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The Farm Animal Genotype-Tissue Expression (FarmGTEx) project has been established to develop a public resource of genetic regulatory variants in livestock, which is essential for linking genetic polymorphisms to variation in phenotypes, helping fundamental biological discovery and exploitation in animal breeding and human biomedicine. Here we show results from the pilot phase of PigGTEx by processing 5,457 RNA-sequencing and 1,602 whole-genome sequencing samples passing quality control from pigs. We build a pig genotype imputation panel and associate millions of genetic variants with five types of transcriptomic phenotypes in 34 tissues. We evaluate tissue specificity of regulatory effects and elucidate molecular mechanisms of their action using multi-omics data. Leveraging this resource, we decipher regulatory mechanisms underlying 207 pig complex phenotypes and demonstrate the similarity of pigs to humans in gene expression and the genetic regulation behind complex phenotypes, supporting the importance of pigs as a human biomedical model.

The Farm Animal Genotype-Tissue Expression (FarmGTEx) project has been established to develop a public resource of genetic regulatory variants in livestock, which is essential for linking genetic polymorphisms to variation in phenotypes, helping fundamental biological discovery and exploitation in animal breeding and human biomedicine. Here we show results from the pilot phase of PigGTEx by processing 5,457 RNA-sequencing and 1,602 whole-genome sequencing samples passing quality control from pigs. We build a pig genotype imputation panel and associate millions of genetic variants with five types of transcriptomic phenotypes in 34 tissues. We evaluate tissue specificity of regulatory effects and elucidate molecular mechanisms of their action using multi-omics data. Leveraging this resource, we decipher regulatory mechanisms underlying 207 pig complex phenotypes and demonstrate the similarity of pigs to humans in gene expression and the genetic regulation behind complex phenotypes, supporting the importance of pigs as a human biomedical model

Additional file 1: QIIME2 pipeline used to process and combine the two 16S sequence datasets.

Additional file 2: Fig. S1. Evolution of heritability estimates along iterations in an holobiont model in CRP to illustrate convergence of the MCMC chain. Fig. S2. PCA for the individual batches and the merged dataset of CLR-transformed ASV abundances. Fig. S3. Distribution of reads per sample in the merged 16S sequencing data after quality control. Fig. S4. Distribution of CLR-transformed ASV abundances, averaged across samples. Fig. S5. Distribution of the ASV heritability estimates.

Additional file 3: Table S1. Correlation of the Euclidean distances between samples in each pair of datasets. Table S2. ASV responsible for 30% of the estimated b2 under the best predictive model for LYM_PHAGO_FITC.

Gut microbial composition plays an important role in numerous traits, including immune response. Integration of host genomic information with microbiome data is a natural step in the prediction of complex traits, although methods to optimize this are still largely unexplored. In this paper, we assess the impact of different modelling strategies on the predictive capacity for six porcine immunocompetence traits when both genotype and microbiota data are available.

In general, the relationship between the predicted functional consequences of missense mutations mapping to genes known to be involved in human diseases and the severity of disease manifestations is weak. In this study, we tested in pigs whether missense single nucleotide polymorphisms (SNPs), predicted to have consequences on the function of genes related to lipid metabolism are associated with lipid phenotypes. Association analysis demonstrated that nine out of 72 nominally associated SNPs were classified as "highly" or "very highly consistent" in silico-predicted functional mutations and did not show association with lipid traits expected to be affected by inactivation of the corresponding gene. Although the lack of endophenotypes and the limited sample size of certain genotypic classes might have limited to some extent the reach of the current study, our data indicate that present-day bioinformatic tools have a modest ability to predict the impact of missense mutations on complex phenotypes.

In general, the relationship between the predicted functional consequences of missense mutations mapping to genes known to be involved in human diseases and the severity of disease manifestations is weak. In this study, we tested in pigs whether missense single nucleotide polymorphisms (SNPs), predicted to have consequences on the function of genes related to lipid metabolism are associated with lipid phenotypes. Association analysis demonstrated that nine out of 72 nominally associated SNPs were classified as “highly” or “very highly consistent” in silico-predicted functional mutations and did not show association with lipid traits expected to be affected by inactivation of the corresponding gene. Although the lack of endophenotypes and the limited sample size of certain genotypic classes might have limited to some extent the reach of the current study, our data indicate that present-day bioinformatic tools have a modest ability to predict the impact of missense mutations on complex phenotypes.

Intense selection of modern pig breeds has resulted in genetic improvement of production traits while the performance of local pig breeds has remained lower. As local pig breeds have been bred in extensive systems, they have adapted to specific environmental conditions, resulting in a rich genotypic and phenotypic diversity. This study is based on European local pig breeds that have been genetically characterized using DNA-pool sequencing data and phenotypically characterized using breed level phenotypes related to stature, fatness, growth, and reproductive performance traits. These data were analyzed using a dedicated approach to detect signatures of selection linked to phenotypic traits in order to uncover potential candidate genes that may underlie adaptation to specific environments.

ROHs are long stretches of DNA homozygous at each polymorphic position. The proportion of genome covered by ROHs and their length are indicators of the level and origin of inbreeding. Frequent common ROHs within the same population define ROH islands and indicate hotspots of selection. In this work, we investigated ROHs in a total of 1131 pigs from 20 European local pig breeds and in three cosmopolitan breeds, genotyped with the GGP Porcine HD Genomic Profiler. plink software was used to identify ROHs. Size classes and genomic inbreeding parameters were evaluated. ROH islands were defined by evaluating different thresholds of homozygous SNP frequency. A functional overview of breed-specific ROH islands was obtained via over-representation analyses of GO biological processes. Mora Romagnola and Turopolje breeds had the largest proportions of genome covered with ROH (~1003 and ~955 Mb respectively), whereas Nero Siciliano and Sarda breeds had the lowest proportions (~207 and 247 Mb respectively). The highest proportion of long ROH (>16 Mb) was in Apulo-Calabrese, Mora Romagnola and Casertana. The largest number of ROH islands was identified in the Italian Landrace (n = 32), Cinta Senese (n = 26) and Lithuanian White Old Type (n = 22) breeds. Several ROH islands were in regions encompassing genes known to affect morphological traits. Comparative ROH structure analysis among breeds indicated the similar genetic structure of local breeds across Europe. This study contributed to understanding of the genetic history of the investigated pig breeds and provided information to manage these pig genetic resources.

