossbred pigs with extreme values for n-6/n-3 FA ratio (10 high vs 10 low), and longissimus dorsi muscle samples were used to identify differentially expressed mRNAs and miRNAs. The observed differentially expressed mRNAs were associated to biological pathways related to muscle growth and immunomodulation, while the differentially expressed microRNAs (ssc-miR-30a-3p, ssc-miR-30e-3p, ssc-miR-15b and ssc-miR-7142-3p) were correlated to adipogenesis and immunity. Relevant miRNA-to-mRNA regulatory networks were also predicted (i.e., mir15b to ARRDC3; mir-7142-3p to METTL21C), and linked to lipolysis, obesity, myogenesis, and protein degradation. The n-6/n-3 PUFA ratio differences in pig skeletal muscle revealed genes, miRNAs and enriched pathways involved in lipid metabolism, cell proliferation and inflammation.

Exon-intron split analysis reveals posttranscriptional regulatory signals induced by high and low n-6/n-3 polyunsaturated fatty acid ratio diets in piglets.

Polyunsaturated fatty acids (PUFA), such as omega-6 (n-6) and omega-3 (n-3), play a vital role in nutrient metabolism, inflammatory response, and gene regulation. microRNAs (miRNA), which can potentially degrade targeted messenger RNAs (mRNA) and/or inhibit their translation, might play a relevant role in PUFA-related changes in gene expression. Although differential expression analyses can provide a comprehensive picture of gene expression variation, they are unable to disentangle when in the mRNA life cycle the regulation of expression is taking place, including any putative functional miRNA-driven repression. To capture this, we used an exon-intron split analysis (EISA) approach to account for posttranscriptional changes in response to extreme values of n-6/n-3 PUFA ratio. Longissimus dorsi muscle samples of male and female piglets from sows fed with n-6/n-3 PUFA ratio of 13:1 (SOY) or 4:1 (LIN), were analyzed in a bidirectional contrast (LIN vs. SOY, SOY vs. LIN). Our results allowed the identification of genes showing strong posttranscriptional downregulation signals putatively targeted by significantly upregulated miRNA. Moreover, we identified genes primarily involved in the regulation of lipid-related metabolism and immune response, which may be associated with the pro- and anti-inflammatory functions of the n-6 and n-3 PUFA, respectively. EISA allowed us to uncover regulatory networks complementing canonical differential expression analyses, thus providing a more comprehensive view of muscle metabolic changes in response to PUFA concentration.

The relationship between dietary lipids, such as omega-6 and omega-3 polyunsaturated fatty acids (PUFA), and gene expression regulation was explored in piglet muscle. While these PUFA can influence nutrient metabolism and inflammatory response, small regulatory molecules called microRNAs (miRNA) can also influence the activity of genes. In this experiment, we used a computational approach dubbed exon­intron split analysis (EISA) to fully understand the role of miRNA in this context and how the genes and miRNA respond to changes in PUFA levels. Our findings demonstrated that some genes involved in lipid metabolism and immune response were affected by different PUFA concentrations and that EISA provides a more comprehensive view of how genes are regulated throughout their life cycle.

Global analysis of the association between pig muscle fatty acid composition and gene expression using RNA-Seq.

Fatty acids (FAs) play an essential role as mediators of cell signaling and signal transduction, affecting metabolic homeostasis and determining meat quality in pigs. However, FAs are transformed by the action of several genes, such as those encoding desaturases and elongases of FAs in lipogenic tissues. The aim of the current work was to identify candidate genes, biological processes, and pathways involved in the modulation of intramuscular FA profile from longissimus dorsi muscle. FA profile by gas chromatography of methyl esters and gene expression by RNA-Seq were determined in 129 Iberian × Duroc backcrossed pigs. An association analysis between the muscle transcriptome and its FA profile was performed, followed by a concordance and functional analysis. Overall, a list of well-known (e.g., PLIN1, LEP, ELOVL6, SC5D, NCOA2, ACSL1, MDH1, LPL, LGALS12, TFRC, GOT1, and FBP1) and novel (e.g., TRARG1, TANK, ENSSSCG00000011196, and ENSSSCG00000038429) candidate genes was identified, either in association with specific or several FA traits. Likewise, several of these genes belong to biological processes and pathways linked to energy, lipid, and carbohydrate metabolism, which seem determinants in the modulation of FA compositions. This study can contribute to elucidate the complex relationship between gene expression and FA profile in pig muscle.

Copy Number Variation on ABCC2-DNMBP Loci Affects the Diversity and Composition of the Fecal Microbiota in Pigs.

Genetic variation in the pig genome partially modulates the composition of porcine gut microbial communities. Previous studies have been focused on the association between single nucleotide polymorphisms (SNPs) and the gut microbiota, but little is known about the relationship between structural variants and fecal microbial traits. The main goal of this study was to explore the association between porcine genome copy number variants (CNVs) and the diversity and composition of pig fecal microbiota. For this purpose, we used whole-genome sequencing data to undertake a comprehensive identification of CNVs followed by a genome-wide association analysis between the estimated CNV status and the fecal bacterial diversity in a commercial Duroc pig population. A CNV predicted as gain (DUP) partially harboring ABCC2-DNMBP loci was associated with richness (P = 5.41 × 10-5, false discovery rate [FDR] = 0.022) and Shannon α-diversity (P = 1.42 × 10-4, FDR = 0.057). The in silico predicted gain of copies was validated by real-time quantitative PCR (qPCR), and its segregation, and positive association with the richness and Shannon α-diversity of the porcine fecal bacterial ecosystem was confirmed in an unrelated F1 (Duroc × Iberian) cross. Our results advise the relevance of considering the role of host-genome structural variants as potential modulators of microbial ecosystems and suggest the ABCC2-DNMBP CNV as a host-genetic factor for the modulation of the diversity and composition of the fecal microbiota in pigs. IMPORTANCE A better understanding of the environmental and host factors modulating gut microbiomes is a topic of greatest interest. Recent evidence suggests that genetic variation in the pig genome partially controls the composition of porcine gut microbiota. However, since previous studies have been focused on the association between single nucleotide polymorphisms and the fecal microbiota, little is known about the relationship between other sources of genetic variation, like the structural variants and microbial traits. Here, we identified, experimentally validated, and replicated in an independent population a positive link between the gain of copies of ABCC2-DNMBP loci and the diversity and composition of pig fecal microbiota. Our results advise the relevance of considering the role of host-genome structural variants as putative modulators of microbial ecosystems and open the possibility of implementing novel holobiont-based management strategies in breeding programs for the simultaneous improvement of microbial traits and host performance.