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mir-33a and mir-33b are co-transcribed with the SREBF2 and SREBF1 transcription factors, respectively. The main role of SREBF1 is the regulation of genes involved in fatty acid metabolism, while SREBF2 regulates genes participating in cholesterol biosynthesis and uptake. Our objective was to study the expression of both miR-33a and miR-33b, together with their host SREBF genes, in liver, adipose tissue and muscle to better understand the role of miR-33a/b in the lipid metabolism of pigs. In our study, the expression of miR-33a, miR-33b and SREBF2 in liver, adipose tissue, and muscle was studied in 42 BC1_LD (25% Iberian x 75% Landrace backcross) pigs by RT-qPCR. In addition, the expression of in-silico predicted target genes and fatty acid composition traits were correlated with the miR-33a/b expression. We observed different tissue expression patterns for both miRNAs. In adipose tissue and muscle a high correlation between miR-33a and miR-33b expression was found, whereas a lower correlation was observed in liver. The expression analysis of in-silico predicted target-lipid related genes showed negative correlations between miR-33b and CPT1A expression in liver. Conversely, positive correlations between miR-33a and PPARGC1A and USF1 gene expression in liver were observed. Lastly, positive and negative correlations between miR-33a/b expression and saturated fatty acid (SFA) and polyunsaturated fatty acid (PUFA) content, respectively, were identified. Overall, our results suggested that both miRNAs are differentially regulated and have distinct functions in liver, in contrast to muscle and adipose tissue. Furthermore, the correlations between miR-33a/b expression both with the expression of in-silico predicted target-lipid related genes and with fatty acid composition, opens new avenues to explore the role of miR33a/b in the regulation of lipid metabolism., This work was supported by the Spanish Ministerio de Economía y Competitividad (MINECO) with project AGL2017-82641-R (https://portal.mineco.gob.es/es-es/Paginas/default.aspx) and the Fondo Europeo de Desarrollo Regional (FEDER, http://ec.europa.eu/regional_policy/es/funding/erdf/). LCM was financially supported by an FPI grant from the AGL2014-56369-C2 project. MB was funded with a “Ramón y Cajal” contract (RYC-2013-12573) from the Spanish Ministerio de Economía y Competitividad. DCP was funded by a “Formació i Contractació de Personal Investigador Novell” (FI-DGR) Ph.D grant from the Generalitat de Catalunya (ECO/1788/2014). MP was funded by an FPI grant from the AGL2017-82641-R project. We acknowledge the support of the Spanish Ministerio de Economía y Competitividad for the “Severo Ochoa Programme for Centres of Excellence in R&D” 2016-2019 (SEV-2015-0533) to the Centre for Research in Agricultural Genomics and the CERCA Programme / Generalitat de Catalunya.

Iberian wild goats (Capra pyrenaica, also known as Iberian ibex, Spanish ibex, and Spanish wild goat) underwent strong genetic bottlenecks during the 19th and 20th centuries due to overhunting and habitat destruction. From the 1970s to 1990s, augmentation translocations were frequently carried out to restock Iberian wild goat populations (very often with hunting purposes), but they were not systematically planned or recorded. On the other hand, recent data suggest the occurrence of hybridization events between Iberian wild goats and domestic goats (Capra hircus). Augmentation translocations and interspecific hybridization might have contributed to increase the diversity of Iberian wild goats. With the aim of investigating this issue, we have genotyped 118 Iberian wild goats from Tortosa-Beceite, Sierra Nevada, Muela de Cortes, Gredos, Batuecas, and Ordesa and Monte Perdido by using the Goat SNP50 BeadChip (Illumina). The analysis of genotypic data indicated that Iberian wild goat populations are strongly differentiated and display low diversity. Only three Iberian wild goats out from 118 show genomic signatures of mixed ancestry, a result consistent with a scenario in which past augmentation translocations have had a limited impact on the diversity of Iberian wild goats. Besides, we have detected eight Iberian wild goats from Tortosa-Beceite with signs of domestic goat introgression. Although rare, hybridization with domestic goats could become a potential threat to the genetic integrity of Iberian wild goats; hence, measures should be taken to avoid the presence of uncontrolled herds of domestic or feral goats in mountainous areas inhabited by this iconic wild ungulate., This research was funded by the European Regional Development Fund (FEDER)/Ministerio de Ciencia e Innovación—Agencia Estatal de Investigación/Project Reference grant: PID2019-105805RB-I00 and by the CERCA Programme/Generalitat de Catalunya. We acknowledge the support of the Spanish Ministry of Economy and Competitiveness for the Center of Excellence Severo Ochoa 2020–2023 (CEX2019-000902-S) grant awarded to the Centre for Research in Agricultural Genomics (CRAG). We also acknowledge José Folch, of the Centro de Investigación y Tecnología Agroalimentaria de Aragón for his help in the development of his research. MGLS was funded with a FPI Ph.D. grant from the Spanish Ministry of Education (BES-C-2017-079079). TFC was funded with a fellowship from the CAPES Foundation-Coordination of Improvement of Higher Education, Ministry of Education of the Federal Government of Brazil. EMS was funded with a FPU Ph.D. grant from the Spanish Ministry of Education (FPU15/01733). GM is a Serra Húnter fellow (professor) of the Generalitat de Catalunya. Thanks also to the CERCA Programme of the Generalitat de Catalunya for their support. Sample collection benefitted from the research grants CGL2012-40043-C02-01, CGL2012-40043-C02-02, and CGL2016-80543-P, also from the Spanish Ministry of Economy and Competitiveness.

