FAANG, establishing metadata standards, validation and best practices for the farmed and companion animal community.

The Functional Annotation of ANimal Genomes (FAANG) project aims, through a coordinated international effort, to provide high quality functional annotation of animal genomes with an initial focus on farmed and companion animals. A key goal of the initiative is to ensure high quality and rich supporting metadata to describe the project's animals, specimens, cell cultures and experimental assays. By defining rich sample and experimental metadata standards and promoting best practices in data descriptions, deposition and openness, FAANG champions higher quality and reusability of published datasets. FAANG has established a Data Coordination Centre, which sits at the heart of the Metadata and Data Sharing Committee. It continues to evolve the metadata standards, support submissions and, crucially, create powerful and accessible tools to support deposition and validation of metadata. FAANG conforms to the findable, accessible, interoperable, and reusable (FAIR) data principles, with high quality, open access and functionally interlinked data. In addition to data generated by FAANG members and specific FAANG projects, existing datasets that meet the main-or more permissive legacy-standards are incorporated into a central, focused, functional data resource portal for the entire farmed and companion animal community. Through clear and effective metadata standards, validation and conversion software, combined with promotion of best practices in metadata implementation, FAANG aims to maximise effectiveness and inter-comparability of assay data. This supports the community to create a rich genome-to-phenotype resource and promotes continuing improvements in animal data standards as a whole.

Isolation of subtelomeric sequences of porcine chromosomes for translocation screening reveals errors in the pig genome assembly.

Balanced chromosomal aberrations have been shown to affect fertility in most species studied, often leading to hypoprolificacy (reduced litter size) in domestic animals such as pigs. With an increasing emphasis in modern food production on the use of a small population of high quality males for artificial insemination, the potential economic and environmental costs of hypoprolific boars, bulls, rams etc. are considerable. There is therefore a need for novel tools to facilitate rapid, cost-effective chromosome translocation screening. This has previously been achieved by standard karyotype analysis; however, this approach relies on a significant level of expertise and is limited in its ability to identify subtle, cryptic translocations. To address this problem, we developed a novel device and protocol for translocation screening using subtelomeric probes and fluorescence in situ hybridisation. Probes were designed using BACs (bacterial artificial chromosomes) from the subtelomeric region of the short (p-arm) and long (q-arm) of each porcine chromosome. They were directly labelled with FITC or Texas Red (p-arm and q-arm respectively) prior to application of a 'Multiprobe' device, thereby enabling simultaneous detection of each individual porcine chromosome on a single slide. Initial experiments designed to isolate BACs in subtelomeric regions led to the discovery of a series of incorrectly mapped regions in the porcine genome assembly (from a total of 82 BACs, only 45 BACs mapped correctly). Our work therefore highlights the importance of accurate physical mapping of newly sequenced genomes. The system herein described allows for robust and comprehensive analysis of the porcine karyotype, an adjunct to classical cytogenetics that provides a valuable tool to expedite efficient, cost effective food production.

Efficiency of genomic prediction for boar taint reduction in Danish Landrace pigs.

Genetic selection against boar taint, which is caused by high skatole and androstenone concentrations in fat, is a more acceptable alternative than is the current practice of castration. Genomic predictors offer an opportunity to overcome the limitations of such selection caused by the phenotype being expressed only in males at slaughter, and this study evaluated different approaches to obtain such predictors. Samples from 1000 pigs were included in a design which was dominated by 421 sib pairs, each pair having one animal with high and one with low skatole concentration (≥0.3 µg/g). All samples were measured for both skatole and androstenone and genotyped using the Illumina SNP60 porcine BeadChip for 62 153 single nucleotide polymorphisms. The accuracy of predicting phenotypes was assessed by cross-validation using six different genomic evaluation methods: genomic best linear unbiased prediction (GBLUP) and five Bayesian regression methods. In addition, this was compared to the accuracy of predictions using only QTL that showed genome-wide significance. The range of accuracies obtained by different prediction methods was narrow for androstenone, between 0.29 (Bayes Lasso) and 0.31 (Bayes B), and wider for skatole, between 0.21 (GBLUP) and 0.26 (Bayes SSVS). Relative accuracies, corrected for h(2) , were 0.54-0.56 and 0.75-0.94 for androstenone and skatole respectively. The whole-genome evaluation methods gave greater accuracy than using only the QTL detected in the data. The results demonstrate that GBLUP for androstenone is the simplest genomic technology to implement and was also close to the most accurate method. More specialised models may be preferable for skatole.