Natural and artificial directional selection in cosmopolitan and autochthonous pig breeds and wild boars have shaped their genomes and resulted in a reservoir of animal genetic diversity. Signatures of selection are the result of these selection events that have contributed to the adaptation of breeds to different environments and production systems. In this study, we analysed the genome variability of 19 European autochthonous pig breeds (Alentejana, Bísara, Majorcan Black, Basque, Gascon, Apulo-Calabrese, Casertana, Cinta Senese, Mora Romagnola, Nero Siciliano, Sarda, Krškopolje pig, Black Slavonian, Turopolje, Moravka, Swallow-Bellied Mangalitsa, Schwäbisch-Hällisches Schwein, Lithuanian indigenous wattle and Lithuanian White old type) from nine countries, three European commercial breeds (Italian Large White, Italian Landrace and Italian Duroc), and European wild boars, by mining whole-genome sequencing data obtained by using a DNA-pool sequencing approach. Signatures of selection were identified by using a single-breed approach with two statistics [within-breed pooled heterozygosity (HP) and fixation index (FST)] and group-based FST approaches, which compare groups of breeds defined according to external traits and use/specialization/type.

In this study, we identified copy number variants (CNVs) in 19 European autochthonous pig breeds and in two commercial breeds (Italian Large White and Italian Duroc) that represent important genetic resources for this species. The genome of 725 pigs was sequenced using a breed-specific DNA pooling approach (30–35 animals per pool) obtaining an average depth per pool of 42×. This approach maximised CNV discovery as well as the related copy number states characterising, on average, the analysed breeds. By mining more than 17.5 billion reads, we identified a total of 9592 CNVs (~683 CNVs per breed) and 3710 CNV regions (CNVRs; 1.15% of the reference pig genome), with an average of 77 CNVRs per breed that were considered as private. A few CNVRs were analysed in more detail, together with other information derived from sequencing data. For example, the CNVR encompassing the KIT gene was associated with coat colour phenotypes in the analysed breeds, confirming the role of the multiple copies in determining breed-specific coat colours. The CNVR covering the MSRB3 gene was associated with ear size in most breeds. The CNVRs affecting the ELOVL6 and ZNF622 genes were private features observed in the Lithuanian Indigenous Wattle and in the Turopolje pig breeds respectively. Overall, the genome variability unravelled here can explain part of the genetic diversity among breeds and might contribute to explain their origin, history and adaptation to a variety of production systems.

In previous GWAS carried out in a Duroc commercial line (Lipgen population), we detected on pig chromosomes 3, 4 and 14 several QTL for gluteus medius muscle redness (GM a*), electric conductivity in the longissimus dorsi muscle (LD CE) and vaccenic acid content in the LD muscle (LD C18:1 n − 7), respectively. We have genotyped, in the Lipgen population, 19 SNPs mapping to 14 genes located within these QTL. Subsequently, association analyses have been performed. After correction for multiple testing, two SNPs in the TGFBRAP1 (rs321173745) and SELENOI (rs330820437) genes were associated with GM a*, whereas ACADSB (rs81449951) and GPR26 (rs343087568) genotypes displayed significant associations with LD vaccenic content. Moreover, the polymorphisms located at the ATP1A2 (rs344748241), ATP8B2 (rs81382410) and CREB3L4 (rs321278469 and rs330133789) genes showed significant associations with LD CE. We made a second round of association analyses including the SNPs mentioned above as well as other SNPs located in the chromosomes to which they map. After performing a correction for multiple testing, the only association that remained significant at the chromosome‐wide level was that between the ATP1A2 genotype and LD CE. From a functional point of view, this association is meaningful because this locus encodes a subunit of the Na+/K+‐ATPase responsible for maintaining an electrochemical gradient across the plasma membrane.

The contribution of microRNAs (miRNAs) to mRNA post-transcriptional regulation has often been explored by the post hoc selection of downregulated genes and determining whether they harbor binding sites for miRNAs of interest. This approach, however, does not discriminate whether these mRNAs are also downregulated at the transcriptional level. Here, we have characterized the transcriptional and post-transcriptional changes in mRNA expression in two porcine tissues: gluteus medius muscle of fasted and fed Duroc gilts and adipose tissue of lean and obese Duroc–Göttingen minipigs. Exon–intron split analysis of RNA-seq data allowed us to identify downregulated mRNAs with high post-transcriptional signals in fed or obese states, and we assessed whether they harbor binding sites for upregulated miRNAs in any of these two physiological states. We found 26 downregulated mRNAs with high post-transcriptional signals in the muscle of fed gilts and 21 of these were predicted targets of miRNAs upregulated in fed pigs. For adipose tissue, 44 downregulated mRNAs in obese minipigs displayed high post-transcriptional signals, and 25 of these were predicted targets of miRNAs upregulated in the obese state. These results suggest that the contribution of miRNAs to mRNA repression is more prominent in the skeletal muscle system. Finally, we identified several genes that may play relevant roles in the energy homeostasis of the pig skeletal muscle (DKK2 and PDK4) and adipose (SESN3 and ESRRG) tissues. By differentiating transcriptional from post-transcriptional changes in mRNA expression, exon–intron split analysis provides a valuable view of the regulation of gene expression, complementary to canonical differential expression analyses.

The contribution of microRNAs (miRNAs) to mRNA post-transcriptional regulation has often been explored by the post hoc selection of downregulated genes and determining whether they harbor binding sites for miRNAs of interest. This approach, however, does not discriminate whether these mRNAs are also downregulated at the transcriptional level. Here, we have characterized the transcriptional and post-transcriptional changes in mRNA expression in two porcine tissues: gluteus medius muscle of fasted and fed Duroc gilts and adipose tissue of lean and obese Duroc–Göttingen minipigs. Exon–intron split analysis of RNA-seq data allowed us to identify downregulated mRNAs with high post-transcriptional signals in fed or obese states, and we assessed whether they harbor binding sites for upregulated miRNAs in any of these two physiological states. We found 26 downregulated mRNAs with high post-transcriptional signals in the muscle of fed gilts and 21 of these were predicted targets of miRNAs upregulated in fed pigs. For adipose tissue, 44 downregulated mRNAs in obese minipigs displayed high post-transcriptional signals, and 25 of these were predicted targets of miRNAs upregulated in the obese state. These results suggest that the contribution of miRNAs to mRNA repression is more prominent in the skeletal muscle system. Finally, we identified several genes that may play relevant roles in the energy homeostasis of the pig skeletal muscle (DKK2 and PDK4) and adipose (SESN3 and ESRRG) tissues. By differentiating transcriptional from post-transcriptional changes in mRNA expression, exon–intron split analysis provides a valuable view of the regulation of gene expression, complementary to canonical differential expression analyses.