The agouti signaling protein (ASIP) gene has a crucial role in pigmentation by encoding a protein that binds the melanocortin 1 receptor and stimulates the synthesis of pheomelanin rather than eumelanin. Several studies have suggested that an increased copy number of the ASIP gene might explain the white pigmentation of certain goat breeds, as previously demonstrated in sheep. In the current work, we have identified the segregation of the ASIP CNV in Murciano-Granadina (black or brown coat), Malagueña (brown, blond or white coat) and Saanen (white coat) goats with available Illumina Goat SNP50 BeadChip (Illumina Inc., San Diego, CA) data by using the PennCNV v1.0.5 and QuantiSNP v2 tools. This result shows that the ASIP CNV segregates in non-white breeds. To gain new insights into this issue, we have estimated the copy number of the ASIP gene in 83 goats from 8 breeds with different coloration patterns using a real-time quantitative PCR approach. Our results showed an increased ASIP copy number not only in Saanen (3.50 ± 0.23 copies relative to the calibrator) and white Malagueña (3.51 ± 0.51 copies) goats, but also in the Murciano-Granadina breed (3.33 ± 0.58 copies) as well as in blond/brown individuals from the Malagueña (3.58 ± 0.73 copies) breed. The number of ASIP copies was not significantly different in these four caprine populations (P > 0.05). Moreover, we did not observe a trend towards increased ASIP copy number in breeds with predominantly white colors, such as Maltese (2.85 ± 0.28 copies), Jonica (2.82 ± 0.39 copies) and Blanca de Rasquera (2.37 ± 0.33 copies). Our results, combined with recent findings demonstrating the high structural complexity of the ASIP locus, indicate that additional functional and expression studies should be performed in order to fully understand the role of ASIP structural variation in goat pigmentation., This study was funded by the European Regional Development Fund (FEDER)/Ministerio de Ciencia, Innovación y Universidades - Agencia Estatal de Investigación/Project Reference grant: AGL2016-76108-R and by the CERCA Programme/Generalitat de Catalunya. We also acknowledge the support of the Spanish Ministry of Economy and Competitivity for the Center of Excellence Severo Ochoa 2016–2019 (SEV-2015-0533) grant awarded to the Centre for Research in Agricultural Genomics (CRAG, Bellaterra, Spain). Dailu Guan was funded by a PhD fellowship from the China Scholarship Council (CSC). María Gracia Luigi-Sierra was funded with an FPI Ph.D. grant (BES-2017-079709) awarded by the Spanish Ministry of Economy and Competitivity.

Artificial selection is one of the major forces modifying the genetic composition of livestock populations. Identifying genes under selection could be useful to elucidate their impact on phenotypic variation. We aimed to identify genomic regions targeted by selection for dairy and pigmentation traits in Murciano-Granadina goats. Performance of a selection scan based on the integrated haplotype score test in a population of 1183 Murciano-Granadina goats resulted in the identification of 77 candidate genomic regions/SNPs. The most significant selective sweeps mapped to chromosomes 1 (69.86 Mb), 4 (41.80–49.95 Mb), 11 (65.74 Mb), 12 (31.24 and 52.51 Mb), 17 (34.76–37.67 Mb), 22 (31.75 Mb), and 26 (26.69–31.05 Mb). By using previously generated RNA-Seq data, we built a catalogue of 6414 genes that are differentially expressed across goat lactation (i.e. 78 days post-partum, early lactation; 216 days post-partum, late lactation; 285 days post-partum, dry period). Interestingly, 183 of these genes mapped to selective sweeps and several of them display functions related with lipid, protein, and carbohydrate metabolism, insulin signaling, cell proliferation, as well as mammary development and involution. Of particular interest are the CSN3 and CSN1S2 genes, which encode two major milk proteins. Additionally, we found three pigmentation genes (GLI3, MC1R, and MITF) co-localizing with selective sweeps. Performance of a genome-wide association study and Sanger sequencing and TaqMan genotyping experiments revealed that the c.801C>G (p.Cys267Trp) polymorphism in the melanocortin 1 receptor (MC1R) gene is the main determinant of the black (GG or GC genotypes) and brown (CC genotypes) colorations of Murciano-Granadina goats., This research was funded by the European Regional Development Fund (FEDER)/Ministerio de Ciencia e Innovación – Agencia Estatal de Investigación/Project Reference grant: PID2019-105805RB-I00 and by the CERCA Programme/Generalitat de Catalunya. We also acknowledge the support of the Spanish Ministry of Economy and Competitivity for the Center of Excellence Severo Ochoa 2020–2023 (CEX2019-000902-S) grant awarded to the Centre for Research in Agricultural Genomics (CRAG, Bellaterra, Spain). Dailu Guan was funded by a PhD fellowship from the China Scholarship Council (CSC). María Gracia Luigi-Sierra was funded with a fellowship Formación de Personal Investigador (BES-C-2017-079709) awarded by the Spanish Ministry of Economy and Competitivity.

