Unmapped sequencing reads in livestock often contain valuable information about pathogens but are typically discarded. We analyzed blood-derived DNA and RNA from chickens and pigs kept under high- and low-biosecurity conditions, focusing on unmapped reads. In chickens, low-biosecurity farms harbored substantially more viral sequences, primarily plant viruses, indicating environmental contamination. In pigs, Mycoplasmoides pneumoniae and several pig-specific viruses were detected. Our bioinformatics pipeline, involving host read removal, assembly, BLAST, and taxonomic filtering, efficiently identified candidate pathogens and contaminants. This approach demonstrates the potential of sequencing-based environmental DNA monitoring to track microbial and viral presence, assess farm biosecurity, and support animal health surveillance.

Abstract Background Recessive lethal alleles causing pre- or postnatal death in homozygous mutant animals, could lead to reduced fertility success. The Friesian horse breed has signs of reduced fertility and has faced high inbreeding rates in the past (∆F > 1%). Consequently, by genetic drift lethal alleles may have reached moderate to high frequencies in the population. Our aim was to identify lethal recessive alleles that — when homozygous — may cause pre- or postnatal death in the Friesian horse. Results We analyzed genotypes (70 K SNP) of over 8,000 Friesian horses, looking for haplotypes with a homozygous deficiency, and used available sequence data of 50 Friesian sires to pinpoint the likely causal variant. A deficit in homozygous animals suggests a lethal allele, because individuals inheriting two copies of such an allele likely die before birth or die before being genotyped, creating a detectable imbalance in genotype frequencies. We found ten candidate haplotypes in the Friesian horse with carrier frequencies ranging from 8.0 to 22.1%. We identified candidate causal variants of six haplotypes, of which two were associated with the already known genetic disorders dwarfism and hydrocephalus. The other candidate variants were a 261-kilobase-pair deletion affecting several non-coding RNA’s, and a 14-base-pair frameshift deletion in the MET gene. Three haplotypes in LD comprised the deletion in MET and were associated with a 25% reduction (P < 0.001) in insemination success in risk matings, likely caused by early embryonic lethality. Conclusions In general, considering the population characteristics of domestic horse breeds, we strongly recommend performing such analyses in other horse breeds using the increasingly available genotype data. Such analyses could provide important contribution to the improvement of fertility rates in horse populations.

In horses, genetic diversity is predominantly observed between breeds, with little variation within breeds. The studbooks of the two largest horse populations in the Netherlands, the Dutch Warmblood horse and Friesian horse population, have ongoing conservation projects including collecting large-scale genotype and sequence data. The current reference genome, derived from a Thoroughbred horse can lead to bias in genetic analyses of other horse breeds. Therefore, the aim of this study was to create high-quality breed-specific reference genomes of Dutch Warmblood and Friesian horses. We performed nanopore long-read sequencing (R10.4, Q20+) of an F1 cross between a Dutch Warmblood horse and a Friesian horse to create two breed-specific reference genomes by trio binning. This resulted in high-quality, haplotype-resolved reference genomes with contig N50 of 37 and 35 Mb and single copy gene completeness of 99.2 and 99.3

Native cattle breeds in Europe are vital to agricultural heritage and livestock production, combining adaptation to diverse environments with desirable traits such as high-quality beef and milk. To investigate genetic diversity, local adaptation, and productivity-related characteristics, we generated whole-genome sequences from 289 cattle representing 11 native breeds and the commercial Holstein-Friesian breed across Finland, the Netherlands, and Portugal. These breeds span diverse climates and management systems, from cold northern regions to Mediterranean environments in southern Europe. The dataset comprises over 11 terabytes of paired-end Illumina NovaSeq6000 sequencing data, with an average depth of 10 × and an alignment rate of 99.7

Most deleterious variants are recessive and segregate at relatively low frequency. Therefore, high sample sizes are required to identify these variants. In this study we report a large-scale sequence based genome-wide association study (GWAS) in pigs, with a total of 120,000 Large White and 80,000 Synthetic breed animals imputed to sequence using a reference population of approximately 1,100 whole genome sequenced pigs. We imputed over 20 million variants with high accuracies (R2>0.9) even for low frequency variants (1-5% minor allele frequency). This sequence-based analysis revealed a total of 14 additive and 9 non-additive significant quantitative trait loci (QTLs) for growth rate and backfat thickness. With the non-additive (recessive) model, we identified a deleterious missense SNP in the CDHR2 gene reducing growth rate and backfat in homozygous Large White animals. For the Synthetic breed, we revealed a QTL on chromosome 15 with a frameshift variant in the OBSL1 gene. This QTL has a major impact on both growth rate and backfat, resembling human 3M-syndrome 2 which is related to the same gene. With the additive model, we confirmed known QTLs on chromosomes 1 and 5 for both breeds, including variants in the MC4R and CCND2 genes. On chromosome 1, we disentangled a complex QTL region with multiple variants affecting both traits, harboring 4 independent QTLs in the span of 5 Mb. Together we present a large scale sequence-based association study that provides a key resource to scan for novel variants at high resolution for breeding and to further reduce the frequency of deleterious alleles at an early stage in the breeding program.