Preserving diversity of indigenous pig (Sus scrofa) breeds is a key factor to (i) sustain the pork chain (both at local and global scales) including the production of high-quality branded products, (ii) enrich the animal biobanking and (iii) progress conservation policies. Single nucleotide polymorphism (SNP) chips offer the opportunity for whole-genome comparisons among individuals and breeds. Animals from twenty European local pigs breeds, reared in nine countries (Croatia: Black Slavonian, Turopolje; France: Basque, Gascon; Germany: Schwabisch-Hällisches Schwein; Italy: Apulo Calabrese, Casertana, Cinta Senese, Mora Romagnola, Nero Siciliano, Sarda; Lithuania: Indigenous Wattle, White Old Type; Portugal: Alentejana, Bísara; Serbia: Moravka, Swallow-Bellied Mangalitsa; Slovenia: Krškopolje pig; Spain: Iberian, Majorcan Black), and three commercial breeds (Duroc, Landrace and Large White) were sampled and genotyped with the GeneSeek Genomic Profiler (GGP) 70 K HD porcine genotyping chip. A dataset of 51 Wild Boars from nine countries was also added, summing up to 1186 pigs (~ 49 pigs/breed). The aim was to: (i) investigate individual admixture ancestries and (ii) assess breed traceability via discriminant analysis on principal components (DAPC). Albeit the mosaic of shared ancestries found for Nero Siciliano, Sarda and Moravka, admixture analysis indicated independent evolvement for the rest of the breeds. High prediction accuracy of DAPC mark SNP data as a reliable solution for the traceability of breed-specific pig products.

Preserving diversity of indigenous pig (Sus scrofa) breeds is a key factor to (i) sustain the pork chain (both at local and global scales) including the production of high-quality branded products, (ii) enrich the animal biobanking and (iii) progress conservation policies. Single nucleotide polymorphism (SNP) chips ofer the opportunity for whole-genome comparisons among individuals and breeds. Animals from twenty European local pigs breeds, reared in nine countries (Croatia: Black Slavonian, Turopolje; France: Basque, Gascon; Germany: Schwabisch-Hällisches Schwein; Italy: Apulo Calabrese, Casertana, Cinta Senese, Mora Romagnola, Nero Siciliano, Sarda; Lithuania: Indigenous Wattle, White Old Type; Portugal: Alentejana, Bísara; Serbia: Moravka, Swallow-Bellied Mangalitsa; Slovenia: Krškopolje pig; Spain: Iberian, Majorcan Black), and three commercial breeds (Duroc, Landrace and Large White) were sampled and genotyped with the GeneSeek Genomic Profler (GGP) 70 K HD porcine genotyping chip. A dataset of 51 Wild Boars from nine countries was also added, summing up to 1186 pigs (~49 pigs/ breed). The aim was to: (i) investigate individual admixture ancestries and (ii) assess breed traceability via discriminant analysis on principal components (DAPC). Albeit the mosaic of shared ancestries found for Nero Siciliano, Sarda and Moravka, admixture analysis indicated independent evolvement

Coronaviruses silently circulate in human and animal populations, causing mild to severe diseases. Therefore, livestock are important components of a “One Health” perspective aimed to control these viral infections. However, at present there is no example that considers pig genetic resources in this context. In this study, we investigated the variability of four genes (ACE2, ANPEP and DPP4 encoding for host receptors of the viral spike proteins and TMPRSS2 encoding for a host proteinase) in 23 European (19 autochthonous and three commercial breeds and one wild boar population) and two Asian Sus scrofa populations. A total of 2229 variants were identified in the four candidate genes: 26% of them were not previously described; 29 variants affected the protein sequence and might potentially interact with the infection mechanisms. The results coming from this work are a first step towards a “One Health” perspective that should consider conservation programs of pig genetic resources with twofold objectives: (i) genetic resources could be reservoirs of host gene variability useful to design selection programs to increase resistance to coronaviruses; (ii) the described variability in genes involved in coronavirus infections across many different pig populations might be part of a risk assessment including pig genetic resources

The gut microbiota influences host performance playing a relevant role in homeostasis and function of the immune system. The aim of the present work was to identify microbial signatures linked to immunity traits and to characterize the contribution of host-genome and gut microbiota to the immunocompetence in healthy pigs. To achieve this goal, we undertook a combination of network, mixed model and microbial-wide association studies (MWAS) for 21 immunity traits and the relative abundance of gut bacterial communities in 389 pigs genotyped for 70K SNPs. The heritability (h 2 ; proportion of phenotypic variance explained by the host genetics) and microbiability (m 2 ; proportion of variance explained by the microbial composition) showed similar values for most of the analyzed immunity traits, except for both IgM and IgG in plasma that was dominated by the host genetics, and the haptoglobin in serum which was the trait with larger m 2 (0.275) compared to h 2 (0.138). Results from the MWAS suggested a polymicrobial nature of the immunocompetence in pigs and revealed associations between pigs gut microbiota composition and 15 of the analyzed traits. The lymphocytes phagocytic capacity (quantified as mean fluorescence) and the total number of monocytes in blood were the traits associated with the largest number of taxa (6 taxa). Among the associations identified by MWAS, 30% were confirmed by an information theory network approach. The strongest confirmed associations were between Fibrobacter and phagocytic capacity of lymphocytes (r = 0.37), followed by correlations between Streptococcus and the percentage of phagocytic lymphocytes (r = -0.34) and between Megasphaera and serum concentration of haptoglobin (r = 0.26). In the interaction network, Streptococcus and percentage of phagocytic lymphocytes were the keystone bacterial and immune-trait, respectively. Overall, our findings reveal an important connection between gut microbiota composition and immunity traits in pigs