A genome-wide linkage analysis for reproductive traits in F2 Large White × Meishan cross gilts.

Female reproductive performance traits in pigs have low heritabilities thus limiting improvement through traditional selective breeding programmes. However, there is substantial genetic variation found between pig breeds with the Chinese Meishan being one of the most prolific pig breeds known. In this study, three cohorts of Large White × Meishan F2 cross-bred pigs were analysed to identify quantitative trait loci (QTL) with effects on reproductive traits, including ovulation rate, teat number, litter size, total born alive and prenatal survival. A total of 307 individuals were genotyped for 174 genetic markers across the genome. The genome-wide analysis of the trait-recorded F2 gilts in their first parity/litter revealed one QTL for teat number significant at the genome level and a total of 12 QTL, which are significant at the chromosome-wide level, for: litter size (three QTL), total born alive (two QTL), ovulation rate (four QTL), prenatal survival (one QTL) and teat number (two QTL). Further support for eight of these QTL is provided by results from other studies. Four of these 12 QTL were mapped for the first time in this study: on SSC15 for ovulation rate and on SSC18 for teat number, ovulation rate and litter size.

CpG islands of chicken are concentrated on microchromosomes.

The chicken karyotype comprises 39 chromosome pairs of which at least 29 are 'microchromosomes'. Microchromosomes account for about 25% of the genomic DNA, but they are cytologically indistinguishable from one another (1). Due to technical limitations there is a strong bias of mapped genes within the chicken genome database ChickGBASE (2) towards macrochromosomes 1-6 and Z, with specific assignments to only one microchromosome (3,4). Several genes have, however, been assigned to the microchromosome group as a whole (3,5-9), demonstrating that these tiny chromosomes do not represent genetically inert DNA. To determine the overall chromosomal distribution of genes, as well as to provide a mapping resource, we prepared a CpG island library from chicken using differential binding to a methyl-CpG chicken using differential binding to a methyl-CpG binding column before and after de novo methylation (10). Surprisingly, we found that chicken CpG islands are highly concentrated on the microchromosomes, whereas macrochromosomes 1-6 are comparatively gene-poor by this assay. Our results raise the possibility that gene density on chicken microchromosomes approaches the maximum value known for vertebrates.

Mapping QTL in the porcine MHC region affecting fatness and growth traits in a Meishan/Large White composite population.

A number of studies have mapped QTL regulating porcine fatness and growth traits to the region of the major histocompatibility complex (MHC) on porcine chromosome 7 using various experimental crosses. The QTL results from crosses using the Chinese Meishan (MS) (slow growing and fat) are particularly interesting because the MS alleles have been found to be associated with increased growth rate and reduced backfat depth. We investigated these QTL further in a composite population derived previously over eight generations by intercrossing Meishan and the European Large White breeds. Genotype information from 32 markers in a 15cM target region was used in linkage and association analyses. A two-step variance component analysis identified QTL for three growth-related traits, explaining 19 ∼ 24% of the phenotypic variance with a confidence interval of 4 cM in the target region. SNP association analyses found that ss181128966 and ss181128924 within the QTL interval were strongly associated with the growth traits. Only weak signals for an effect on backfat depth were found in the association and linkage analyses, possibly because of past directional selection in the composite population.

An intronic polymorphism in the porcine IRF7 gene is associated with better health and immunity of the host during Sarcocystis infection, and affects interferon signalling.

