Age Effects Aggressive Behavior: RNA-Seq Analysis in Cattle with Implications for Studying Neoteny Under Domestication.

The reactive type of aggression is regulated mostly by the brain's prefrontal cortex; however, the molecular changes underlying aggressiveness in adults have not been fully characterized. We used an RNA-seq approach to investigate differential gene expression in the prefrontal cortex of bovines from the aggressive Lidia breed at different ages: young three-year old and adult four-year-old bulls. A total of 50 up and 193 down-regulated genes in the adult group were identified. Furthermore, a cross-species comparative analysis retrieved 29 genes in common with previous studies on aggressive behaviors, representing an above-chance overlap with the differentially expressed genes in adult bulls. We detected changes in the regulation of networks such as synaptogenesis, involved in maintenance and refinement of synapses, and the glutamate receptor pathway, which acts as excitatory driver in aggressive responses. The reduced reactive aggression typical of domestication has been proposed to form part of a retention of juvenile traits as adults (neoteny).

A novel missense variant in endothelin-2 (EDN2) causes a growth and respiratory lethal syndrome in bovine.

The high level of fragmentation of the Spanish Lidia cattle breed, divided into lineages called 'castas' and into herds within lineages based on reproductive isolation, increases the risk of homozygosity and the outbreak of recessive genetic defects. Since 2004, an increasing number of calves have been identified in a Lidia herd with signs of severe growth retardation, respiratory alterations and juvenile lethality, which constitutes a novel inherited syndrome in cattle and was subsequently termed growth and respiratory lethal syndrome. We performed a genome-wide association study on a cohort of 13 affected calves and 24 putative non-carrier parents, mapping the disease to a wide 6 cM region on bovine chromosome 3 (p < 10-7 ). Whole genome re-sequencing of three affected calves and three putative non-carrier parents identified a novel missense variant (c.149G>A|p.Cys50Tyr) in exon 2 of the endothelin 2 (EDN2) gene. Bioinformatic analyses of p.Cys50Tyr effects predicted them to be damaging for both the structure and the function of the edn2 protein, and to create a new site of splicing that may also affect the pattern of pre-mRNA splicing and exon definition. Sanger sequencing of this variant on the rest of the sample set confirmed the segregation pattern obtained with whole genome re-sequencing. The identification of the causative variant and the development of a diagnostic genetic test enable the efficient design of matings to keep the effective population size as high as possible, as well as providing insights into the first EDN2-associated hereditary disease in cattle or other species.

Gene expression profiles underlying aggressive behavior in the prefrontal cortex of cattle.

BACKGROUND: Aggressive behavior is an ancient and conserved trait, habitual for most animals in order to eat, protect themselves, compete for mating and defend their territories. Genetic factors have been shown to play an important role in the development of aggression both in animals and humans, displaying moderate to high heritability estimates. Although such types of behaviors have been studied in different animal models, the molecular architecture of aggressiveness remains poorly understood. This study compared gene expression profiles of 16 prefrontal cortex (PFC) samples from aggressive and non-aggressive cattle breeds: Lidia, selected for agonistic responses, and Wagyu, selected for tameness. RESULTS: A total of 918 up-regulated and 278 down-regulated differentially expressed genes (DEG) were identified, representing above-chance overlap with genes previously identified in studies of aggression across species, as well as those implicated in recent human evolution. The functional interpretation of the up-regulated genes in the aggressive cohort revealed enrichment of pathways such as Alzheimer disease-presenilin, integrins and the ERK/MAPK signaling cascade, all implicated in the development of abnormal aggressive behaviors and neurophysiological disorders. Moreover, gonadotropins, are up-regulated as natural mechanisms enhancing aggression. Concomitantly, heterotrimeric G-protein pathways, associated with low reactivity mental states, and the GAD2 gene, a repressor of agonistic reactions associated with PFC activity, are down-regulated, promoting the development of the aggressive responses selected for in Lidia cattle. We also identified six upstream regulators, whose functional activity fits with the etiology of abnormal behavioral responses associated with aggression. CONCLUSIONS: These transcriptional correlates of aggression, resulting, at least in part, from controlled artificial selection, can provide valuable insights into the complex architecture that underlies naturally developed agonistic behaviors. This analysis constitutes a first important step towards the identification of the genes and metabolic pathways that promote aggression in cattle and, providing a novel model species to disentangle the mechanisms underlying variability in aggressive behavior.