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. 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. 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 predi

ROHs are long stretches of DNA homozygous at each polymorphic position. The proportion of genome covered by ROHs and their length are indicators of the level and origin of inbreeding. Frequent common ROHs within the same population define ROH islands and indicate hotspots of selection. In this work, we investigated ROHs in a total of 1131 pigs from 20 European local pig breeds and in three cosmopolitan breeds, genotyped with the GGP Porcine HD Genomic Profiler. PLINK software was used to identify ROHs. Size classes and genomic inbreeding parameters were evaluated. ROH islands were defined by evaluating different thresholds of homozygous SNP frequency. A functional overview of breed-specific ROH islands was obtained via overrepresentation analyses of GO biological processes. Mora Romagnola and Turopolje breeds had the largest proportions of genome covered with ROH (~1003 and ~955 Mb respectively), whereas Nero Siciliano and Sarda breeds had the lowest proportions (~207 and 247 Mb respectively). The highest proportion of long ROH (>16 Mb) was in Apulo-Calabrese, Mora Romagnola and Casertana. The largest number of ROH islands was identified in the Italian Landrace (n=32), Cinta Senese (n=26) and LithuanianWhite Old Type (n=22) breeds. Several ROH islands were in regions encompassing genes known to affect morphological traits. Comparative ROH structure analysis among breeds indicated the similar genetic structure of local breeds across Europe. This study contributed to understanding of the genetic history of the investigated pig breeds and provided information to manage these pig genetic resources.

Coronaviruses silently circulate in human and animal populations, causing mild to severe diseases. Therefore, livestock are important components of a "One Health" perspective aimed to control these viral infections. However, at present there is no example that considers pig genetic resources in this context. In this study, we investigated the variability of four genes (ACE2, ANPEP and DPP4 encoding for host receptors of the viral spike proteins and TMPRSS2 encoding for a host proteinase) in 23 European (19 autochthonous and three commercial breeds and one wild boar population) and two Asian Sus scrofa populations. A total of 2229 variants were identified in the four candidate genes: 26% of them were not previously described; 29 variants affected the protein sequence and might potentially interact with the infection mechanisms. The results coming from this work are a first step towards a "One Health" perspective that should consider conservation programs of pig genetic resources with twofold objectives: (i) genetic resources could be reservoirs of host gene variability useful to design selection programs to increase resistance to coronaviruses; (ii) the described variability in genes involved in coronavirus infections across many different pig populations might be part of a risk assessment including pig genetic resources.

Coronaviruses silently circulate in human and animal populations, causing mild to severe diseases. Therefore, livestock are important components of a “One Health” perspective aimed to control these viral infections. However, at present there is no example that considers pig genetic resources in this context. In this study, we investigated the variability of four genes (ACE2, ANPEP and DPP4 encoding for host receptors of the viral spike proteins and TMPRSS2 encoding for a host proteinase) in 23 European (19 autochthonous and three commercial breeds and one wild boar population) and two Asian Sus scrofa populations. A total of 2229 variants were identified in the four candidate genes: 26% of them were not previously described; 29 variants affected the protein sequence and might potentially interact with the infection mechanisms. The results coming from this work are a first step towards a “One Health” perspective that should consider conservation programs of pig genetic resources with twofold objectives: (i) genetic resources could be reservoirs of host gene variability useful to design selection programs to increase resistance to coronaviruses; (ii) the described variability in genes involved in coronavirus infections across many different pig populations might be part of a risk assessment including pig genetic resources

[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.

[Background]: Analyses of gut microbiome composition in livestock species have shown its potential to contribute to the regulation of complex phenotypes. However, little is known about the host genetic control over the gut microbial communities. In pigs, previous studies are based on classical “single-gene-single-trait” approaches and have evaluated the role of host genome controlling gut prokaryote and eukaryote communities separately., [Results]: In order to determine the ability of the host genome to control the diversity and composition of microbial communities in healthy pigs, we undertook genome-wide association studies (GWAS) for 39 microbial phenotypes that included 2 diversity indexes, and the relative abundance of 31 bacterial and six commensal protist genera in 390 pigs genotyped for 70 K SNPs. The GWAS results were processed through a 3-step analytical pipeline comprised of (1) association weight matrix; (2) regulatory impact factor; and (3) partial correlation and information theory. The inferred gene regulatory network comprised 3561 genes (within a 5 kb distance from a relevant SNP–P < 0.05) and 738,913 connections (SNP-to-SNP co-associations). Our findings highlight the complexity and polygenic nature of the pig gut microbial ecosystem. Prominent within the network were 5 regulators, PRDM15, STAT1, ssc-mir-371, SOX9 and RUNX2 which gathered 942, 607, 588, 284 and 273 connections, respectively. PRDM15 modulates the transcription of upstream regulators of WNT and MAPK-ERK signaling to safeguard naive pluripotency and regulates the production of Th1- and Th2-type immune response. The signal transducer STAT1 has long been associated with immune processes and was recently identified as a potential regulator of vaccine response to porcine reproductive and respiratory syndrome. The list of regulators was enriched for immune-related pathways, and the list of predicted targets includes candidate genes previously reported as associated with microbiota profile in pigs, mice and human, such as SLIT3, SLC39A8, NOS1, IL1R2, DAB1, TOX3, SPP1, THSD7B, ELF2, PIANP, A2ML1, and IFNAR1. Moreover, we show the existence of host-genetic variants jointly associated with the relative abundance of butyrate producer bacteria and host performance., [Conclusions]: Taken together, our results identified regulators, candidate genes, and mechanisms linked with microbiome modulation by the host. They further highlight the value of the proposed analytical pipeline to exploit pleiotropy and the crosstalk between bacteria and protists as significant contributors to host-microbiome interactions and identify genetic markers and candidate genes that can be incorporated in breeding program to improve host-performance and microbial traits.