Interferon regulatory factor 7 (IRF7), as a key regulator of type I interferon response, plays an important role during innate response against viral infection. Although well conserved across species, the structure of IRF7 and its function during parasite infection are not well documented in farm animals, such as the pig. To bridge this gap, we have determined the porcine IRF7 gene structure and identified two intronic single nucleotide polymorphisms (SNPs), SNP g.748G>C and SNP g.761A>G, in commercial pig breeds. The distribution of SNP g.761A>G in multiple breeds suggested that it was in Hardy-Weinberg equilibrium and allowed us to map it at the top of SSC2. We found that during Sarcocystis miescheriana infection, the G allele was associated with high lymphocyte levels (P < 0.02), reduced drop in platelet levels (P < 0.002) and IgG1-Th2-dominated response (P < 0.05). This suggests that the G allele was associated with better health and immunity of the host during Sarcocystis infection. Furthermore, we have also provided suggestive evidence that the G allele of SNPc.761A>G enhances the transactivation activity of IRF7, possibly by improving IRF7 transcript splicing of intron-3. These findings would suggest that IRF7, as a transcriptional regulator, is involved in the defence mechanism against a larger spectrum of pathogens, and in more host species, than initially anticipated.

Refined candidate region specified by haplotype sharing for Escherichia coli F4ab/F4ac susceptibility alleles in pigs.

Infection of the small intestine by enterotoxigenic Escherichia coli F4ab/ac is a major welfare problem and financial burden for the pig industry. Natural resistance to this infection is inherited as a Mendelian recessive trait, and a polymorphism in the MUC4 gene segregating for susceptibility/resistance is presently used in a selection programme by the Danish pig breeding industry. To elucidate the genetic background involved in E. coli F4ab/ac susceptibility in pigs, a detailed haplotype map of the porcine candidate region was established. This region covers approximately 3.7 Mb. The material used for the study is a three generation family, where the founders are two Wild boars and eight Large White sows. All pigs have been phenotyped for susceptibility to F4ab/ac using an adhesion assay. Their haplotypes are known from segregation analysis using flanking markers. By a targeted approach, the candidate region was subjected to screening for polymorphisms, mainly focusing on intronic sequences. A total of 18 genes were partially sequenced, and polymorphisms were identified in GP5, CENTB2, APOD, PCYT1A, OSTalpha, ZDHHC19, TFRC, ACK1, MUC4, MUC20, KIAA0226, LRCH3 and MUC13. Overall, 227 polymorphisms were discovered in the founder generation. The analysis revealed a large haplotype block, spanning at least 1.5 Mb around MUC4, to be associated with F4ab/ac susceptibility.

The sheep genome reference sequence: a work in progress.

Until recently, the construction of a reference genome was performed using Sanger sequencing alone. The emergence of next-generation sequencing platforms now means reference genomes may incorporate sequence data generated from a range of sequencing platforms, each of which have different read length, systematic biases and mate-pair characteristics. The objective of this review is to inform the mammalian genomics community about the experimental strategy being pursued by the International Sheep Genomics Consortium (ISGC) to construct the draft reference genome of sheep (Ovis aries). Component activities such as data generation, sequence assembly and annotation are described, along with information concerning the key researchers performing the work. This aims to foster future participation from across the research community through the coordinated activities of the consortium. The review also serves as a 'marker paper' by providing information concerning the pre-publication release of the reference genome. This ensures the ISGC adheres to the framework for data sharing established at the recent Toronto International Data Release Workshop and provides guidelines for data users.

Refined localization of the Escherichia coli F4ab/F4ac receptor locus on pig chromosome 13.

Diarrhoea in newborn and weaned pigs caused by enterotoxigenic Escherichia coli (ETEC) expressing F4 fimbriae leads to considerable losses in pig production. In this study, we refined the mapping of the receptor locus for ETEC F4ab/F4ac adhesion (F4bcR) by joint analysis of Nordic and Swiss data. A total of 236 pigs from a Nordic experimental herd, 331 pigs from a Swiss experimental herd and 143 pigs from the Swiss performing station were used for linkage analysis. Genotyping data of six known microsatellite markers, two newly developed markers (MUC4gt and HSA125gt) and an intronic SNP in MUC4 (MUC4-8227) were used to create the linkage map. The region for F4bcR was refined to the interval SW207-S0075 on pig chromosome 13. The most probable position of F4bcR was in the SW207-MUC4 region. The order of six markers was supported by physical mapping on the BAC fingerprint contig from the Wellcome Trust Sanger Institute. Thus, the region for F4bcR could be reduced from 26 to 14 Mb.

Mapping of the pig genome.