Near-Infrared Reflectance Spectroscopy for Predicting the Phospholipid Fraction and the Total Fatty Acid Composition of Freeze-Dried Beef.

Research on fatty acids (FA) is important because their intake is related to human health. NIRS can be a useful tool to estimate the FA of beef but due to the high moisture and the high absorbance of water makes it difficult to calibrate the analyses. This work evaluated near-infrared reflectance spectroscopy as a tool to assess the total fatty acid composition and the phospholipid fraction of fatty acids of beef using freeze-dried meat. An average of 22 unrelated pure breed young bulls from 15 European breeds were reared on a common concentrate-based diet. A total of 332 longissimus thoracis steaks were analysed for fatty acid composition and a freeze-dried sample was subjected to near-infrared spectral analysis. 220 samples (67%) were used as a calibration set with the remaining 110 (33%) being used for validation of the models obtained. There was a large variation in the total FA concentration across the animals giving a good data set for the analysis and whilst the coefficient of variation was nearly 68% for the monounsaturated FA it was only 27% for the polyunsaturated fatty acids (PUFA). PLS method was used to develop the prediction models. The models for the phospholipid fraction had a low R2p and high standard error, while models for neutral lipid had the best performance, in general. It was not possible to obtain a good prediction of many individual PUFA concentrations being present at low concentrations and less variable than other FA. The best models were developed for Total FA, saturated FA, 9c18:1 and 16:1 with R2p greater than 0.76. This study indicates that NIRS is a feasible and useful tool for screening purposes and it has the potential to predict most of the FA of freeze-dried beef.

Analysis of conservation priorities of Iberoamerican cattle based on autosomal microsatellite markers.

BACKGROUND: Determining the value of livestock breeds is essential to define conservation priorities, manage genetic diversity and allocate funds. Within- and between-breed genetic diversity need to be assessed to preserve the highest intra-specific variability. Information on genetic diversity and risk status is still lacking for many Creole cattle breeds from the Americas, despite their distinct evolutionary trajectories and adaptation to extreme environmental conditions. METHODS: A comprehensive genetic analysis of 67 Iberoamerican cattle breeds was carried out with 19 FAO-recommended microsatellites to assess conservation priorities. Contributions to global diversity were investigated using alternative methods, with different weights given to the within- and between-breed components of genetic diversity. Information on Iberoamerican plus 15 worldwide cattle breeds was used to investigate the contribution of geographical breed groups to global genetic diversity. RESULTS: Overall, Creole cattle breeds showed a high level of genetic diversity with the highest level found in breeds admixed with zebu cattle, which were clearly differentiated from all other breeds. Within-breed kinships revealed seven highly inbred Creole breeds for which measures are needed to avoid further genetic erosion. However, if contribution to heterozygosity was the only criterion considered, some of these breeds had the lowest priority for conservation decisions. The Weitzman approach prioritized highly differentiated breeds, such as Guabalá, Romosinuano, Cr. Patagonico, Siboney and Caracú, while kinship-based methods prioritized mainly zebu-related breeds. With the combined approaches, breed ranking depended on the weights given to the within- and between-breed components of diversity. Overall, the Creole groups of breeds were generally assigned a higher priority for conservation than the European groups of breeds. CONCLUSIONS: Conservation priorities differed significantly according to the weight given to within- and between-breed genetic diversity. Thus, when establishing conservation programs, it is necessary to also take into account other features. Creole cattle and local isolated breeds retain a high level of genetic diversity. The development of sustainable breeding and crossbreeding programs for Creole breeds, and the added value resulting from their products should be taken into consideration to ensure their long-term survival.

Colombian Creole horse breeds: Same origin but different diversity.