The advent of next-generation sequencing technologies allowed relative quantification of microbiome communities and their spatial and temporal variation. In recent years, supervised learning (i.e., prediction of a phenotype of interest) from taxonomic abundances has become increasingly common in the microbiome field. However, a gap exists between supervised and classical unsupervised analyses, based on computing ecological dissimilarities for visualization or clustering. Despite this, both approaches face common challenges, like the compositional nature of next-generation sequencing data or the integration of the spatial and temporal dimensions. Here we propose a kernel framework to place on a common ground the unsupervised and supervised microbiome analyses, including the retrieval of microbial signatures (taxa importances). We define two compositional kernels (Aitchison-RBF and compositional linear) and discuss how to transform non-compositional beta-dissimilarity measures into kernels. Spatial data is integrated with multiple kernel learning, while longitudinal data is evaluated by specific kernels. We illustrate our framework through a single point soil dataset, a human dataset with a spatial component, and a previously unpublished longitudinal dataset concerning pig production. The proposed framework and the case studies are freely available in the kernInt package at https://github.com/elies-ramon/kernInt.

The paper reports the results of a survey on the demographic and phenotypic characterization of 20 European local pig breeds involved in the H2020 TREASURE project including information on: demographic parameters, main morphological features, reproductive information, additional information collected at herd-level (i.e. temperament, holding, mating practices), origins and development of the breeds. Almost all the breeds (18 out of 20) possess a herd book even if the starting year is highly variable (from 1980 to 2006). Number of breeding females ranged from 24 (Moravka) to over 200,000 heads (Ibérico). Male/female ratio varied greatly with the highest values for the Italian breeds probably due to the different policy of animal recording. Almost all the breeds undergo a conservation program whereas really few are interested by other conservation techniques and for less than five breeds data related to effective number and inbreeding coefficient are easily available. Average values for teat number, litter size and weaned piglets are 12, 8 and 6 respectively with a great potential for their improvement. The depicted scenario is highly diversified and the data collected represent the starting point for the achievement of a collective trademark under the umbrella of the TREASURE project.

In this study, we identified copy number variants (CNVs) in 19 European autochthonous pig breeds and in two commercial breeds (Italian Large White and Italian Duroc) that represent important genetic resources for this species. The genome of 725 pigs was sequenced using a breed-specific DNA pooling approach (30-35 animals per pool) obtaining an average depth per pool of 42x. This approach maximised CNV discovery as well as the related copy number states characterising, on average, the analysed breeds. By mining more than 17.5 billion reads, we identified a total of 9592 CNVs (similar to 683 CNVs per breed) and 3710 CNV regions (CNVRs; 1.15% of the reference pig genome), with an average of 77 CNVRs per breed that were considered as private. A few CNVRs were analysed in more detail, together with other information derived from sequencing data. For example, the CNVR encompassing the KIT gene was associated with coat colour phenotypes in the analysed breeds, confirming the role of the multiple copies in determining breed-specific coat colours. The CNVR covering the MSRB3 gene was associated with ear size in most breeds. The CNVRs affecting the ELOVL6 and ZNF622 genes were private features observed in the Lithuanian Indigenous Wattle and in the Turopolje pig breeds respectively. Overall, the genome variability unravelled here can explain part of the genetic diversity among breeds and might contribute to explain their origin, history and adaptation to a variety of production systems.

Despite the broad variety of available microRNA (miRNA) prediction tools, their application to the discovery and annotation of novel miRNA genes in domestic species is still limited. In this study we designed a comprehensive pipeline (eMIRNA) for miRNA identification in the yet poorly annotated porcine genome and demonstrated the usefulness of implementing a motif search positional refinement strategy for the accurate determination of precursor miRNA boundaries. The small RNA fraction from gluteus medius skeletal muscle of 48 Duroc gilts was sequenced and used for the prediction of novel miRNA loci. Additionally, we selected the human miRNA annotation for a homology-based search of porcine miRNAs with orthologous genes in the human genome. A total of 20 novel expressed miRNAs were identified in the porcine muscle transcriptome and 27 additional novel porcine miRNAs were also detected by homology-based search using the human miRNA annotation. The existence of three selected novel miRNAs (ssc-miR-483, ssc-miR484 and ssc-miR-200a) was further confirmed by reverse transcription quantitative real-time PCR analyses in the muscle and liver tissues of Göttingen minipigs. In summary, the eMIRNA pipeline presented in the current work allowed us to expand the catalogue of porcine miRNAs and showed better performance than other commonly used miRNA prediction approaches. More importantly, the flexibility of our pipeline makes possible its application in other yet poorly annotated non-model species.

[Background]: The role of non-coding RNAs in the porcine muscle metabolism is poorly understood, with few studies investigating their expression patterns in response to nutrient supply. Therefore, we aimed to investigate the changes in microRNAs (miRNAs), long intergenic non-coding RNAs (lincRNAs) and mRNAs muscle expression before and after food intake., [Results]: We measured the miRNA, lincRNA and mRNA expression levels in the gluteus medius muscle of 12 gilts in a fasting condition (AL-T0) and 24 gilts fed ad libitum during either 5 h. (AL-T1, N = 12) or 7 h. (AL-T2, N = 12) prior to slaughter. The small RNA fraction was extracted from muscle samples retrieved from the 36 gilts and sequenced, whereas lincRNA and mRNA expression data were already available. In terms of mean and variance, the expression profiles of miRNAs and lincRNAs in the porcine muscle were quite different than those of mRNAs. Food intake induced the differential expression of 149 (AL-T0/AL-T1) and 435 (AL-T0/AL-T2) mRNAs, 6 (AL-T0/AL-T1) and 28 (AL-T0/AL-T2) miRNAs and none lincRNAs, while the number of differentially dispersed genes was much lower. Among the set of differentially expressed miRNAs, we identified ssc-miR-148a-3p, ssc-miR-22-3p and ssc-miR-1, which play key roles in the regulation of glucose and lipid metabolism. Besides, co-expression network analyses revealed several miRNAs that putatively interact with mRNAs playing key metabolic roles and that also showed differential expression before and after feeding. One case example was represented by seven miRNAs (ssc-miR-148a-3p, ssc-miR-151-3p, ssc-miR-30a-3p, ssc-miR-30e-3p, ssc-miR-421-5p, ssc-miR-493-5p and ssc-miR-503) which putatively interact with the PDK4 mRNA, one of the master regulators of glucose utilization and fatty acid oxidation., [Conclusions]: As a whole, our results evidence that microRNAs are likely to play an important role in the porcine skeletal muscle metabolic adaptation to nutrient availability.

