Marie-José Mercat

Summary: The late-stage development of the blastocyst before implantation is a unique feature of ungulates. During this period, the epiblast proliferates and remains pluripotent for several days before gastrulation begins. Simultaneously, extra-embryonic tissues undergo significant growth, elongating to several tens of centimeters. However, the mechanisms that regulate and synchronize these processes remain poorly understood. In this study, we analyzed transcriptomic and epigenomic data at the single-cell level from early and late porcine blastocyst cells, spanning from the hatched blastocyst stage (E7) to early (E9) and late ovoid blastocyst (E11). We characterized 15,370 blastocyst cells, clustering them into distinct embryonic and extra-embryonic populations with specific chromatin accessibility landscapes. For each population, we inferred gene regulatory networks based on enhancer-driven gene regulation modules (eRegulons) and validated them through motif occupancy visualization. Our findings reveal strong dynamics in gene regulatory module activity within extra-embryonic tissues at the onset of blastocyst elongation, while gene regulatory activity in the epiblast remains relatively stable.

Gene expression is a dynamic phenotype influenced by tissue-specific regulatory mechanisms, which can modulate expression directly or indirectly through cis or trans factors. Identifying genetic variants in these regulatory regions can improve both expression quantitative trait locus (eQTL) mapping and gene expression prediction. Whole genome sequences offer the possibility for enhanced eQTL mapping accuracy, but detecting causal variants remains challenging. Here, we evaluate the potential added-value of integrating tissue-specific epigenetic annotations, such as chromatin accessibility and methylation status, into within-breed genomic predictions of expression for three pig breeds. Functional annotations from early developmental stages improved eQTL mapping interpretability as shown by the enrichment of trait-relevant QTLs. However, despite the use of functional annotations, predictions across breeds remain challenging due to differences in genetic architectures. Our work contributes to the understanding of gene expression regulation in livestock and highlights the value of functional annotations, despite continued challenges for predictions across breeds. Highlights: Matched whole-genome sequences and expressions reveal transcriptional regulatory variants. Early epigenetic marks can guide transcriptome prediction and eQTL mapping. Annotation-guided models can enhance the interpretability of eQTL mapping. Porting predictions across pig breeds remains challenging even with annotations. A case study linked IGF2 liver eQTLs with annotations and cholesterol QTLs.

In humans and livestock species, genome-wide association studies (GWAS) have been applied to study the association between variants distributed across the genome and a phenotype of interest. To discover genetic polymorphisms affecting the duodenum, liver, and muscle transcriptomes of 300 pigs from 3 different breeds (Duroc, Landrace, and Large White), we performed expression GWAS between 25,315,878 polymorphisms and the expression of 13,891 genes in duodenum, 12,748 genes in liver, and 11,617 genes in muscle. More than 9.68 × 10¹¹ association tests were performed, yielding 14,096,080 significantly associated variants, which were grouped in 26,414 expression quantitative trait locus (eQTL) regions. Over 56% of the variants were within 1 Mb of their associated gene. In addition to the 100-kb region upstream of the transcription start site, we identified the importance of the 100-kb region downstream of the 3′UTR for gene regulation, as most of the cis-regulatory variants were located within these 2 regions. We also observed 39,874 hotspot regulatory polymorphisms associated with the expression of 10 or more genes that could modify the protein structure or the expression of a regulator gene. In addition, 2 motifs (5′-GATCCNGYGTTGCYG-3′ and a poly(A) sequence) were enriched across the 3 tissues within the neighboring sequences of the most significant single-nucleotide polymorphisms in each cis-eQTL region. The 14 million significant associations obtained in this study are publicly available and have enabled the identification of expression-associated cis-, trans-, and hotspot regulatory variants within and across tissues, thus shedding light on the molecular mechanisms of regulatory variations that shape end-trait phenotypes.