Transmission Ratio Distortion (TRD), the uneven transmission of an allele from a parent to its offspring, can be caused by allelic differences affecting gametogenesis, fertilization or embryogenesis. However, TRD remains vaguely studied at a genomic scale. We sequenced the diploid and haploid genomes of three boars from leukocytes and spermatozoa at 50x to shed light into the genetic basis of spermatogenesis-caused Allelic Ratio Distortion (ARD). We first developed a Binomial model to identify ARD by simultaneously analysing all three males. This led to the identification of 55 ARD SNPs, most of which were animal-specific. We then evaluated ARD individually within each pig by a Fisher’s exact test and identified two shared genes (TOP3A and UNC5B) and four shared genomic regions harbouring distinct ARD SNPs in the three boars. The shared genomic regions contained candidate genes with functions related to spermatogenesis including AK7, ARID4B, BDKRB2, GSK3B, NID1, NSMCE1, PALB2, VRK1 and ZC3H13. Using the Fisher’s test, we also identified 378 genes containing variants with protein damaging potential in at least one boar, a high proportion of which, including FAM120B, TDRD15, JAM2 or AOX4 among others, are associated to spermatogenesis. Overall, our results show that sperm is subjected to ARD with variants associated to a wide variety of genes involved in different stages of spermatogenesis., This work was supported by the Spanish Ministry of Economy and Competitiveness (MINECO) under grant AGL2013-44978-R and grant AGL2017-86946-R and by the CERCA Programme/Generalitat de Catalunya. AGL2017-86946-R was also funded by the Spanish State Research Agency (AEI) and the European Regional Development Fund (ERDF). We thank the Agency for Management of University and Research Grants (AGAUR) of the Generalitat de Catalunya (Grant Numbers 2014 SGR 1528 and 2017 SGR 01060). We also acknowledge the support of the Spanish Ministry of Economy and Competitivity for the Center of Excellence Severo Ochoa 2016–2019 (Grant Number SEV-2015-0533) grant awarded to the Centre for Research in Agricultural Genomics (CRAG). MG acknowledges a Ph.D. studentship from MINECO (Grant Number BES-2014-070560)., We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).

Morphological traits are of great importance to dairy goat production given their effect on phenotypes of economic interest. However, their underlying genomic architecture has not yet been extensively characterized. Herein, we aimed to identify genomic regions associated with body, udder, and leg conformation traits recorded in 825 Murciano-Granadina goats. We genotyped this resource population using the GoatSNP50 BeadChip (Illumina Inc., San Diego, CA) and performed genome-wide association analyses using the GEMMA software. We found 2 genome-wide significant associations between markers rs268273468 [Capra hircus (CHI) 16:69617700] and rs268249346 (CHI 28:18321523) and medial suspensory ligament. In contrast, we did not detect any genome-wide significant associations for body and leg traits. Moreover, we found 12, 19, and 7 chromosome-wide significant associations for udder, body, and leg traits, respectively. Comparison of our data with previous studies revealed a low level of positional concordance between regions associated with morphological traits. In addition to technical factors, this lack of concordance could be due to a substantial level of genetic heterogeneity among breeds or to the strong polygenic background of morphological traits, which makes it difficult to detect genetic factors that have small phenotypic effects., This research was funded by the European Fund for Regional Development/Ministerio de Ciencia, Innovación y Universidades - Agencia Estatal de Investigación/ Project Reference (AGL2016-76108-R). We acknowledge the financial support from the Spanish Ministry of Economy and Competitiveness, through the “Severo Ochoa Programme for Centres of Excellence in R&D” 2016-2019 (SEV-2015-0533), and from the CERCA programme of the Generalitat de Catalunya. Emilio Mármol-Sánchez was funded with an FPU PhD grant awarded by the Spanish Ministry of Education (FPU15/01733). Maria Gracia Luigi-Sierra was funded with an FPI PhD grant from the Spanish Ministry of Economy and Competitivity (BES-2017-079709).

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., This work was supported by the Spanish Ministerio de Economía y Competitividad (MINECO) and the Fondo Europeo de Desarrollo Regional (FEDER) with Project References: AGL2014-56369-C2 and AGL2017-82641-R. D. Crespo-Piazuelo was funded by a “Formació i Contractació de Personal Investigador Novell” (FI-DGR) Ph.D grant from the Generalitat de Catalunya (ECO/1788/2014). L. Criado-Mesas was funded with a FPI grant from the AGL2014-56369-C2 project. M. Revilla was also funded by a FI-DGR (ECO/1639/2013). M. Ballester was financially supported by a “Ramón y Cajal” Contract (RYC-2013-12573) from the Spanish Ministerio de Economía y Competitividad. We acknowledge the support of the Spanish Ministerio de Economía y Competitividad for the “Severo Ochoa Programme for Centres of Excellence in R&D” 2016–2019 (SEV-2015-0533) to the Centre for Research in Agricultural Genomics and the CERCA Programme / Generalitat de Catalunya.