BackgroundIntegration of high throughput DNA genotyping and RNA-sequencing data enables the discovery of genomic regions that regulate gene expression, known as expression quantitative trait loci (eQTL). In pigs, efforts to date have been mainly focused on purebred lines for traits with commercial relevance as such growth and meat quality. However, little is known on genetic variants and mechanisms associated with the robustness of an animal, thus its overall health status. Here, the liver, lung, spleen, and muscle transcriptomes of 100 three-way crossbred female finishers were studied, with the aim of identifying novel eQTL regulatory regions and transcription factors (TFs) associated with regulation of porcine metabolism and health-related traits.ResultsAn expression genome-wide association study with 535,896 genotypes and the expression of 12,680 genes in liver, 13,310 genes in lung, 12,650 genes in spleen, and 12,595 genes in muscle resulted in 4,293, 10,630, 4,533, and 6,871 eQTL regions for each of these tissues, respectively. Although only a small fraction of the eQTLs were annotated as cis-eQTLs, these presented a higher number of polymorphisms per region and significantly stronger associations with their target gene compared to trans-eQTLs. Between 20 and 115 eQTL hotspots were identified across the four tissues. Interestingly, these were all enriched for immune-related biological processes. In spleen, two TFs were identified: ERF and ZNF45, with key roles in regulation of gene expression.ConclusionsThis study provides a comprehensive analysis with more than 26,000 eQTL regions identified that are now publicly available. The genomic regions and their variants were mostly associated with tissue-specific regulatory roles. However, some shared regions provide new insights into the complex regulation of genes and their interactions that are involved with important traits related to metabolism and immunity.

The pig IPEC-J2 and chicken SL-29 cell lines are of interest because of their untransformed nature and wide use in functional studies. Molecular characterization of these cell lines is important to gain insight into possible molecular aberrations. The aim of this paper is to provide a molecular and epigenetic characterization of the IPEC-J2 and SL-29 cell lines, a cell-line reference for the FAANG community, and future biomedical research. Whole genome sequencing, gene expression, DNA methylation, chromatin accessibility, and ChIP-seq of four histone marks (H3K4me1, H3K4me3, H3K27ac, H3K27me3) and an insulator (CTCF) are used to achieve these aims. Heteroploidy (aneuploidy) of various chromosomes was observed from whole genome sequencing analysis in both cell lines. Furthermore, higher gene expression for genes located on chromosomes with aneuploidy in comparison to diploid chromosomes was observed. Regulatory complexity of gene expression, DNA methylation, and chromatin accessibility was investigated through an integrative approach.

Understanding the genetic basis of similar phenotypes shared between lineages is a long-lasting research interest. Even though animal evolution offers many examples of parallelism, for many phenotypes little is known about the underlying genes and mutations. We here use a combination of whole-genome sequencing, expression analyses, and comparative genomics to study the parallel genetic origin of ptilopody (Pti) in chicken. Ptilopody (or foot feathering) is a polygenic trait that can be observed in domesticated and wild avian species and is characterized by the partial or complete development of feathers on the ankle and feet. In domesticated birds, ptilopody is easily selected to fixation, though extensive variation in the type and level of feather development is often observed. By means of a genome-wide association analysis, we identified two genomic regions associated with ptilopody. At one of the loci, we identified a 17-kb deletion affecting PITX1 expression, a gene known to encode a transcription regulator of hindlimb identity and development. Similarly to pigeon, at the second loci, we observed ectopic expression of TBX5, a gene involved in forelimb identity and a key determinant of foot feather development. We also observed that the trait evolved only once as foot-feathered birds share the same haplotype upstream TBX5. Our findings indicate that in chicken and pigeon ptilopody is determined by the same set of genes that affect similar molecular pathways. Our study confirms that ptilopody has evolved through parallel evolution in chicken and pigeon.