In order to understand the genetic ancestry and mitochondrial DNA (mtDNA) diversity of current Colombian horse breeds we sequenced a 364-bp fragment of the mitocondrial DNA D-loop in 116 animals belonging to five Spanish horse breeds and the Colombian Paso Fino and Colombian Creole cattle horse breeds. Among Colombian horse breeds, haplogroup D had the highest frequency (53%), followed by haplogroups A (19%), C (8%) and F (6%). The higher frequency of haplogroup D in Colombian horse breeds supports the theory of an ancestral Iberian origin for these breeds. These results also indicate that different selective pressures among the Colombian breeds could explain the relatively higher genetic diversity found in the Colombian Creole cattle horse when compared with the Colombian Paso Fino.

Syringohydromyelia in Dogs: The Genomic Component Underlying a Complex Neurological Disease.

Syringohydromyelia (SHM) is a neurological disorder characterized by the appearance of fluid-containing cavities within the spinal cord. Although SHM is thought to be under multigenic control, the molecular basis of this disease remains poorly defined. A genome-wide association study (GWAS) was carried out comparing the whole genome sequences (WGS) from 12 dogs with SHM and 2 panels of 26 dogs (either older than 5 years and showing the absence of SHM or belonging to breeds not susceptible to SHM) to identify candidate genes associated with the development of SHM. Seven candidate genes were identified. Of these, five genes were determined to be involved in bone development (PLXNA2, HHAT, MBOAT2, ITGAV) and calcium homeostasis (HPCAL1). Although further validation is needed at the transcript level, it is worth highlighting the association of a possible pathogenic variant which generated a new intronic branch-site sequence in PLXNA2 (T/C, CFA7:7043294). Considering previous studies in dogs that show SHM related to craniocervical junction (CCJ) malformations, these genes can be considered good candidates for the development of this disease. This report dissects the genomic component of SHM in dogs, which paves the way for further research on this complex disease found both in canine and human species.

Genetic Structure of the Ca Rater Mallorquí Dog Breed Inferred by Microsatellite Markers.

Ca Rater Mallorquí is a dog breed from the Island of Mallorca (Spain) traditionally used as a hunting and ratting dog to prevent disease spread and economic losses related to rodent activities on farms. However, the census data shows a population decline that should be addressed by implementing a conservation program. The first step to implementing a conservation plan is knowing the genetic situation of the Ca Rater Mallorquí population. Therefore, we aimed to genetically characterise the breed in our study. We analysed 33 microsatellites recommended by the International Society of Animal Genetics (ISAG) in 77 samples. Data were obtained from 13 samples of Balearic, Spanish, and international dog breeds to study the genetic diversity among breeds. The population did not significantly deviate from the Hardy-Weinberg equilibrium with heterozygosity (Ho) of 0.655 and expected heterozygosity (He) of 0.685. The Wright's fixation indices, the Factorial Correspondence Analysis (FCA), a dendrogram representing Reynolds genetic distance between populations, and the pairwise FST values establish the Ca Rater Mallorquí as an independent breed distinct from the Balearic, Spanish, and international breeds.

Modulation of Host Immune Response during Leishmania infantum Natural Infection: A Whole-Transcriptome Analysis of the Popliteal Lymph Nodes in Dogs.