Circular RNAs (circRNAs) are emerging as a novel class of noncoding RNAs which potential role as gene regulators is quickly gaining interest. circRNAs have been studied in different tissues and cell types across several animal species. However, a thorough characterization of the circRNAome in ejaculated sperm remains unexplored. In this study, we profiled the sperm circRNA catalogue using 40 porcine ejaculates. A complex population of 1,598 circRNAs was shared in at least 30 of the 40 samples. Generally speaking, the predicted circRNAs presented low abundances and were tissue-specific. Around 80% of the circRNAs identified in the boar sperm were reported as novel. Results from abundance correlation between circRNAs and miRNAs together with the prediction of microRNA (miRNA) target sites in circRNAs suggested that circRNAs may act as miRNA sponges. Moreover, we found significant correlations between the abundance of 148 exonic circRNAs and sperm motility parameters. Two of these correlations, involving ssc_circ_1458 and ssc_circ_1321, were confirmed by RT-qPCR using 36 additional samples with extreme and opposite sperm motility values. Our study provides a thorough characterization of circRNAs in sperm and suggests that circRNAs hold potential as noninvasive biomarkers for sperm quality and male fertility., This work was supported by the Spanish Ministry of Economy and Competitiveness (MINECO) under grant AGL2013-44978-R and grant AGL2017-86946-R and by the CERCA Programme/Generalitat de Catalunya. AGL2017-86946-R was also funded by the Spanish State Research Agency (AEI) and the European Regional Development Fund (ERDF). We thank the Agency for Management of University and Research Grants (AGAUR) of the Generalitat de Catalunya (Grant Numbers 2014 SGR 1528 and 2017 SGR 1060). We also acknowledge the support of the Spanish Ministry of Economy and Competitivity for the Center of Excellence Severo Ochoa 2016–2019 (Grant Number SEV-2015-0533) grant awarded to the Centre for Research in Agricultural Genomics (CRAG). MG acknowledges a Ph.D. studentship from MINECO (Grant Number BES-2014-070560).

[Background]: In this study, we aimed to investigate the molecular basis of lactation as well as to identify the genetic factors that influence milk yield and composition in goats. To achieve these two goals, we have analyzed how the mRNA profile of the mammary gland changes in seven Murciano-Granadina goats at each of three different time points, i.e. 78 d (T1, early lactation), 216 d (T2, late lactation) and 285 d (T3, dry period) after parturition. Moreover, we have performed a genome-wide association study (GWAS) for seven dairy traits recorded in the 1st lactation of 822 Murciano-Granadina goats., [Results]: The expression profiles of the mammary gland in the early (T1) and late (T2) lactation were quite similar (42 differentially expressed genes), while strong transcriptomic differences (more than one thousand differentially expressed genes) were observed between the lactating (T1/T2) and non-lactating (T3) mammary glands. A large number of differentially expressed genes were involved in pathways related with the biosynthesis of amino acids, cholesterol, triglycerides and steroids as well as with glycerophospholipid metabolism, adipocytokine signaling, lipid binding, regulation of ion transmembrane transport, calcium ion binding, metalloendopeptidase activity and complement and coagulation cascades. With regard to the second goal of the study, the performance of the GWAS allowed us to detect 24 quantitative trait loci (QTLs), including three genome-wide significant associations: QTL1 (chromosome 2, 130.72-131.01 Mb) for lactose percentage, QTL6 (chromosome 6, 78.90-93.48 Mb) for protein percentage and QTL17 (chromosome 17, 11.20 Mb) for both protein and dry matter percentages. Interestingly, QTL6 shows positional coincidence with the casein genes, which encode 80% of milk proteins., [Conclusions]: The abrogation of lactation involves dramatic changes in the expression of genes participating in a broad array of physiological processes such as protein, lipid and carbohydrate metabolism, calcium homeostasis, cell death and tissue remodeling, as well as immunity. We also conclude that genetic variation at the casein genes has a major impact on the milk protein content of Murciano-Granadina goats., This research was funded by the European Fund for Regional Development/Ministerio de Ciencia, Innovación y Universidades-Agencia Estatal de Investigación/Project Reference AGL2016–76108-R. We acknowledge the financial support from the Spanish Ministry of Economy and Competitiveness, through the “Severo Ochoa Programme for Centres of Excellence in R&D” 2016–2019 (SEV-2015-0533), and from the CERCA programme of the Generalitat de Catalunya. Dailu Guan was funded by a PhD fellowship from the China Scholarship Council (CSC). Maria Luigi-Sierra was funded with a PhD fellowship “Formación de Personal Investigador” (BES-C-2017-0024) awarded by the Spanish Ministry of Economy and Competitivity. Emilio Mármol-Sánchez was funded with a PhD fellowship (FPU15/01733) awarded by the Spanish Ministry of Education and Culture (MECD).

[Background]: Genetic pressure in animal breeding is sparking the interest of breeders for selecting elite boars with higher sperm quality to optimize ejaculate doses and fertility rates. However, the molecular basis of sperm quality is not yet fully understood. Our aim was to identify candidate genes, pathways and DNA variants associated to sperm quality in swine by analysing 25 sperm-related phenotypes and integrating genome-wide association studies (GWAS) and RNA-seq under a systems biology framework., [Results]: By GWAS, we identified 12 quantitative trait loci (QTL) associated to the percentage of head and neck abnormalities, abnormal acrosomes and motile spermatozoa. Candidate genes included CHD2, KATNAL2, SLC14A2 and ABCA1. By RNA-seq, we identified a wide repertoire of mRNAs (e.g. PRM1, OAZ3, DNAJB8, TPPP2 and TNP1) and miRNAs (e.g. ssc-miR-30d, ssc-miR-34c, ssc-miR-30c-5p, ssc-miR-191, members of the let-7 family and ssc-miR-425-5p) with functions related to sperm biology. We detected 6128 significant correlations (P-value ≤ 0.05) between sperm traits and mRNA abundances. By expression (e)GWAS, we identified three trans-expression QTL involving the genes IQCJ, ACTR2 and HARS. Using the GWAS and RNA-seq data, we built a gene interaction network. We considered that the genes and interactions that were present in both the GWAS and RNA-seq networks had a higher probability of being actually involved in sperm quality and used them to build a robust gene interaction network. In addition, in the final network we included genes with RNA abundances correlated with more than four semen traits and miRNAs interacting with the genes on the network. The final network was enriched for genes involved in gamete generation and development, meiotic cell cycle, DNA repair or embryo implantation. Finally, we designed a panel of 73 SNPs based on the GWAS, eGWAS and final network data, that explains between 5% (for sperm cell concentration) and 36% (for percentage of neck abnormalities) of the phenotypic variance of the sperm traits., [Conclusions]: By applying a systems biology approach, we identified genes that potentially affect sperm quality and constructed a SNP panel that explains a substantial part of the phenotypic variance for semen quality in our study and that should be tested in other swine populations to evaluate its relevance for the pig breeding sector., This work was supported by the Spanish Ministry of Economy and Competitiveness (MINECO) under grant AGL2013-44978-R and grant AGL2017-86946-R and by the CERCA Programme/Generalitat de Catalunya. AGL2017-86946-R was also funded by the Spanish State Research Agency (AEI) and the European Regional Development Fund (ERDF). We thank the Agency for Management of University and Research Grants (AGAUR) of the Generalitat de Catalunya (Grant Numbers 2014 SGR 1528 and 2017 SGR 1060). We also acknowledge the support of the Spanish Ministry of Economy and Competitivity for the Center of Excellence Severo Ochoa 2016–2019 (Grant Number SEV-2015-0533) grant awarded to the Centre for Research in Agricultural Genomics (CRAG). MG acknowledges a Ph.D. studentship from MINECO (Grant Number BES-2014-070560) and a Short-Stay fellowship from MINECO (EEBB-I-18-12860) at AR’s group.