The geographic distributions of some coastal marine species have appeared as cosmopolitan ever since they were first scientifically documented. In particular, for many benthic species that are associated with anthropogenic substrata, there is much speculation as to whether or not their broad distributions can be explained by natural mechanisms of dispersal. Here, we focused on two congeneric coastal crustaceans with cosmopolitan distributions—the tube-dwelling amphipods Jassa marmorata and Jassa slatteryi. Both species are common elements of marine biofouling on nearly all kinds of artificial hard substrata in temperate to warm seas. We hypothesized that the two species’ modern occurrences across the oceans are the result of human shipping activities that started centuries ago. Mitochondrial DNA sequences of the CO1 fragment of specimens from distinct marine regions around the world were analysed, evaluating genetic structure and migration models and making inferences on putative native ranges of the two Jassa species. Populations of both species exhibited considerable genetic diversity with differing levels of geographic structure. For both species, at least two dominant haplotypes were shared among several geographic populations. Rapid demographic expansion and high migration rates between geographically distant regions support a scenario of ongoing dispersal all over the world. Our findings indicate that the likely former native range of J. marmorata is the Northwest Atlantic, whereas the likely former native range of J. slatteryi is the Northern Pacific region. As corroborated by the genetic connectivity between populations, shipping still appears to be the more successful vector of the two species’ dispersal when compared to natural mechanisms. Historical invasion events that likely started centuries ago, along with current ongoing dispersal, confirm these species’ identities as true “neocosmopolitans”.

Abstract Chromosome inversions have clear effects on genome evolution and have been associated with speciation, adaptation, and the evolution of the sex chromosomes. In birds, these inversions may play an important role in hybridization of species and disassortative mating. We identified a large (≈64 Mb) inversion polymorphism in the great tit (Parus major) that encompasses almost 1,000 genes and more than 90% of Chromosome 1A. The inversion occurs at a low frequency in a set of over 2,300 genotyped great tits in the Netherlands with only 5% of the birds being heterozygous for the inversion. In an additional analysis of 29 resequenced birds from across Europe, we found two heterozygotes. The likely inversion breakpoints show considerable genomic complexity, including multiple copy number variable segments. We identified different haplotypes for the inversion, which differ in the degree of recombination in the center of the chromosome. Overall, this remarkable genetic variant is widespread among distinct great tit populations and future studies of the inversion haplotype, including how it affects the fitness of carriers, may help to understand the mechanisms that maintain it.

Piglet mortality is a complex phenotype that depends on the environment, selection on piglet health, but also on the interaction between the piglet and sow. However, also monogenic recessive defects contribute to piglet mortality. Selective breeding has decreased overall piglet mortality by improving both mothering abilities and piglet viability. However, variants underlying recessive monogenic defects are usually not well captured within the breeding values, potentially drifting to higher frequency as a result of intense selection or genetic drift. This study describes the identification by whole-genome sequencing of a recessive 16-bp deletion in the SPTBN4 gene causing postnatal mortality in a pig breeding line. The deletion induces a frameshift and a premature stop codon, producing an impaired and truncated spectrin beta non-erythrocytic 4 protein (SPTBN4). Applying medium density single nucleotide polymorphism (SNP) data available for all breeding animals, a pregnant carrier sow sired by a carrier boar was identified. Of the resulting piglets, two confirmed homozygous piglets suffered from severe myopathy, hind-limb paralysis, and tremors. Histopathological examination showed dispersed degeneration and decrease of cross-striations in the dorsal and hind-limb muscle fibers of the affected piglets. Hence, the affected piglets are unable to walk or drink, usually resulting in death within a few hours after birth. This study demonstrates how growing genomic resources in pig breeding can be applied to identify rare syndromes in breeding populations, that are usually poorly documented and often are not even known to have a genetic basis. The study allows to prevent carrier-by-carrier matings, thereby gradually decreasing the frequency of the detrimental allele and avoiding the birth of affected piglets, improving animal welfare. Finally, these "natural knockouts" increase our understanding of gene function within the mammalian clade, and provide a potential model for human disease.

qPCR validation of CNVs. Genomic regions validated, qPCR-PennCNV state correspondence and applied primers. (CSV 192 kb)