The sequencing of the pig genome revealed the existence of homozygous individuals for a nonsense mutation in the argininosuccinate synthase 1 (ASS1) gene (rs81212146, c.944T>A, L315X). Paradoxically, an AA homozygous genotype for this polymorphism is expected to abolish the function of the ASS1 enzyme that participates in the urea cycle, leading to citrullinemia, hyperammonemia, coma and death. Sequencing of five Duroc boars that sired a population of 350 Duroc barrows revealed the segregation of the c.944T>A polymorphism, so we aimed to investigate its phenotypic consequences. Genotyping of this mutation in the 350 Duroc barrows revealed the existence of seven individuals homozygous (AA) for the nonsense mutation. These AA pigs had a normal weight despite the fact that mild citrullinemia often involves impaired growth. Sequencing of the region surrounding the mutation in TT, TA and AA individuals revealed that the A substitution in the second position of the codon (c.944T>A) is in complete linkage disequilibrium with a C replacement (c.943T>C) in the first position of the codon. This second mutation would compensate for the potentially damaging effect of the c.944T>A replacement. In fact, this is the most probable reason why pigs with homozygous AA genotypes at the 944 site of the ASS1 coding region are alive. Our results illustrate the complexities of predicting the consequences of nonsense mutations on gene function and phenotypes, not only because of annotation issues but also owing to the existence of genetic mechanisms that sometimes limit the penetrance of highly harmful mutations.

Despite the broad variety of available microRNA (miRNA) prediction tools, their application to the discovery and annotation of novel miRNA genes in domestic species is still limited. In this study we designed a comprehensive pipeline (eMIRNA) for miRNA identification in the yet poorly annotated porcine genome and demonstrated the usefulness of implementing a motif search positional refinement strategy for the accurate determination of precursor miRNA boundaries. The small RNA fraction from gluteus medius skeletal muscle of 48 Duroc gilts was sequenced and used for the prediction of novel miRNA loci. Additionally, we selected the human miRNA annotation for a homology-based search of porcine miRNAs with orthologous genes in the human genome. A total of 20 novel expressed miRNAs were identified in the porcine muscle transcriptome and 27 additional novel porcine miRNAs were also detected by homology-based search using the human miRNA annotation. The existence of three selected novel miRNAs (ssc-miR-483, ssc-miR484 and ssc-miR-200a) was further confirmed by reverse transcription quantitative real-time PCR analyses in the muscle and liver tissues of Göttingen minipigs. In summary, the eMIRNA pipeline presented in the current work allowed us to expand the catalogue of porcine miRNAs and showed better performance than other commonly used miRNA prediction approaches. More importantly, the flexibility of our pipeline makes possible its application in other yet poorly annotated non-model species.

In this study, we identified copy number variants (CNVs) in 19 European autochthonous pig breeds and in two commercial breeds (Italian Large White and Italian Duroc) that represent important genetic resources for this species. The genome of 725 pigs was sequenced using a breed-specific DNA pooling approach (30-35 animals per pool) obtaining an average depth per pool of 42x. This approach maximised CNV discovery as well as the related copy number states characterising, on average, the analysed breeds. By mining more than 17.5 billion reads, we identified a total of 9592 CNVs (similar to 683 CNVs per breed) and 3710 CNV regions (CNVRs; 1.15% of the reference pig genome), with an average of 77 CNVRs per breed that were considered as private. A few CNVRs were analysed in more detail, together with other information derived from sequencing data. For example, the CNVR encompassing the KIT gene was associated with coat colour phenotypes in the analysed breeds, confirming the role of the multiple copies in determining breed-specific coat colours. The CNVR covering the MSRB3 gene was associated with ear size in most breeds. The CNVRs affecting the ELOVL6 and ZNF622 genes were private features observed in the Lithuanian Indigenous Wattle and in the Turopolje pig breeds respectively. Overall, the genome variability unravelled here can explain part of the genetic diversity among breeds and might contribute to explain their origin, history and adaptation to a variety of production systems.

BACKGROUND: Natural and artificial directional selection in cosmopolitan and autochthonous pig breeds and wild boars have shaped their genomes and resulted in a reservoir of animal genetic diversity. Signatures of selection are the result of these selection events that have contributed to the adaptation of breeds to different environments and production systems. In this study, we analysed the genome variability of 19 European autochthonous pig breeds (Alentejana, Bisara, Majorcan Black, Basque, Gascon, Apulo-Calabrese, Casertana, Cinta Senese, Mora Romagnola, Nero Siciliano, Sarda, Krskopolje pig, Black Slavonian, Turopolje, Moravka, Swallow-Bellied Mangalitsa, Schwabisch-Hallisches Schwein, Lithuanian indigenous wattle and Lithuanian White old type) from nine countries, three European commercial breeds (Italian Large White, Italian Landrace and Italian Duroc), and European wild boars, by mining whole-genome sequencing data obtained by using a DNA-pool sequencing approach. Signatures of selection were identified by using a single-breed approach with two statistics [within-breed pooled heterozygosity (H-P) and fixation index (F-ST)] and group-based F(ST)approaches, which compare groups of breeds defined according to external traits and use/specialization/type. Results We detected more than 22 million single nucleotide polymorphisms (SNPs) across the 23 compared populations and identified 359 chromosome regions showing signatures of selection. These regions harbour genes that are already known or new genes that are under selection and relevant for the domestication process in this species, and that affect several morphological and physiological traits (e.g. coat colours and patterns, body size, number of vertebrae and teats, ear size and conformation, reproductive traits, growth and fat deposition traits). Wild boar related signatures of selection were detected across all the genome of several autochthonous breeds, which suggests that crossbreeding (accidental or deli

Genetic characterization of local breeds is essential to preserve their genomic variability, to advance conservation policies and to contribute to their promotion and sustainability. Genomic diversity of twenty European local pig breeds and a small sample of Spanish wild pigs was assessed using high density SNP chips. A total of 992 DNA samples were analyzed with the GeneSeek Genomic Profiler (GGP) 70 K HD porcine genotyping chip. Genotype data was employed to compute genetic diversity, population differentiation and structure, genetic distances, linkage disequilibrium and effective population size. Our results point out several breeds, such as Turopolje, Apulo Calabrese, Casertana, Mora Romagnola and Lithuanian indigenous wattle, having the lowest genetic diversity, supported by low heterozygosity and very small effective population size, demonstrating the need of enhanced conservation strategies. Principal components analysis showed the clustering of the individuals of the same breed, with few breeds being clearly isolated from the rest. Several breeds were partially overlapped, suggesting genetic closeness, which was particularly marked in the case of Iberian and Alentejana breeds. Spanish wild boar was also narrowly related to other western populations, in agreement with recurrent admixture between wild and domestic animals. We also searched across the genome for loci under diversifying selection based on F-S(T) outlier tests. Candidate genes that may underlie differences in adaptation to specific environments and productive systems and phenotypic traits were detected in potentially selected genomic regions.