[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, [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., Part of the research presented in this publication was funded by grants AGL2013–48742-C2–1-R and AGL2013–48742-C2–2-R awarded by the Spanish Ministry of Economy and Competitivity. We also acknowledge the support of the Spanish Ministry of Economy and Competitivity for the Center of Excellence Severo Ochoa 2016–2019 (SEV-2015-0533) grant awarded to the Centre for Research in Agricultural Genomics. Ángela Cánovas was funded under the Juan de la Cierva program awarded by the Spanish Ministry of Science. Taina F Cardoso was funded with a fellowship from the CAPES Foundation-Coordination of Improvement of Higher Education, Ministry of Education (MEC) of the Federal Government of Brazil. Rayner González-Prendes was funded by a FPU Ph.D. grant from the Spanish Ministry of Education (FPU12/00860). Emilio Mármol-Sánchez was funded by a FPU Ph.D. grant from the Spanish Ministry of Education (FPU15/01733).

Circular RNAs (circRNAs) are emerging as a novel class of noncoding RNAs which potential role as gene regulators is quickly gaining interest. Although circRNAs have been studied in several tissues and cell types across several animal species, the characterization of the circRNAome in ejaculated sperm remains unexplored. In this study, we profiled the sperm circRNA catalogue in 40 boar samples. A complex population of 1,598 circRNAs was shared in at least 30 samples. The predicted circRNAs presented in general low abundances and were highly tissue-specific. Circa 80% of the circRNAs identified in the boar sperm were reported as novel. We also constructed a circRNA-miRNA interaction network based on experimental and predictive microRNA (miRNA) binding sites. Moreover, we found significant correlation between the abundance of some circRNAs and sperm motility parameters and confirmed two of these correlations by RT-qPCR in 36 samples with extreme sperm quality. Our study provides a thorough characterization of a novel cell type for the circRNA encyclopedia collection and suggests that circRNAs are potential noninvasive biomarkers for male sperm quality and thus, might also hold potential to predict male fertility.

[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, [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., Part of the research presented in this publication was funded by grants AGL2013–48742-C2–1-R and AGL2013–48742-C2–2-R awarded by the Spanish Ministry of Economy and Competitiveness and grant 2014 SGR 1528 from the Agency for Management of University and Research Grants of the Generalitat de Catalunya. We also acknowledge the support of the Spanish Ministry of Economy and Competitiveness for the Center of Excellence Severo Ochoa 2016–2019 (SEV-2015-0533) grant awarded to the Center for Research in Agricultural Genomics. Tainã F Cardoso was funded with a fellowship from the CAPES Foundation-Coordination of Improvement of Higher Education, Ministry of Education (MEC) of the Federal Government of Brazil. Rayner González-Prendes and Emilio Mármol-Sánchez were funded with FPU Ph.D. grants from the Spanish Ministry of Education (FPU12/00860 and FPU15/01733). Maria Ballester is financially supported by a Ramon y Cajal contract (RYC-2013-12573) from the Spanish Ministry of Economy and Competitiveness. Thanks also to the CERCA Programme of the Generalitat de Catalunya for their support.