Leishmania infantum, the etiological agent of canine leishmaniosis (CanL) in Europe, was responsible of the largest outbreak of human leishmaniosis in Spain. The parasite infects and survives within myeloid lineage cells, causing a potentially fatal disease if left untreated. The only treatment option relies on chemotherapy, although immunotherapy strategies are being considered as novel approaches to prevent progression of the disease. To this aim, a deeper characterization of the molecular mechanisms behind the immunopathogenesis of leishmaniosis is necessary. Thus, we evaluated, for the first time, the host immune response during L. infantum infection through transcriptome sequencing of the popliteal lymph nodes aspirates of dogs with CanL. Differential expression and weighted gene co-expression network analyses were performed, resulting in the identification of 5,461 differentially expressed genes (DEGs) and four key modules in sick dogs, compared to controls. As expected, defense response was the highest enriched biological process in the DEGs, with six genes related to immune response against pathogens (CHI3L1, SLPI, ACOD1, CCL5, MPO, BPI) included among the ten most expressed genes; and two of the key co-expression modules were associated with regulation of immune response, which also positively correlated with clinical stage and blood monocyte concentration. In particular, sick dogs displayed significant changes in the expression of Th1, Th2, Th17 and Tr1 cytokines (e. g. TNF-α, IFN-γ, IL-21, IL-17, IL-15), markers of T cell and NK cell exhaustion (e. g. LAG3, CD244, Blimp-1, JUN), and B cell, monocyte and macrophage disrupted functionality (e. g. CD40LG, MAPK4, IL-1R, NLRP3, BCMA). In addition, we found an overexpression of XBP1 and some other genes involved in endoplasmic reticulum stress and the IRE1 branch of the unfolded protein response, as well as one co-expression module associated with these processes, which could be induced by L. infantum to prevent host cell apoptosis and modulate inflammation-induced lymphangiogenesis at lymph nodes. Moreover, 21 lncRNAs were differentially expressed in sick dogs, and one key co-expression module was associated with chromatin organization, suggesting that epigenetic mechanisms could also contribute to dampening host immune response during natural L. infantum infection in the lymph nodes of dogs suffering from clinical leishmaniosis.

Gene Banks as Reservoirs to Detect Recent Selection: The Example of the Asturiana de los Valles Bovine Breed.

Gene banks, framed within the efforts for conserving animal genetic resources to ensure the adaptability of livestock production systems to population growth, income, and climate change challenges, have emerged as invaluable resources for biodiversity and scientific research. Allele frequency trajectories over the few last generations contain rich information about the selection history of populations, which cannot be obtained from classical selection scan approaches based on present time data only. Here we apply a new statistical approach taking advantage of genomic time series and a state of the art statistic (nSL) based on present time data to disentangle both old and recent signatures of selection in the Asturiana de los Valles cattle breed. This local Spanish originally multipurpose breed native to Asturias has been selected for beef production over the last few generations. With the use of SNP chip and whole-genome sequencing (WGS) data, we detect candidate regions under selection reflecting the effort of breeders to produce economically valuable beef individuals, e.g., by improving carcass and meat traits with genes such as MSTN, FLRT2, CRABP2, ZNF215, RBPMS2, OAZ2, or ZNF609, while maintaining the ability to thrive under a semi-intensive production system, with the selection of immune (GIMAP7, GIMAP4, GIMAP8, and TICAM1) or olfactory receptor (OR2D2, OR2D3, OR10A4, and 0R6A2) genes. This kind of information will allow us to take advantage of the invaluable resources provided by gene bank collections from local less competitive breeds, enabling the livestock industry to exploit the different mechanisms fine-tuned by natural and human-driven selection on different populations to improve productivity.

Genomic Characterization of a Set of Iberian Peninsula Bovine Local Breeds at Risk of Extinction: Morenas Gallegas.

A set of five local bovine breeds in danger of extinction named Cachena, Caldelá, Limiá, Frieiresa, and Vianesa and included in the group of Morenas Gallegas are located in the Autonomous Community of Galicia at the Northwest of Spain. Local authorities launched a conservation plan at the end of the 21th century in order to preserve this important genetic reservoir. However, Morenas Gallegas bovine breeds never have been analyzed with genomic tools and this information may be crucial to develop conservation plans. The aim of the study was to analyze their genetic diversity and genetic relationships with a set of local and cosmopolitan European bovine breeds using single nucleotide polymorphisms. Our results show own genetic signatures for the Morenas Gallegas breeds which form a separate cluster when compared to the Spanish breeds analyzed, with the exception of the Cachena breed. The genetic diversity levels of the Morenas Gallegas were intermediate or high, and low inbreeding coefficients can be found except for the Frieiresa breed (11%). Vianesa breed evidenced two lineages depending on the Frieiresa component influence. The Morenas Gallegas bovine breeds group represent an important Spanish bovine genetic reservoir and despite their classification within a single generic group, the five breeds show their own genetic uniqueness.