[Background]: Feed efficiency (FE) has a major impact on the economic sustainability of pig production. We used a systems-based approach that integrates single nucleotide polymorphism (SNP) co-association and gene-expression data to identify candidate genes, biological pathways, and potential predictors of FE in a Duroc pig population., [Results]: We applied an association weight matrix (AWM) approach to analyse the results from genome-wide association studies (GWAS) for nine FE associated and production traits using 31K SNPs by defining residual feed intake (RFI) as the target phenotype. The resulting co-association network was formed by 829 SNPs. Additive effects of this SNP panel explained 61% of the phenotypic variance of RFI, and the resulting phenotype prediction accuracy estimated by cross-validation was 0.65 (vs. 0.20 using pedigree-based best linear unbiased prediction and 0.12 using the 31K SNPs). Sixty-eight transcription factor (TF) genes were identified in the co-association network; based on the lossless approach, the putative main regulators were COPS5, GTF2H5, RUNX1, HDAC4, ESR1, USP16, SMARCA2 and GTF2F2. Furthermore, gene expression data of the gluteus medius muscle was explored through differential expression and multivariate analyses. A list of candidate genes showing functional and/or structural associations with FE was elaborated based on results from both AWM and gene expression analyses, and included the aforementioned TF genes and other ones that have key roles in metabolism, e.g. ESRRG, RXRG, PPARGC1A, TCF7L2, LHX4, MAML2, NFATC3, NFKBIZ, TCEA1, CDCA7L, LZTFL1 or CBFB. The most enriched biological pathways in this list were associated with behaviour, immunity, nervous system, and neurotransmitters, including melatonin, glutamate receptor, and gustation pathways. Finally, an expression GWAS allowed identifying 269 SNPs associated with the candidate genes’ expression (eSNPs). Addition of these eSNPs to the AWM panel of 829 SNPs did not improve the accuracy of genomic predictions., [Conclusions]: Candidate genes that have a direct or indirect effect on FE-related traits belong to various biological processes that are mainly related to immunity, behaviour, energy metabolism, and the nervous system. The pituitary gland, hypothalamus and thyroid axis, and estrogen signalling play fundamental roles in the regulation of FE in pigs. The 829 selected SNPs explained 61% of the phenotypic variance of RFI, which constitutes a promising perspective for applying genetic selection on FE relying on molecular-based prediction.

The genetic factors determining the phenotypic variation of porcine fatness phenotypes are still largely unknown. We investigated whether the polymorphism of eight genes (MIGA2, CRY2, NPAS2, CIART, ARNTL2, PER1, PER2 and PCK1), which display differential expression in the skeletal muscle of fasted and fed sows, is associated with the variation of lipid and mRNA expression phenotypes in Duroc pigs. The performance of an association analysis with the GEMMA software demonstrated that the rs330779504 SNP in the MIGA2 gene is associated with LDL concentration at 190 days (LDL2, corrected P-value = 0.057). Moreover, the rs320439526 SNP of the CRY2 gene displayed a significant association with stearic acid content in the longissimus dorsi muscle (LD C18:0, corrected P-value = 0.015). Both SNPs were also associated with the mRNA levels of the corresponding genes in the gluteus medius skeletal muscle. From a biological perspective these results are meaningful because MIGA2 protein plays an essential role in mitochondrial fusion, a process tightly connected with the energy status of the cell, while CRY2 is a fundamental component of the circadian clock. However, inclusion of these two SNPs in chromosome-wide association analyses demonstrated that they are not located at the peaks of significance for the two traits under study (LDL2 for rs330779504 and LD C18:0 for rs320439526), thus implying that these two SNPs do not have causal effects.

[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.

[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.

Genetic characterization of local breeds is essential to preserve their genomic variability, to advance conservation policies and to contribute to their promotion and sustainability. Genomic diversity of twenty European local pig breeds and a small sample of Spanish wild pigs was assessed using high density SNP chips. A total of 992 DNA samples were analyzed with the GeneSeek Genomic Profiler (GGP) 70 K HD porcine genotyping chip. Genotype data was employed to compute genetic diversity, population differentiation and structure, genetic distances, linkage disequilibrium and effective population size. Our results point out several breeds, such as Turopolje, Apulo Calabrese, Casertana, Mora Romagnola and Lithuanian indigenous wattle, having the lowest genetic diversity, supported by low heterozygosity and very small effective population size, demonstrating the need of enhanced conservation strategies. Principal components analysis showed the clustering of the individuals of the same breed, with few breeds being clearly isolated from the rest. Several breeds were partially overlapped, suggesting genetic closeness, which was particularly marked in the case of Iberian and Alentejana breeds. Spanish wild boar was also narrowly related to other western populations, in agreement with recurrent admixture between wild and domestic animals. We also searched across the genome for loci under diversifying selection based on F-S(T) outlier tests. Candidate genes that may underlie differences in adaptation to specific environments and productive systems and phenotypic traits were detected in potentially selected genomic regions.