The gut microbiota has been evolving with its host along the time creating a symbiotic relationship. In this study, we assess the role of the host genome in the modulation of the microbiota composition in pigs. Gut microbiota compositions were estimated through sequencing the V3-V4 region of the 16S rRNA gene from rectal contents of 285 pigs. A total of 1,261 operational taxonomic units were obtained and grouped in 18 phyla and 101 genera. Firmicutes (45.36%) and Bacteroidetes (37.47%) were the two major phyla obtained, whereas at genus level Prevotella (7.03%) and Treponema (6.29%) were the most abundant. Pigs were also genotyped with a high-throughput method for 45,508 single nucleotide polymorphisms that covered the entire pig genome. Subsequently, genome-wide association studies were made among the genotypes of these pigs and their gut microbiota composition. A total of 52 single-nucleotide polymorphisms distributed in 17 regions along the pig genome were associated with the relative abundance of six genera; Akkermansia, CF231, Phascolarctobacterium, Prevotella, SMB53, and Streptococcus. Our results suggest 39 candidate genes that may be modulating the microbiota composition and manifest the association between host genome and gut microbiota in pigs., This work was funded by the Fondo Europeo de Desarrollo Regional (FEDER) and the Spanish Ministerio de Economía y Competitividad (MINECO AGL2014-56369-C2 and AGL2017-82641-R). D. Crespo-Piazuelo was funded by a “Formació i Contractació de Personal Investigador Novell” (FI-DGR) Ph.D grant from the Generalitat de Catalunya (ECO/1788/2014) and by the PiGutNet COST Action (www.pigutnet.eu) for a Short Term Scientific Mission at the GABI laboratory (INRA, France) under the supervision of J. Estellé. Contract of L. Migura-Garcia was supported by INIA and the European Social Fund. L. Criado-Mesas was funded with a FPI grant from the AGL2014-56369-C2 project. M. Revilla was also funded by a FI-DGR (ECO/1639/2013). M. Ballester was financially supported by a “Ramón y Cajal” contract (RYC-2013-12573) from the Spanish Ministry of Economy and Competitiveness. We acknowledge the support of the Spanish Ministry of Economy and Competitiveness for the “Severo Ochoa Programme for Centres of Excellence in R&D” 2016-2019 (SEV-2015-0533) grant awarded to the Centre for Research in Agricultural Genomics and the CERCA Programme/Generalitat de Catalunya.

The selection in commercial swine breeds for meat-production efficiency has been increasing among the past decades, reducing the intramuscular fat content, which has changed the sensorial and technological properties of pork. Through processes of natural adaptation and selective breeding, the accumulation of mutations has driven the genetic divergence between pig breeds. The most common and well-studied mutations are single-nucleotide polymorphisms (SNPs). However, insertions and deletions (indels) usually represents a fifth part of the detected mutations and should also be considered for animal breeding. In the present study, three different programs (Dindel, SAMtools mpileup, and GATK) were used to detect indels from Whole Genome Sequencing data of Iberian boars and Landrace sows. A total of 1,928,746 indels were found in common with the three programs. The VEP tool predicted that 1,289 indels may have a high impact on protein sequence and function. Ten indels inside genes related with lipid metabolism were genotyped in pigs from three different backcrosses with Iberian origin, obtaining different allelic frequencies on each backcross. Genome-Wide Association Studies performed in the Longissimus dorsi muscle found an association between an indel located in the C1q and TNF related 12 (C1QTNF12) gene and the amount of eicosadienoic acid (C20:2(n-6))., This work was funded by the Ministerio de Economía y Competitividad (MINECO) and the Fondo Europeo de Desarrollo Regional (FEDER) projects AGL2014-56369-C2-2-R and AGL2017-82641-R. DCP was funded by a “Formacio i Contractacio de Personal Investigador Novell”(FIDGR) Ph.D grant from the Generalitat de Catalunya (ECO/1788/2014). LCM was funded with a FPI grant from the AGL2014-56369-C2 project. MR was also funded by a FI-DGR (ECO/1639/2013). MB was financially supported by a “Ramo´n y Cajal” contract (RYC-2013-12573) from the Spanish Ministry of Economy and Competitiveness. We acknowledge the support of the Spanish Ministry of Economy and Competitiveness for the “Severo Ochoa Programme for Centres of Excellence in R&D” 2016-2019 (SEV-2015-0533) grant awarded to the Centre for Research in Agricultural Genomics and the CERCA Programme / Generalitat de Catalunya.

Hypothesis: The probiotic strain Bacillus toyonensis (Toyo) may interfere with the quorum-sensing (QS) mechanisms of Escherichia coli (ETEC) K88. Three different in vitro approaches were performed. Materials and methods: Trial 1: The adhesiveness of ETEC-K88 was evaluated using porcine intestinal epithelial cells (IPEC-J2). Bacteria were grown in the ileal and the colonic sterile digesta supernatants (SN) obtained from 32 weaned piglets supplemented (TREAT) or not (CTR) with Toyo (109 CFU/g) and subsequently added to IPEC-J2. Trial 2: Using a similar model to Trial 1, E. coli was grown in media including sterile SN obtained from cultures of E. coli coincubated or not with Toyo or sterile SN from Toyo incubated or not with acyl-homoserine lactone (AHL). Trial 3: The gene expression of the fimbrial adhesin (F4) and the heat-labile enterotoxin (LT) of ETEC-K88 was also determined after different treatments. Results: The incubation of bacteria with the ileal TREAT leads to a reduction in the adhesion of E. coli (P = 0.06). As expected, ETEC-K88 grown with its own SN increased its ability to colonize the epithelium (P = 0.023), which was slightly reduced by Toyo. Gene expression results suggest that Toyo was able to reduce the expression of F4 induced by the ETEC-K88 SN (0.64-fold changes). Moreover, when ETEC-K88 is grown in the supernatant of a previous culture of Toyo with AHL or it is cocultured with Toyo in the presence of AHL, the expressions of F4 (0.61-fold changes) or LT (0.67-fold changes) were also reduced, respectively. Conclusion: These results suggest the ability of B. toyonensis to act on the QS systems of ETEC-K88 by different complex mechanisms.