Background: Intensive selection of modern pig breeds resulted in genetic improvement of productive traits while local pig breeds remained less performant. As they have been bred in extensive systems, they have adapted to specifical environmental conditions resulting in a rich genotypic and phenotypic diversity. This study is based on European local pig breeds 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 selection signatures linked to phenotypic traits in order to uncover potential candidate genes that may be under adaptation to specific environments. Results: Genetic data analysis of European pig breeds revealed four main axes of genetic variation represented by Iberian and modern breeds (i.e. Large White, Landrace, and Duroc). In addition, breeds clustered according to their geographical origin, for example French Gascon and Basque breeds, Italian Apulo Calabrese and Casertana breeds, Spanish Iberian and Portuguese Alentejano breeds. Principal component analysis of phenotypic data distinguished between larger and leaner breeds with better growth potential and reproductive performance on one hand and breeds that were smaller, fatter, and had low growth and reproductive efficiency on the other hand. Linking selection signatures with phenotype identified 16 significant genomic regions associated with stature, 24 with fatness, 2 with growth and 192 with reproduction. Among them, several regions contained candidate genes with possible biological effect on stature, fatness, growth and reproduction performance traits. For example, strong associations were found for stature in two regions containing the ANXA4 and ANTXR1 genes, for fatness containing the DNMT3A and POMC genes and for reproductive performance containing the HSD17B7 gene. Conclusions: The present study on European local pig breeds used a dedicated approach for searching selection signatures supported by phenotypic data at the breed level to identify potential candidate genes that may have adapted to different living environments and production systems. Results can be useful to define conservation programs of local pig breeds.

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, 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 (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 deliberate) occurred with wild boars. Conclusions: Our findings provide a catalogue of genetic variants of many European pig populations and identify genome regions that can explain, at least in part, the phenotypic diversity of these genetic resources.

Local pig breeds are adapted to the specific local environment and fed with various locally available feedstuffs. Besides their genetic merit for agro-biodiversity, they represent the basis for sustainable local pork chains. The present chapter aims to present history and current status of the Basque pig breed, its exterior phenotypic characteristics, geographical location, production system and main products. This French autochthonous breed of pigs, which almost disappeared few decades ago, seems now consolidated, thanks to a chain organization and recognised high-quality products (Protected Designation of Origin). Reproductive performance data reviewed from the literature and estimated from the LIGERAL database (herdbook) are presented. Available data on production traits including growth (early, middle, late and overall growth), feed intake, body composition and carcass traits are also summarized. Meat quality traits (pH, colour, intramuscular fat content and fatty acid composition) and back fat tissue characteristics (fatty acid profile) are also described. Studies on the Basque pig breed are scarce. Different production systems, feeding regimes and feed composition used among studies can explain differences observed between studies, especially for productive traits. However, the current review gives insight into the reproduction, production and carcass and meat quality traits of this local pig breed.

The present chapter aims to present history and current status of Gascon pig breed, one of the local pig breeds investigated in the project TREASURE. This French autochthonous breed of pigs, which almost disappeared, now enjoys a new boom. The quality of its product is recognized by the consumers and by official quality labels (Protected Designation of Origin). Exterior phenotypic characteristics of the breed, geographical location, production system and main products are described. Reproductive performance data available in the literature and estimated from the LIGERAL database (herdbook) are presented. Literature data on production traits are also summarized for growth (early, middle, late and overall growth), feed intake, body composition and carcass traits. Meat quality traits (pH, colour, intramuscular fat content and composition) and fat tissue characteristics (fatty acid profile) are also described. Studies on Gascon pig breed are scarce and variability between studies, especially regarding productive traits, can be explained by differences in production systems, feeding regimes and feed composition according to studies. Nevertheless, the current review gives updated insights into the reproduction, production and quality traits of this local pig breed.

Weaning is a critical transition phase in swine production in which piglets must cope with different stressors that may affect their health. During this period, the prophylactic use of antibiotics is still frequent to limit piglet morbidity, which raises both economic and public health concerns such as the appearance of antimicrobial-resistant microbes. With the interest of developing tools for assisting health and management decisions around weaning, it is key to provide robustness indexes that inform on the animals capacity to endure the challenges associated to weaning. This work aimed at developing a modelling approach for facilitating the quantification of piglet resilience to weaning. We monitored 325 Large White pigs weaned at 28 days of age and further housed and fed conventionally during the post-weaning period without antibiotic administration. Body weight and diarrhoea scores were recorded before and after weaning, and blood was sampled at weaning and one week later for collecting haematological data. We constructed a dynamic model based on the Gompertz-Makeham law to describe live weight trajectories during the first 75 days after weaning following the rationale that the animal response is partitioned in two time windows (a perturbation and a recovery window). Model calibration was performed for each animal. Our results show that the transition time between the two time windows, as well as the weight trajectories are characteristic for each individual. The model captured the weight dynamics of animals at different degrees of perturbation, with an average coefficient of determination of 0.99, and a concordance correlation coefficient of 0.99. The utility of the model is that it provides biological parameters that inform on the amplitude and length of perturbation, and the rate of animal recovery. Our rationale is that the dynamics of weight inform on the capability of the animal to cope with the weaning disturbance. Indeed, there were significant correlations between model parameters and individual diarrhoea scores and haematological traits. Overall, the parameters of our model can be useful for constructing weaning robustness indexes by using exclusively the growth curves. We foresee that this modelling approach will provide a step forward in the quantitative characterization of robustness. Implications: The quantitative characterization of animal robustness at weaning is a key step for management strategies to improve health and welfare. This characterization is also instrumental for the further design of selection strategies for productivity and robustness. Within a precision livestock farming optic, this study develops a mathematical modelling approach to describe the body weight of piglets from weaning with the rationale that weight trajectories provide central information to quantify the capability of the animal to cope with the weaning disturbance.