Rapid identification and interpretation of gene-environment associations using the new R.SamBada landscape genomics pipeline.

samßada is a genome-environment association software, designed to search for signatures of local adaptation. However, pre- and postprocessing of data can be labour-intensive, preventing wider uptake of the method. We have now developed R.SamBada, an r-package providing a pipeline for landscape genomic analysis based on samßada, spanning from the retrieval of environmental conditions at sampling locations to gene annotation using the Ensembl genome browser. As a result, R.SamBada standardizes the landscape genomics pipeline and eases the search for candidate genes of local adaptation, enhancing reproducibility of landscape genomic studies. The efficiency and power of the pipeline is illustrated using two examples: sheep populations from Morocco with no evident population structure and Lidia cattle from Spain displaying population substructuring. In both cases, R.SamBada enabled rapid identification and interpretation of candidate genes, which are further discussed in the light of local adaptation. The package is available in the r CRAN package repository and on GitHub (github.com/SolangeD/R.SamBada).

Evaluation of suitable reference genes for gene expression studies in bovine muscular tissue.

BACKGROUND: Real-time reverse transcriptase quantitative polymerase chain reaction (real-time RTqPCR) is a technique used to measure mRNA species copy number as a way to determine key genes involved in different biological processes. However, the expression level of these key genes may vary among tissues or cells not only as a consequence of differential expression but also due to different factors, including choice of reference genes to normalize the expression levels of the target genes; thus the selection of reference genes is critical for expression studies. For this purpose, ten candidate reference genes were investigated in bovine muscular tissue. RESULTS: The value of stability of ten candidate reference genes included in three groups was estimated: the so called 'classical housekeeping' genes (18S, GAPDH and ACTB), a second set of genes used in expression studies conducted on other tissues (B2M, RPII, UBC and HMBS) and a third set of novel genes (SF3A1, EEF1A2 and CASC3). Three different statistical algorithms were used to rank the genes by their stability measures as produced by geNorm, NormFinder and Bestkeeper. The three methods tend to agree on the most stably expressed genes and the least in muscular tissue. EEF1A2 and HMBS followed by SF3A1, ACTB, and CASC3 can be considered as stable reference genes, and B2M, RPII, UBC and GAPDH would not be appropriate. Although the rRNA-18S stability measure seems to be within the range of acceptance, its use is not recommended because its synthesis regulation is not representative of mRNA levels. CONCLUSION: Based on geNorm algorithm, we propose the use of three genes SF3A1, EEF1A2 and HMBS as references for normalization of real-time RTqPCR in muscle expression studies.

Steroid pathway and oestrone sulphate production in canine inflammatory mammary carcinoma.

Spontaneous canine mammary inflammatory carcinoma (IMC) shares epidemiologic, histopathologic and clinical characteristics with the inflammatory breast carcinoma (IBC) disease in humans. We have analysed the steroids levels in serum and in tissue homogenates of IMC, the expression of two of their receptors (androgen and beta-estrogen) and of three enzymes included in the steroidogenesis pathway (aromatase (CYP19A1), steroid sulphatase (STS) and estrogen sulfotransferase (EST)) trying to explain the specific accumulation of steroids in IMC tissues generating deposits in the form of lipid droplets whose presence can be attributed to steroids secreted by IMC cells. According to our working hypothesis, oestrone sulphate would be the main component of these lipid droplets. The presence of these steroid deposits would contribute to the intense proliferation and invasive behaviour of IMC and IBC, although their involvement in angiogenesis is yet to be demonstrated.

Polymorphisms in ten candidate genes are associated with conformational and locomotive traits in Spanish Purebred horses.