Understanding the molecular basis of cell function and ultimate phenotypes is crucial for the development of biological markers. With this aim, several RNA-seq studies have been devoted to characterize the transcriptome of ejaculated spermatozoa in relation to sperm quality and fertility. Semen quality follows a seasonal pattern and decays in the summer months in several animal species. The aim of this study was to deeply profile the transcriptome of the boar sperm and to evaluate its seasonal changes. We sequenced the total and the short fractions of the sperm RNA from 10 Pietrain boars, 5 collected in summer and 5 five sampled in winter, and identified a complex and rich transcriptome with 4,436 coding genes of moderate to high abundance. Transcript fragmentation was high but less obvious in genes related to spermatogenesis, chromatin compaction and fertility. Short non-coding RNAs mostly included piwi-interacting RNAs, transfer RNAs and micro-RNAs. We also compared the transcriptome of the summer and the winter ejaculates and identified 34 coding genes and 7 micro-RNAs with a significantly distinct distribution. These genes were mostly related to oxidative stress, DNA damage and autophagy. This is the deepest characterization of the boar sperm transcriptome and the first study linking the transcriptome and the seasonal variability of semen quality in animals. The annotation described here can be used as a reference for the identification of markers of sperm quality in pigs.

The study of the boar sperm transcriptome by RNA-seq can provide relevant information on sperm quality and fertility and might contribute to animal breeding strategies. However, the analysis of the spermatozoa RNA is challenging as these cells harbor very low amounts of highly fragmented RNA, and the ejaculates also contain other cell types with larger amounts of non-fragmented RNA. Here, we describe a strategy for a successful boar sperm purification, RNA extraction and RNA-seq library preparation. Using these approaches our objectives were: (i) to evaluate the sperm recovery rate (SRR) after boar spermatozoa purification by density centrifugation using the non-porcine-specific commercial reagent BoviPureTM; (ii) to assess the correlation between SRR and sperm quality characteristics; (iii) to evaluate the relationship between sperm cell RNA load and sperm quality traits and (iv) to compare different library preparation kits for both total RNA-seq (SMARTer Universal Low Input RNA and TruSeq RNA Library Prep kit) and small RNA-seq (NEBNext Small RNA and TailorMix miRNA Sample Prep v2) for high-throughput sequencing. Our results show that pig SRR (~22%) is lower than in other mammalian species and that it is not significantly dependent of the sperm quality parameters analyzed in our study. Moreover, no relationship between the RNA yield per sperm cell and sperm phenotypes was found. We compared a RNA-seq library preparation kit optimized for low amounts of fragmented RNA with a standard kit designed for high amount and quality of input RNA and found that for sperm, a protocol designed to work on low-quality RNA is essential. We also compared two small RNA-seq kits and did not find substantial differences in their performance. We propose the methodological workflow described for the RNA-seq screening of the boar spermatozoa transcriptome., This work was supported by the Spanish Ministerio de Economía y Competitividad (MINECO) under grant AGL2013-44978-R, (Grant Number 2014 SGR 1528) from the Agency for Management of University and Research Grants of the Generalitat de Catalunya. We also acknowledge the support of the Spanish Ministry of Economy and Competitivity for the Center of Excellence Severo Ochoa 2016–2019 (Grant Number SEV-2015-0533) grant awarded to the Centre for Research in Agricultural Genomics (CRAG). We are also thankful to the CERCA Programme of the Generalitat de Catalunya. Fabiana Quoos Mayer was recipient of a post-doctoral scholarship from the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Bolsista CAPES Proc. Grant no. BEX 6707/14-9), Brazil. Marta Gòdia acknowledges a PhD studentship from MINECO (Grant Number BES-2014-070560). Alex Clop acknowledges a MINECO’s Ramon y Cajal research fellow (Grant Number RYC-2011-07763).

The aim of this work was to study the genetic basis of the backfat expression of lipid-related genes associated with meat quality traits in pigs. We performed a genome-wide association study with the backfat gene expression measured in 44 genes by qPCR and the PorcineSNP60 BeadChip genotypes in 115 Iberian x Landrace backcross animals. A total of 193 expression-associated SNPs located in 19 chromosomal regions were associated with expression levels of ACSM5, ELOVL6, FABP4, FADS2, and SLC27A4 genes. Three expression quantitative trail loci (eQTLs) corresponding to ACSM5, FABP4, and FADS2 were classified as cis-acting eQTLs, whereas the remaining 16 eQTLs have trans-regulatory effects. Remarkably, a SNP in the ACSM5 promoter region and a SNP in the 3′UTR region of FABP4 were the most associated polymorphisms with the ACSM5 and FABP4 expression levels, respectively. Moreover, relevant lipid-related genes mapped in the trans-eQTLs regions associated with the ACSM5, FABP4, FADS2, and SLC27A4 genes. Interestingly, a trans-eQTL hotspot on SSC13 regulating the gene expression of ELOVL6, ELOLV5, and SCD, three important genes implicated in the elongation and desaturation of fatty acids, was identified. These findings provide new data to further understand the functional regulatory mechanisms implicated in the variation of fatty acid composition in pigs., This work has been funded by the MINECO AGL2014-56369-C2-R and AGL2017-82641-R grants. MR was funded by a Formació i Contractació de Personal Investigador Novell (FI-DGR) PhD grant from Generalitat de Catalunya (ECO/1639/2013). MB was financially supported by a Ramon y Cajal contract (RYC-2013-12573) from the Spanish Ministry of Economy and Competitiveness, APO was funded by a Personal Investigador en Formación (PIF) PhD grant from the Universitat Autònoma de Barcelona (458-01-1/2011), DCP by a Formació i Contractació de Personal Investigador Novell (FI-DGR) PhD grant from Generalitat de Catalunya (ECO/1788/2014) and LCM was funded by a FPI PhD grant from the Spanish Ministry of Economy and Competitiveness (BES-2015-075403). Funded by grant INIA CPE03-010-C3. We acknowledge financial support from the “Severo Ochoa Programme for Centres of Excellence in R&D” 2016-2019 (SEV‐2015‐0533) and the CERCA Programme / Generalitat de Catalunya.