The past year has seen major developments in both the genetic (linkage) and physical maps of the porcine genome. Landmark loci have been established on all pig chromosomes and outline genetic maps have been elaborated. The use of pigs as models of human genetic diseases is likely to expand through the use of transgenic pigs.

Quantitative trait loci for production traits in pigs: a combined analysis of two Meishan x Large White populations.

Combined analysis of data from two or more resource populations can improve the power and accuracy of QTL mapping and allow some cross-validation of results. In this study, we performed a genome-wide scan using combined data from two F(2) populations derived from a cross between Large White and Chinese Meishan pigs. A total of 739 pigs were included in the analysis. In total 187 markers were genotyped in the two populations, including 115 markers genotyped in both populations, and these markers covered 2282 cM of the pig genome with an average of 13.58 cM between markers. Seven traits (teat number, birth weight, weaning weight, test-end weight, fat depth at shoulder, fat depth at mid back and fat depth at loin) were analysed for both individual populations and the combined population. There were 9 (2, 10), 1 (4, 4) and 14 (5, 18) QTL that achieved 1% genome-wide, 5% genome-wide and suggestive significance levels respectively in population 1 (population 2, combined population). Additive effects of QTL detected in the two populations at all significance levels were largely consistent suggesting that the QTL represent real genetic effects, but this was not the case for dominance or imprinting effects. There were also a number of significant interactions between detected QTL effects and population.

The cholecystokinin type A receptor g.179A>G polymorphism affects feeding rate.

A polymorphism within the 5' untranslated region of the cholecystokinin type A receptor (CCKAR) gene has been shown to affect feed intake and growth in commercial pig lines. To further investigate the phenotype of animals carrying alternative alleles at this polymorphism, we genotyped animals from a distinct segregating commercial line and an experimental cross F(2) population, both with electronically recorded feeding pattern data. The data indicate that the daily feed intake increasing effect of the DQ496228:g.179G allele is mediated through a faster rate of feed intake, without evidence for an effect on other feeding behaviour traits.

Characterization of the porcine KIT ligand gene: expression analysis, genomic structure, polymorphism detection and association with coat colour traits.

Kit ligand (KITLG) is the ligand for the type III receptor tyrosine kinase KIT. Studies of the KIT/KITLG pathway in a number of mammalian species have shown that it is important for the development of stem cell populations in haematopoietic tissues, germ cells in reproductive organs and the embryonic migrating melanoblasts that give rise to melanocytes. Consequently, mutations in the pathway may result in a range of defects including anaemia, sterility and de-pigmentation. The cDNA sequence of the porcine KITLG gene has been reported previously, and is an attractive candidate locus for moderating coat colour in pigs. In this paper we report the gene structure and physical mapping of the porcine gene. We also report the identification of polymorphisms in the gene, one of which was used to confirm linkage to chromosome 5. Preliminary RNA expression studies using a panel of tissues have shown that in addition to the known variant lacking exon 6, there is alternative splicing of exon 4. However, little evidence was found for the KITLG gene being linked to variation in colour in a Meishan x Large White cross.

Dynamic differential regulation of innate immune transcripts during the infection of alveolar macrophages by the porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome virus (PRRSV), an arterivirus, is the etiologic agent of an infectious disease of that name, characterized by respiratory disorders, abortion in pregnant sows and high mortality in piglets, resulting in significant economic losses in the pig industry worldwide. In order to identify whether genetic differences in PRRSV response may exist in pigs, alveolar macrophages were used to assess the progression of the type-I interferon (IFN) transcript response in porcine alveolar macrophages infected by PRRSV. Our results suggest that a dynamic differential regulation of the type-I IFN and chemokine transcripts may operate during the first hours of infection with and entry of the virus in alveolar macrophages, and provide a compelling mechanism for the establishment of PRRSV replication in susceptible cells.

A novel inactivated vaccine against Lawsonia intracellularis induces rapid induction of humoral immunity, reduction of bacterial shedding and provides robust gut barrier function.