The Spanish Purebred horses, also known as Andalusian horses, compete to the highest standards in international dressage events. Gait and conformation could be used as early selection criteria to detect young horses with promising dressage ability. Although the genetic background of equine size variation has been recently uncovered, the genetic basis of horse conformational and locomotive traits is not known, hampered by the complex genetic architecture underlying quantitative traits and the lack of phenotypic data. The aim of this study was to validate the loci associated with size in 144 Spanish Purebred horses, and to seek novel associations between loci previously associated with the development of osteochondrosis (OC) lesions and 20 conformational and locomotive traits. Ten loci were associated with different conformational and locomotive traits (LCORL/NCAPG, HMGA2, USP31, MECR, COL24A1, MGP, FAM184B, PTH1R, KLF3 and SGK1), and the LCORL/NCAPG association with size in the Spanish Purebred horse was validated. Except for HMGA2, all polymorphisms seem to influence both the prevalence of OC lesions and morphological characters, supporting the link between conformation and OC. Also, the implication of most genes in either immune and inflammatory responses and cellular growth, or ossification processes, reinforces the role that these mechanisms have in the aetiology of OC, as well as their reflection on the general conformation of the individual. These polymorphisms could be used in marker-assisted selection (MAS) programmes to improve desirable conformational traits, but taking into account their possible detrimental effect on OC prevalence.

Red-legged partridge (Alectoris rufa) de-novo transcriptome assembly and identification of gene-related markers.

The red-legged partridge (Alectoris rufa) has a great socio-economic importance as a game species and is reared by millions in farms in several European countries. The ability to respond to a wide spectrum of pathogens and environmental changes is key for farm-reared animals that, as such, face even higher pathogen exposure and specifically for those submitted to restocking programs. In this study, RNA-sequencing and de-novo assembly of genes expressed in different immune tissues were performed. The raw FASTQ files were submitted to the NCBI SRA database with accession number PRJNA289204. A total of 94.2 million reads were obtained and assembled into 51,403 contigs using OASES software. The final annotated partridge immune transcriptome comprises almost 7000 unigenes, available as FASTA in the supplementary material. A total of 12,828 microsatellites and 33,857 Single Nucleotide Polymorphisms (SNPs) were identified. The candidate gene sequences and the large number of potential genetic markers from the red-legged partridge transcriptome reliably identified through the use for the first time of a high coverage 100-bp paired-end RNA-seq protocol, provide new tools for future studies in this and related species, thus contributing to the ongoing development of genomic resources in avian species. Further investigation into candidate genes and gene-associated markers will help to uncover individual variability in the resistance to infections and other external aggressions in partridges.

Distribution and Functionality of Copy Number Variation across European Cattle Populations.

Copy number variation (CNV), which is characterized by large-scale losses or gains of DNA fragments, contributes significantly to genetic and phenotypic variation. Assessing CNV across different European cattle populations might reveal genetic changes responsible for phenotypic differences, which have accumulated throughout the domestication history of cattle as consequences of evolutionary forces that act upon them. To explore pattern of CNVs across European cattle, we genotyped 149 individuals, that represent different European regions, using the Illumina Bovine HD Genotyping array. A total of 9,944 autosomal CNVs were identified in 149 samples using a Hidden Markov Model (HMM) as employed in PennCNV. Animals originating from several breeds of British Isles, and Balkan and Italian regions, on average, displayed higher abundance of CNV counts than Dutch or Alpine animals. A total of 923 CNV regions (CNVRs) were identified by aggregating CNVs overlapping in at least two animals. The hierarchical clustering of CNVRs indicated low differentiation and sharing of high-frequency CNVRs between European cattle populations. Various CNVRs identified in the present study overlapped with olfactory receptor genes and genes related to immune system. In addition, we also detected a CNV overlapping the Kit gene in English longhorn cattle which has previously been associated with color-sidedness. To conclude, we provide a comprehensive overview of CNV distribution in genome of European cattle. Our results indicate an important role of purifying selection and genomic drift in shaping CNV diversity that exists between different European cattle populations.

Steroids and receptors in canine mammary cancer.

