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.

Differential gene expression in ovaries of pregnant pigs with high and low prolificacy levels and identification of candidate genes for litter size.

Previous results from a genome scan in an F(2) Iberian × Meishan pig intercross showed several chromosome regions associated with litter size traits in this species. In order to identify candidate genes underlying these quantitative trait loci (QTL), we performed an ovary gene expression analysis during the sow's pregnancy. F(2) sows were ranked by their estimated breeding values for prolificacy: six sows with the highest estimated breeding value (EBV) (i.e., high prolificacy) and six sows with the lowest EBV (low prolificacy) were selected. Samples were hybridized using an Affymetrix GeneChip porcine genome array. Statistical analysis with a mixed model approach identified 221 differentially expressed probes, representing 189 genes. These genes were functionally annotated in order to identify genetic pathways overrepresented in this list. Among the functional groups most represented was, in first position, immune system response activation against external stimulus. The second group consisted of integrated genes that regulate maternal homeostasis by complement and coagulation cascades. A third group was involved in lipid and fatty acid enzymes of metabolic processes, which participate in the steroidogenesis pathway. In order to identify powerful candidate genes for prolificacy, the second approach of this study was to merge microarray data with the QTL positional information affecting litter size, previously detected in the same experimental cross. As a result, we have identified 27 differentially expressed genes colocalizing with QTL for litter size traits, which fulfill the biological, positional, and functional criteria.

Analysis of porcine MUC4 gene as a candidate gene for prolificacy QTL on SSC13 in an Iberian × Meishan F2 population.

BACKGROUND: Reproductive traits, such as prolificacy, are of great interest to the pig industry. Better understanding of their genetic architecture should help to increase the efficiency of pig productivity through the implementation of marker assisted selection (MAS) programmes. RESULTS: The Mucin 4 (MUC4) gene has been evaluated as a candidate gene for a prolificacy QTL described in an Iberian × Meishan (Ib × Me) F2 intercross. For association analyses, two previously described SNPs (DQ124298:g.243A>G and DQ124298:g.344A>G) were genotyped in 347 pigs from the Ib × Me population. QTL for the number of piglets born alive (NBA) and for the total number of piglets born (TNB) were confirmed on SSC13 at positions 44 cM and 51 cM, respectively. The MUC4 gene was successfully located within the confidence intervals of both QTL. Only DQ124298:g.344A>G MUC4 polymorphism was significantly associated with both NBA and TNB (P-value < 0.05) with favourable effects coming from the Meishan origin. MUC4 expression level was determined in F2 sows displaying extreme phenotypes for the number of embryos (NE) at 30-32 days of gestation. Differences in the uterine expression of MUC4 were found between high (NE ≥ 13) and low (NE ≤ 11) prolificacy sows. Overall, MUC4 expression in high prolificacy sows was almost two-fold increased compared with low prolificacy sows. CONCLUSIONS: Our data suggest that MUC4 could play an important role in the establishment of an optimal uterine environment that would increase embryonic survival during pig gestation.

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.

A bi-dimensional genome scan for prolificacy traits in pigs shows the existence of multiple epistatic QTL.

BACKGROUND: Prolificacy is the most important trait influencing the reproductive efficiency of pig production systems. The low heritability and sex-limited expression of prolificacy have hindered to some extent the improvement of this trait through artificial selection. Moreover, the relative contributions of additive, dominant and epistatic QTL to the genetic variance of pig prolificacy remain to be defined. In this work, we have undertaken this issue by performing one-dimensional and bi-dimensional genome scans for number of piglets born alive (NBA) and total number of piglets born (TNB) in a three generation Iberian by Meishan F(2) intercross. RESULTS: The one-dimensional genome scan for NBA and TNB revealed the existence of two genome-wide highly significant QTL located on SSC13 (P < 0.001) and SSC17 (P < 0.01) with effects on both traits. This relative paucity of significant results contrasted very strongly with the wide array of highly significant epistatic QTL that emerged in the bi-dimensional genome-wide scan analysis. As much as 18 epistatic QTL were found for NBA (four at P < 0.01 and five at P < 0.05) and TNB (three at P < 0.01 and six at P < 0.05), respectively. These epistatic QTL were distributed in multiple genomic regions, which covered 13 of the 18 pig autosomes, and they had small individual effects that ranged between 3 to 4% of the phenotypic variance. Different patterns of interactions (a x a, a x d, d x a and d x d) were found amongst the epistatic QTL pairs identified in the current work. CONCLUSIONS: The complex inheritance of prolificacy traits in pigs has been evidenced by identifying multiple additive (SSC13 and SSC17), dominant and epistatic QTL in an Iberian x Meishan F(2) intercross. Our results demonstrate that a significant fraction of the phenotypic variance of swine prolificacy traits can be attributed to first-order gene-by-gene interactions emphasizing that the phenotypic effects of alleles might be strongly modulated by the genetic background where they segregate.

Inbreeding depression load for litter size in Entrepelado and Retinto Iberian pig varieties1.