In a previous study, we observed that circadian clock genes are differentially expressed in the skeletal muscle of fasting and fed sows. The goal of the current work was to investigate if these genes are also differentially expressed in tissues containing the central (hypothalamus) and peripheral (duodenum, dorsal fat, muscle, and liver) clocks. As animal material, we used 12 sows that fasted 12 h before slaughtering (T0) and 12 sows that were fed ad libitum 7 h prior slaughtering (T2). Tissue samples were collected immediately after slaughter and total RNA was subsequently extracted. The expression of the ARNTL, BHLHE40, CRY2, NPAS2, NR1D1, PER1, PER2, and SIK1 genes was measured by quantitative reverse transcription PCR. The numbers of clock genes showing differential expression before and after feeding varied depending on the tissue i.e., four in dorsal fat and duodenum, six in skeletal muscle, and seven in the liver. In contrast, none of the eight analysed genes displayed a significant differential expression in hypothalamus, the tissue where the central clock resides. This result supports that the differential expression of clock genes in the four tissues mentioned above is probably induced by nutrition and not by the central clock entrained by light. Moreover, we have observed that the NPAS2 and ARNTL genes display positive log2(FC) values in the five tissues under analysis, whilst the CRY2, PER1 (except dorsal fat) and PER2 (except hypothalamus) genes generally show negative log2(FC) values. Such result might be explained by the existence of a negative feedback loop between the ARNTL/NPAS2 and CRY/PER genes. Collectively, these results support that nutrition plays an important role in modulating the timing of porcine peripheral circadian clocks. Such regulation could be essential for coordinating the subsequent metabolic response to nutrient supply.

The aim of this work was to analyse the distribution of causal and candidate mutations associated to relevant productive traits in twenty local European pig breeds. Also, the potential of the SNP panel employed for elucidating the genetic structure and relationships among breeds was evaluated. Most relevant genes and mutations associated with pig morphological, productive, meat quality, reproductive and disease resistance traits were prioritized and analyzed in a maximum of 47 blood samples from each of the breeds (Alentejana, Apulo-Calabrese, Basque, Bísara, Majorcan Black, Black Slavonian (Crna slavonska), Casertana, Cinta Senese, Gascon, Iberian, Krškopolje (Krškopoljski), Lithuanian indigenous wattle, Lithuanian White Old Type, Mora Romagnola, Moravka, Nero Siciliano, Sarda, Schwäbisch-Hällisches Schwein (Swabian Hall pig), Swallow-Bellied Mangalitsa and Turopolje). We successfully analyzed allelic variation in 39 polymorphisms, located in 33 candidate genes. Results provide relevant information regarding genetic diversity and segregation of SNPs associated to production and quality traits. Coat color and morphological trait-genes that show low level of segregation, and fixed SNPs may be useful for traceability. On the other hand, we detected SNPs which may be useful for association studies as well as breeding programs. For instance, we observed predominance of alleles that might be unfavorable for disease resistance and boar taint in most breeds and segregation of many alleles involved in meat quality, fatness and growth traits. Overall, these findings provide a detailed catalogue of segregating candidate SNPs in 20 European local pig breeds that may be useful for traceability purposes, for association studies and for breeding schemes. Population genetic analyses based on these candidate genes are able to uncover some clues regarding the hidden genetic substructure of these populations, as the extreme genetic closeness between Iberian and Alentejana breeds and

In a previous study, we observed that circadian clock genes are differentially expressed in the skeletal muscle of fasting and fed sows. The goal of the current work was to investigate if these genes are also differentially expressed in tissues containing the central (hypothalamus) and peripheral (duodenum, dorsal fat, muscle, and liver) clocks. As animal material, we used 12 sows that fasted 12 h before slaughtering (T0) and 12 sows that were fed ad libitum 7 h prior slaughtering (T2). Tissue samples were collected immediately after slaughter and total RNA was subsequently extracted. The expression of the ARNTL, BHLHE40, CRY2, NPAS2, NR1D1, PER1, PER2, and SIK1 genes was measured by quantitative reverse transcription PCR. The numbers of clock genes showing differential expression before and after feeding varied depending on the tissue i.e., four in dorsal fat and duodenum, six in skeletal muscle, and seven in the liver. In contrast, none of the eight analysed genes displayed a significant differential expression in hypothalamus, the tissue where the central clock resides. This result supports that the differential expression of clock genes in the four tissues mentioned above is probably induced by nutrition and not by the central clock entrained by light. Moreover, we have observed that the NPAS2 and ARNTL genes display positive log2(FC) values in the five tissues under analysis, whilst the CRY2, PER1 (except dorsal fat) and PER2 (except hypothalamus) genes generally show negative log2(FC) values. Such result might be explained by the existence of a negative feedback loop between the ARNTL/NPAS2 and CRY/PER genes. Collectively, these results support that nutrition plays an important role in modulating the timing of porcine peripheral circadian clocks. Such regulation could be essential for coordinating the subsequent metabolic response to nutrient supply.

[Background]: The molecular basis of compensatory growth in monogastric animals has not yet been fully explored. Herewith, in this study we aim to determine changes in the pig skeletal muscle transcriptome profile during compensatory growth following a feed restriction period. A RNA-Seq experiment was performed with a total of 24 females belonging to a Duroc commercial line. Half of the animals received either a restricted (RE) or ad libitum (AL) diet during the first fattening period (60–125 d of age). After that, all gilts were fed ad libitum for a further ~30 d until the age of ~155 d, when animals were slaughtered and samples of gluteus medius muscle were harvested to perform RNA-Seq analyses and intramuscular fat content determination., [Results]: During the period following food restriction, RE animals re-fed ad libitum displayed compensatory growth, showed better feed conversion rate and tended to deposit more subcutaneous fat than AL fed animals. Animals were slaughtered in the phase of accelerated growth, when RE animals had not completely compensated the performance of AL group, showing lower live and carcass weights. At intramuscular level, RE gilts showed a higher content of polyunsaturated fatty acids during the compensatory growth phase. The comparison of RE and AL expression profiles allowed the identification of 86 (ǀlog2Fold-Changeǀ > 1, padj < 0.05) differentially expressed (DE) genes. A functional categorization of these DE genes identified AMPK Signaling as the most significantly enriched canonical pathway. This kinase plays a key role in the maintenance of energy homeostasis as well as in the activation of autophagy. Among the DE genes identified as components of AMPK Signaling pathway, five out of six genes were downregulated in RE pigs., [Conclusions]: Animals re-fed after a restriction period exhibited a less oxidative metabolic profile and catabolic processes in muscle than animals fed ad libitum. The downregulation of autophagy observed in the skeletal muscle of pigs undergoing compensatory growth may constitute a mechanism to increase muscle mass thus ensuring an accelerated growth rate. These results reveal that the downregulation of AMPK Signaling plays an important role in compensatory growth 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.

[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.