Efforts to improve sustainability in livestock production systems have focused on two objectives: investigating the genetic control of immune function as it pertains to robustness and disease resistance, and finding predictive markers for use in breeding programs. In this context, the peripheral blood transcriptome represents an important source of biological information about an individual’s health and immunological status, and has been proposed for use as an intermediate phenotype to measure immune capacity. The objective of this work was to study the genetic architecture of variation in gene expression in the blood of healthy young pigs using two approaches: an expression genome-wide association study (eGWAS) and allele-specific expression (ASE) analysis. The blood transcriptomes of 60-day-old Large White pigs were analyzed by expression microarrays for eGWAS (242 animals) and by RNA-Seq for ASE analysis (38 animals). Using eGWAS, the expression levels of 1901 genes were found to be associated with expression quantitative trait loci (eQTLs). We recovered 2839 local and 1752 distant associations (Single Nucleotide Polymorphism or SNP located less or more than 1 Mb from expression probe, respectively). ASE analyses confirmed the extensive cis-regulation of gene transcription in blood, and revealed allelic imbalance in 2286 SNPs, which affected 763 genes. eQTLs and ASE-genes were widely distributed on all chromosomes. By analyzing mutually overlapping eGWAS results, we were able to describe putative regulatory networks, which were further refined using ASE data. At the functional level, genes with genetically controlled expression that were detected by eGWAS and/or ASE analyses were significantly enriched in biological processes related to RNA processing and immune function. Indeed, numerous distant and local regulatory relationships were detected within the major histocompatibility complex region on chromosome 7, revealing ASE for most class I and II genes. This study represents, to the best of our knowledge, the first genome-wide map of the genetic control of gene expression in porcine peripheral blood. These results represent an interesting resource for the identification of genetic markers and blood biomarkers associated with variations in immunity traits in pigs, as well as any other complex traits for which blood is an appropriate surrogate tissue.

In crossbreeding schemes, within-line selection of purebreds is performed mainly to improve the performance of crossbred descendants under field conditions. The genetic correlation between purebred and crossbred performance is an important parameter to be assessed because purebred performance can be a poor predictor of the performance of crossbred offspring. With the availability of high-density markers, the feasibility of using crossbred information to evaluate purebred candidates can be reassessed. This study implements and applies a single-step terminal-cross model (GEN) to real data to estimate the genetic parameters of several production and quality traits in pigs. Piétrain sires were mated with Piétrain and Large White dams to produce purebred and crossbred male half-sib piglets; growth rate, feed conversion ratio, lean meat, pH of longissimus dorsi muscle, drip loss and intramuscular fat content were recorded on all half-sibs. Animals were genotyped using the Illumina Porcine SNP60 BeadChip. The genetic correlation between purebred and crossbred performance was estimated separately for each trait. Purebred animals were evaluated using an animal model, whereas the additive genetic effect of a crossbred individual was decomposed into the additive effects of the sire and dam and a Mendelian sampling effect that was confounded with the residual effect. Genotypes of the Piétrain animals were integrated in the genetic evaluation by using a single-step procedure. As benchmarks, we used a model that was identical to GEN but only accounted for pedigree information (PED) and also two univariate single-step models (GEN_UNI) that took either purebred or crossbred performance into account. Genetic correlations between purebred and crossbred performance were high and positive for all traits (>0.69). Accuracies of estimated breeding values of genotyped sires and purebred offspring that were obtained with the GEN model outperformed both those obtained with the PED and the GEN_UNI models. The use of genomic information increased the predictive ability of the GEN model, but it did not substantially outperform the GEN_UNI models. We present a single-step terminal-cross model that integrates genomic information of purebred and crossbred performance by using available software. It improves the theoretical accuracy of genetic evaluations in breeding programs that are based on crossbreeding.