Individual-specific hidden inbreeding depression load (IDL) can be accounted for in livestock populations by appropriate best linear unbiased prediction approaches. This genetic effect has a recessive pattern and reveals when inherited in terms of identity-by-descent. Nevertheless, IDL inherits as a pure additive genetic background and can be selected using standard breeding values. The main target of this research was to evaluate IDL for litter size in 2 Iberian pig varieties (Entrepelado and Retinto) from a commercial breeding-stock. Analyses were performed on the total number of piglets born (both alive and dead) and used data from 3,200 (8.02 ± 0.04 piglets/litter) Entrepelado and 4,744 Retinto litters (8.40 ± 0.03 piglets/litter). Almost 50% of Entrepelado sows were inbred (1.7% to 25.0%), whereas this percentage reduced to 37.4% in the Retinto variety (0.2% to 25.0%). The analytical model was solved by Bayesian inference and accounted for 2 systematic effects (sow age and breed/variety of the artificial insemination boar), 2 permanent environmental effects (herd-year-season and sow), and 2 genetic effects (IDL and infinitesimal additive). In terms of posterior means (PM), additive genetic and IDL variances were similar in the Entrepelado variety (PM, 0.68 vs. 0.76 piglets2, respectively) and their 95% credibility intervals (95CI) overlapped, although without including zero (0.38 to 0.94 vs. 0.15 to 1.31 piglets2, respectively). The same pattern revealed in the Retinto variety, with IDL variance (PM, 0.41 piglets2; 95CI, 0.07 to 0.88 piglets2) slightly larger than the additive genetic variance (PM, 0.37 piglets2; 95CI, 0.16 to 0.59 piglets2). The relevance of IDL was also checked by a Bayes factor and the deviance information criterion, the model including this effect being clearly favored in both cases. Although the analysis assumed null genetic covariance between IDL and infinitesimal additive effects, a moderate negative correlation (-0.31) was suggested when plotting the PM of breeding values in the Entrepelado variety; a negative genetic trend for IDL was also revealed in this Iberian pig variety (-0.25 piglets for 100% inbred offspring of individuals born in 2014), whereas no trend was detected in Retinto breeding-stock. Those were the first estimates of IDL in a commercial livestock population, they giving evidence of a relevant genetic background with potential consequences on the reproductive performance of Iberian sows.

Mapping of quantitative trait loci for cholesterol, LDL, HDL, and triglyceride serum concentrations in pigs.

The fine mapping of polymorphisms influencing cholesterol (CT), triglyceride (TG), and lipoprotein serum levels in human and mouse has provided a wealth of knowledge about the complex genetic architecture of these traits. The extension of these genetic analyses to pigs would be of utmost importance since they constitute a valuable biological and clinical model for the study of coronary artery disease and myocardial infarction. In the present work, we performed a whole genome scan for serum lipid traits in a half-sib Duroc pig population of 350 individuals. Phenotypic registers included total CT, TG, and low (LDL)- and high (HDL)-density lipoprotein serum concentrations at 45 and 190 days of age. This approach allowed us to identify two genomewide significant quantitative trait loci (QTL) for HDL-to-LDL ratio at 45 days (SSC6, 84 cM) and for TG at 190 days (SSC4, 23 cM) as well as a number of chromosomewide significant QTL. The comparison of QTL locations at 45 and 190 days revealed a notable lack of concordance at these two time points, suggesting that the effects of these QTL are age specific. Moreover, we have observed a considerable level of correspondence among the locations of the most significant porcine lipid QTL and those identified in humans. This finding might suggest that, in mammals, diverse polymorphisms located in a common set of genes are involved in the genetic variation of serum lipid levels.

Association of CA repeat polymorphism at intron 1 of insulin-like growth factor (IGF-I) gene with circulating IGF-I concentration, growth, and fatness in swine.

Evidence is accumulating that intronic polymorphic cytosine-adenosine (CA) repeats may play a role in gene expression. In this work, we investigated whether a polymorphic CA short tandem repeat (STR) located at the first intron of the pig insulin-like growth factor I (IGF-I) gene influences plasma IGF-I concentration in pigs as well as phenotypic variation in growth and fatness traits. We measured plasma IGF-I levels at one to four time points from 35 to 215 days of age in 340 performance-tested Landrace and Duroc pigs previously genotyped for the IGF-I STR. Data were analyzed within breed with a linear mixed model with the number of CA repeats as a covariate. At least five alleles were segregating in each breed, differing in one to seven repeats. The results showed that in each breed, circulating IGF-I at 160 days of age increased with the length of the shortest allele, accounting for an average trend of 4.38 +/- 1.28 ng/ml of IGF-I per additional repeat (P = 0.001). Longer repeats were associated with early growth in Landrace boars (1.92 +/- 0.92 kg per CA at 160 days; P = 0.038) and with back fat thickness (-0.57 +/- 0.20 mm per CA; P = 0.005) and lean content (7.52 +/- 3.00 g/kg per CA at 105 kg; P = 0.013) adjusted for carcass weight in Duroc barrows, as expected from the effect of circulating IGF-I on these traits. The consistency of the results across populations supports the hypothesis that the length of the CA repeats at intron 1 of the IGF-I gene is associated with circulating IGF-I levels, and that this effect is not neutral with respect to growth and fatness.