Porcine proliferative ileitis is a major economic burden for the swine industry, affecting growing pigs and young adult pigs. In this study, the protective efficacy of an inactivated, injectable whole-cell bacteria vaccine against L. intracellularis - Porcilis® Ileitis was evaluated under field conditions. Eighty-five, three-week-old pigs on a commercial farrow-to-finish farm were vaccinated by the intramuscular route, either with a dose of injectable vaccine, or with saline. A subset of vaccinates and control pigs were necropsied at 21 days post-challenge. Incidence and severity of ileitis were evaluated by gross and microscopic observation of ileal tissues. Colonization of the gut after challenge was examined by L. intracellularis-specific immunohistochemistry, and qPCR of ileal scrapings. Integrity of the intestinal barrier was evaluated to quantify a range of intestinal markers including secreted mucin and intestinal alkaline phosphatase, and innate immune markers including Caspase-3 and Calprotectin. A second subset of pigs was monitored for fecal shedding of L. intracellularis, until resolution of shedding. Our investigation indicated that Porcilis Ileitis provided robust protection against ileitis, reduced bacterial shedding 15-fold (p < .05) and preserved normal gut barrier function in the face of an experimental challenge with virulent L. intracellularis.

Livestock genetics. Fat pigs can blame their genes.

Contrary to the predictions of some sceptics, livestock do have genes with significant effects on their economically important traits; isolating these genes should be facilitated by emerging maps of the pig genome.

A polymorphism in the 5'-untranslated region of the porcine cholecystokinin type a receptor gene affects feed intake and growth.

The location and utilization of quantitative trait loci (QTL) and candidate genes with significant effects on economically important traits are becoming increasingly important in livestock breeding programs. The porcine cholecystokinin type A receptor (CCKAR) is a candidate gene for performance traits, due to its known role in the physiological control of feed intake, satiety, and obesity. We investigated the association of CCKAR polymorphisms with feeding, growth, and efficiency traits in an F2 population derived from a cross between Meishan and Large White founder animals and in lines of Large White pigs that had been divergently selected on the basis of lean growth efficiency traits. In the F2 population, CCKAR genotype was significantly associated with daily feed intake and average daily gain. The effects of the polymorphisms were then assessed in a larger-scale analysis of segregating commercial lines. A newly discovered single-nucleotide polymorphism (SNP) within the 5'-untranslated region (5'-UTR) had highly significant effects on feed intake, average daily gain, and days to 110 kg, which were not seen for a previously reported SNP within the CCKAR gene. Furthermore, we provide evidence that the novel SNP disrupts the binding of the YY1 transcription factor, which raises the possibility that it is the causal variant. The 5'-UTR SNP could be utilized as a molecular genetic test for increased feed intake, faster lean growth, and reduced days to market weight in segregating commercial lines.

The ARKdb: genome databases for farmed and other animals.

The ARKdb genome databases provide comprehensive public repositories for genome mapping data from farmed species and other animals (http://www.thearkdb.org) providing a resource similar in function to that offered by GDB or MGD for human or mouse genome mapping data, respectively. Because we have attempted to build a generic mapping database, the system has wide utility, particularly for those species for which development of a specific resource would be prohibitive. The ARKdb genome database model has been implemented for 10 species to date. These are pig, chicken, sheep, cattle, horse, deer, tilapia, cat, turkey and salmon. Access to the ARKdb databases is effected via the World Wide Web using the ARKdb browser and Anubis map viewer. The information stored includes details of loci, maps, experimental methods and the source references. Links to other information sources such as PubMed and EMBL/GenBank are provided. Responsibility for data entry and curation is shared amongst scientists active in genome research in the species of interest. Mirror sites in the United States are maintained in addition to the central genome server at Roslin.

Is inosine the physiological energy source of pig erythrocytes?

Pig erythrocytes are unable to metabolize glucose and their physiological energy source is unknown. These cells have a high-capacity nucleoside transport system with similar properties to that responsible for nucleoside transport in other species. Nucleoside transport is sufficiently rapid to allow the possibility that inosine and/or adenosine may represent major energy substrates for pig erythrocytes in vivo. Normal and adenosine deaminase-deficient pig erythrocytes have similar ATP levels, suggesting that adenosine is not important in this respect. However, it was calculated that an extracellular inosine concentration of only 40 nM could support the cells' entire energy requirement, a value 40-fold lower than plasma levels of this nucleoside.