The aims of this study were to investigate the serum and tissue content of androgens and estrogens in canine inflammatory mammary carcinomas (IMC) as well as in non-inflammatory malignant mammary tumors (MMT), and assessed the immunoexpression of estrogen and androgen receptors using immunohistochemistry. Profiles for the androgens dehydroepiandrosterone (DHEA), androstenedione (A4), and testosterone (T), and for the estrogens 17beta estradiol (E2) and estrone-sulphate (SO4E1) were measured both in tissue homogenates and in serum of MMT and IMC by EIA techniques in 42 non-inflammatory malignant mammary tumors (MMT) and in 14 inflammatory mammary carcinomas (IMC), prospectively collected from 56 female dogs. Androgen receptor (AR) and estrogen receptor alpha (ERalpha) and beta (ERbeta) expression was studied using immunohistochemistry (strepavidin-biotin-peroxidase method) in samples of 32 MMT and 14 IMC, and counted by a computer image analyzer. IMC serum and tissue levels of androgens were significantly higher than MMT levels. Tissue content of estrogens was also significantly higher in IMC than in MMT. Serum values of SO4E1 were significantly higher in IMC, but serum levels of E2 were significantly lower in IMC compared to MMT cases. Medium-high androgen receptor intensity was observed in 64.28% of IMC and 40.62% of MMT. No important differences were found between ERalpha expression in IMC (100% negative) and MMT (90% negative). ERbeta and AR were intensely expressed in highly malignant inflammatory mammary carcinoma cells. To our knowledge, this is the first report relative to AR immunohistochemistry in canine mammary cancer and to estrogens or androgens in serum of dogs with benign or malignant mammary tumors.

Estimation of the genetic admixture composition of Iberian dry-cured ham samples using DNA multilocus genotypes.

Under current Spanish regulations, the pigs that provide the raw material for the preparation of the country's most appreciated meat-derived product, dry-cured Iberian ham, must be of a specific genetic composition. Only the Duroc breed is accepted for crossing with Iberian pigs, and a maximum of 50% of the Duroc genome is permitted in the animals used to make this ham. This paper describes a set of statistical procedures for detecting the 'breed composition' of Iberian ham via the use of multilocus genotypes obtained by the amplification of 25 microsatellite markers. The proposed procedure detected up to 20% of ham samples with a genetic composition incompatible with present legislation - either because the Duroc genome was present in a percentage greater than that permitted, or because of the significant presence (>25%) of white coat pig genomes. The probability of finding an illegal cured ham was greater in restaurants than in retail grocery stores, and in medium-low category restaurants or stores than in higher category establishments.

Transcriptomic Characterization of Innate and Acquired Immune Responses in Red-Legged Partridges (Alectoris rufa): A Resource for Immunoecology and Robustness Selection.

Present and future challenges for wild partridge populations include the resistance against possible disease transmission after restocking with captive-reared individuals, and the need to cope with the stress prompted by new dynamic and challenging scenarios. Selection of individuals with the best immune ability may be a good strategy to improve general immunity, and hence adaptation to stress. In this study, non-infectious challenges with phytohemagglutinin (PHA) and sheep red blood cells allowed the classification of red-legged partridges (Alectoris rufa) according to their overall immune responses (IR). Skin from the area of injection of PHA and spleen, both from animals showing extreme high and low IR, were selected to investigate the transcriptional profiles underlying the different ability to cope with pathogens and external aggressions. RNA-seq yielded 97 million raw reads from eight sequencing libraries and approximately 84% of the processed reads were mapped to the reference chicken genome. Differential expression analysis identified 1488 up- and 107 down-regulated loci in individuals with high IR versus low IR. Partridges displaying higher innate IR show an enhanced activation of host defence gene pathways complemented with a tightly controlled desensitization that facilitates the return to cellular homeostasis. These findings indicate that the immune system ability to respond to aggressions (either diseases or stress produced by environmental changes) involves extensive transcriptional and post-transcriptional regulations, and expand our understanding on the molecular mechanisms of the avian immune system, opening the possibility of improving disease resistance or robustness using genome assisted selection (GAS) approaches for increased IR in partridges by using genes such as AVN or BF2 as markers. This study provides the first transcriptome sequencing data of the Alectoris genus, a resource for molecular ecology that enables integration of genomic tools in further studies.