Exploring alternative models for sex-linked quantitative trait loci in outbred populations: application to an iberian x landrace pig intercross.

We present a very flexible method that allows us to analyze X-linked quantitative trait loci (QTL) in crosses between outbred lines. The dosage compensation phenomenon is modeled explicitly in an identity-by-descent approach. A variety of models can be fitted, ranging from considering alternative fixed alleles within the founder breeds to a model where the only genetic variation is within breeds, as well as mixed models. Different genetic variances within each founder breed can be estimated. We illustrate the method with data from an F(2) cross between Iberian x Landrace pigs for intramuscular fat content and meat color component a*. The Iberian allele exhibited a strong overdominant effect for intramuscular fat in females. There was also limited evidence of one or more regions affecting color component a*. The analysis suggested that the QTL alleles were fixed in the Iberian founders, whereas there was some evidence of segregation in Landrace for the QTL affecting a* color component.

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.

Transcriptional Characterization of Porcine Leptin and Leptin Receptor Genes.

The leptin (LEP) and its receptor (LEPR) regulate food intake and energy balance through hypothalamic signaling. However, the LEP-LEPR axis seems to be more complex and its expression regulation has not been well described. In pigs, LEP and LEPR genes have been widely studied due to their relevance. Previous studies reported significant effects of SNPs located in both genes on growth and fatness traits. The aim of this study was to determine the expression profiles of LEP and LEPR across hypothalamic, adipose, hepatic and muscle tissues in Iberian x Landrace backcrossed pigs and to analyze the effects of gene variants on transcript abundance. To our knowledge, non porcine LEPR isoforms have been described rather than LEPRb. A short porcine LEPR isoform (LEPRa), that encodes a protein lacking the intracellular residues responsible of signal transduction, has been identified for the first time. The LEPRb isoform was only quantifiable in hypothalamus while LEPRa appeared widely expressed across tissues, but at higher levels in liver, suggesting that both isoforms would develop different roles. The unique LEP transcript showed expression in backfat and muscle. The effects of gene variants on transcript expression revealed interesting results. The LEPRc.1987C>T polymorphism showed opposite effects on LEPRb and LEPRa hypothalamic expression. In addition, one out of the 16 polymorphisms identified in the LEPR promoter region revealed high differential expression in hepatic LEPRa. These results suggest a LEPR isoform-specific regulation at tissue level. Conversely, non-differential expression of LEP conditional on the analyzed polymorphisms could be detected, indicating that its regulation is likely affected by other mechanisms rather than gene sequence variants. The present study has allowed a transcriptional characterization of LEP and LEPR isoforms on a range of tissues. Their expression patterns seem to indicate that both molecules develop peripheral roles apart from their known hypothalamic signal transduction function.

On growth, fatness, and form: a further look at porcine chromosome 4 in an Iberian x Landrace cross.

A crossed population between Iberian x Landrace pigs consisting of 321 F2, 87 F3, and 85 backcross individuals has been analyzed to refine the number and positions of quantitative trait loci (QTL) affecting shape, growth, fatness, and meat quality traits in SSC4. A multitrait multi-QTL approach has been used. Our results suggest that carcass length and shoulder weight are affected by two loci. The first one, close to the AFABP gene, has a very strong pleiotropic effect on fatness, whereas the second one, in the interval between S0073 and S0214, also affects live weight, although to a lesser extent. This latter QTL would correspond to the FAT1 locus described initially in pigs. It seems that SSC4's loci play an important role in redistributing total weight, and the Landrace allele increases shoulder weight and carcass length much more than ham or total weight. Furthermore, there is also strong evidence of additional loci influencing pH and color in more distant, telomeric positions.

Detection of QTL affecting fatty acid composition in the pig.

We present a QTL genome scan for fatty acid composition in pigs. An F2 cross between Iberian x Landrace pigs and a regression approach fitting the carcass weight as a covariate for QTL identification was used. Chromosomes (Chrs) 4, 6, 8, 10, and 12 showed highly significant effects. The Chr 4 QTL influenced the linoleic content and both the fatty acid double-bond index and peroxidability index. In Chr 6 we found significant associations with the double-bond index and the unsaturated index of fatty acids. Chr 8 showed clear effects on the percentages of palmitic and palmitoleic fatty acids as well as the average chain length of fatty acids. In Chr 10 we detected a significant QTL for the percentage of myristic fatty acid, with an F value that was slightly above the genomewide threshold. The percentage of linolenic fatty acid was affected by a region on Chr 12. A nearly significant QTL for the content of gadoleic fatty acid was also detected in Chr 12. We also analyzed the genomic QTL distribution by a regression model that fits the backfat thickness as a covariate. Some of the QTL that were detected in our analysis could not be detected when the data were corrected by backfat thickness. This work shows how critical the selection of a covariate can be in the interpretation of results. This is the first report of a genome scan detection of QTL directly affecting fatty acid composition in pigs.