[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, [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., Part of the research presented in this publication was funded by grants AGL2013–48742-C2–1-R and AGL2013–48742-C2–2-R awarded by the Spanish Ministry of Economy and Competitivity and grant 2014 SGR 1528 from the Agency for Management of University and Research Grants of the Generalitat de Catalunya. We also acknowledge the support of the Spanish Ministry of Economy and Competitivity for the Center of Excellence Severo Ochoa 2016–2019 (SEV-2015-0533) grant awarded to the Centre for Research in Agricultural Genomics (CRAG). Tainã Figueiredo Cardoso was funded with a fellowship from the CAPES Foundation-Coordination of Improvement of Higher Education, Ministry of Education of the Federal Government of Brazil. Rayner Gonzalez-Prendes was funded with a FPU Ph.D. grant from the Spanish Ministry of Education (FPU12/00860). Thanks also to the CERCA Programme of the Generalitat de Catalunya.

In the present study, liver co-expression networks and expression Genome Wide Association Study (eGWAS) were performed to identify DNA variants and molecular pathways implicated in the functional regulatory mechanisms of meat quality traits in pigs. With this purpose, the liver mRNA expression of 44 candidates genes related with lipid metabolism was analysed in 111 Iberian x Landrace backcross animals. The eGWAS identified 92 eSNPs located in seven chromosomal regions and associated with eight genes: CROT, CYP2U1, DGAT1, EGF, FABP1, FABP5, PLA2G12A, and PPARA. Remarkably, cis-eSNPs associated with FABP1 gene expression which may be determining the C18:2(n-6)/C18:3(n-3) ratio in backfat through the multiple interaction of DNA variants and genes were identified. Furthermore, a hotspot on SSC8 associated with the gene expression of eight genes was identified and the TBCK gene was pointed out as candidate gene regulating it. Our results also suggested that the PI3K-Akt-mTOR pathway plays an important role in the control of the analysed genes highlighting nuclear receptors as the NR3C1 or PPARA. Finally, sex-dimorphism associated with hepatic lipid metabolism was identified with over-representation of female-biased genes. These results increase our knowledge of the genetic architecture underlying fat composition traits., This work was funded by MINECO AGL2011-29821-C02 and AGL2014-56369-C2 grants. M. Ballester is financially supported by a Ramon y Cajal contract (RYC-2013-12573) from the Spanish Ministry of Economy and Competitiveness. J. Corominas was funded by a FPI PhD grant from the Spanish Ministry of Education (BES-2009-081223). M. Revilla was funded by a Formació i Contractació de Personal Investigador Novell (FI-DGR) PhD grant from Generalitat de Catalunya (ECO/1639/2013).

Copy number variations (CNVs) are important genetic variants complementary to SNPs, and can be considered as biomarkers for some economically important traits in domestic animals. In the present study, a genomic analysis of porcine CNVs based on next-generation sequencing data was carried out to identify CNVs segregating in an Iberian x Landrace backcross population and study their association with fatty acid composition and growth-related traits. A total of 1,279 CNVs, including duplications and deletions, were detected, ranging from 106 to 235 CNVs across samples, with an average of 183 CNVs per sample. Moreover, we detected 540 CNV regions (CNVRs) containing 245 genes. Functional annotation suggested that these genes possess a great variety of molecular functions and may play a role in production traits in commercial breeds. Some of the identified CNVRs contained relevant functional genes (e.g., CLCA4, CYP4X1, GPAT2, MOGAT2, PLA2G2A and PRKG1, among others). The variation in copy number of four of them (CLCA4, GPAT2, MOGAT2 and PRKG1) was validated in 150 BC1_LD (25% Iberian and 75% Landrace) animals by qPCR. Additionally, their contribution regarding backfat and intramuscular fatty acid composition and growth–related traits was analyzed. Statistically significant associations were obtained for CNVR112 (GPAT2) for the C18:2(n-6)/C18:3(n-3) ratio in backfat and carcass length, among others. Notably, GPATs are enzymes that catalyze the first step in the biosynthesis of both triglycerides and glycerophospholipids, suggesting that this CNVR may contribute to genetic variation in fatty acid composition and growth traits. These findings provide useful genomic information to facilitate the further identification of trait-related CNVRs affecting economically important traits in pigs., This work has been funded by the MICINN AGL2014-56369-C2 and MINECO AGL2011-29821-C02. MR was funded by a Formacio i Contractacio de Personal Investigador Catalunya (ECO/1639/2013). APO was funded by a Personal Investigador en Formacion (PIF) PhD grant from the Universitat Autònoma de Barcelona (458-01-1/2011), DCP by a Formacio i Contractacio de Personal Investigador Novell (FIDGR) PhD grant from Generalitat de Catalunya. (ECO/1788/2014) and EP was funded by National Council of Scientific and Technological Development of Brazil (CNPq 202243/2014-1). MB was financially supported by a Ramon y Cajal contract (RYC-2013-12573) from the Spanish Ministry of Economy and Competitiveness. Novell (FI-DGR) PhD grant from Generalitat de