Background Understanding variation in genome structure is essential to understand phenotypic differences within populations and the evolutionary history of species. A promising form of this structural variation is copy number variation (CNV). CNVs can be generated by different recombination mechanisms, such as non-allelic homologous recombination, that rely on specific characteristics of the genome architecture. These structural variants can therefore be more abundant at particular genes ultimately leading to variation in phenotypes under selection. Detailed characterization of CNVs therefore can reveal evolutionary footprints of selection and provide insight in their contribution to phenotypic variation in wild populations. Results Here we use genotypic data from a long-term population of great tits ( Parus major ), a widely studied passerine bird in ecology and evolution, to detect CNVs and identify genomic features prevailing within these regions. We used allele intensities and frequencies from high-density SNP array data from 2,175 birds. We detected 41,029 CNVs concatenated into 8,008 distinct CNV regions (CNVRs). We successfully validated 93.75% of the CNVs tested by qPCR, which were sampled at different frequencies and sizes. A mother-daughter family structure allowed for the evaluation of the inheritance of a number of these CNVs. Thereby, only CNVs with 40 probes or more display segregation in accordance with Mendelian inheritance, suggesting a high rate of false negative calls for smaller CNVs. As CNVRs are a coarse-grained map of CNV loci, we also inferred the frequency of coincident CNV start and end breakpoints. We observed frequency-dependent enrichment of these breakpoints at homologous regions, CpG sites and AT-rich intervals. A gene ontology enrichment analyses showed that CNVs are enriched in genes underpinning neural, cardiac and ion transport pathways. Conclusion Great tit CNVs are present in almost half of the genes and prominent at repetitive-homologous and regulatory regions. Although overlapping genes under selection, the high number of false negatives make neutrality or association tests on CNVs detected here difficult. Therefore, CNVs should be further addressed in the light of their false negative rate and architecture to improve the comprehension of their association with phenotypes and evolutionary history.

CNV regions (CNVRs) in the great tit (Parus major) genome. (CSV 333 kb)

The great tit is a widely studied passerine bird species in ecology that, in the past decades, has provided important insights into speciation, phenology, behavior and microevolution. After completion of the great tit genome sequence, a customized high density 650k SNP array was developed enabling more detailed genomic studies in this species.

Enrichment analysis of genes in CNV breakpoints (frequency â Ľâ 4). (TXT 3 kb)

Our work use the WGS data present the molecular genetic evidence for a novel mutation potentially underlying autosomal dwarfism in chicken. The identification of the adw mutation provides the basis for future studies towards dwarf status in different species, as well as the functional role of TMEM263 in growth and developmental pathways. Autosomal dwarfism (adw) in chicken is known as a growth deficiency caused by a recessive mutation. The features of autosomal dwarfism are known as a proportionally 30% growth reduction with short shank length. The adw variant was first recognized in the Cornell K-strain of White Leghorns but the genetic causal variant remained unknown. To detect the underlying causal variant underlying the trait of adw, fine mapping was conducted based on previous linkage research on chromosome 1. We found a nonsense mutation in the transmembrane protein 263 gene (TMEM263) that is completely associated with the autosomal dwarf phenotype. Variants were detected by comparing whole-genome sequencing data from white leghorns vs adw chicken. Many potential variants were identified but after filtering for the known variant with variant databases, only one potential variant remained associated with autosomal dwarfism. A stop gain variant in TMEM263 is found unique in dwarf chicken and absent in normal-sized controls. In human, TMEM263 is associated with bone mineral density and the protein interacts with growth hormone 1. Therefore the nonsense mutation in TMEM263 likely leads to a protein truncation and therefore affects its function.

BACKGROUND:Many common and relevant diseases affecting equine welfare have yet to be tested regarding structural variants such as copy number variations (CNVs). CNVs make up a substantial proportion of total genetic variability in populations of many species, resulting in more sequence differences between individuals than SNPs. Associations between CNVs and disease phenotypes have been established in several species, but equine CNV studies have been limited. Aim of this study was to identify CNVs and to perform a genome-wide association (GWA) study in Friesian horses to identify genomic loci associated with insect bite hypersensitivity (IBH), a common seasonal allergic dermatitis observed in many horse breeds worldwide.RESULTS:Genotypes were obtained using the Axiom® Equine Genotyping Array containing 670,796 SNPs. After quality control of genotypes, 15,041 CNVs and 5350 CNV regions (CNVRs) were identified in 222 Friesian horses. Coverage of the total genome by CNVRs was 11.2% with 49.2% of CNVRs containing genes. 58.0% of CNVRs were novel (i.e. so far only identified in Friesian horses). A SNP- and CNV-based GWA analysis was performed, where about half of the horses were affected by IBH. The SNP-based analysis showed a highly significant association between the MHC region on ECA20 and IBH in Friesian horses. Associations between the MHC region on ECA20 and IBH were also detected based on the CNV-based analysis. However, CNVs associated with IBH in Friesian horses were not often in close proximity to SNPs identified to be associated with IBH.CONCLUSIONS:CNVs were identified in a large sample of the Friesian horse population, thereby contributing to our knowledge on CNVs in horses and facilitating our understanding of the equine genome and its phenotypic expression. A clear association was identified between the MHC region on ECA20 and IBH in Friesian horses based on both SNP- and CNV-based GWA studies. These results imply that MHC contributes to IBH sensitivity in Friesian horses. Although subsequent analyses are needed for verification, nucleotide differences, as well as more complex structural variations like CNVs, seem to contribute to IBH sensitivity. IBH should be considered as a common disease with a complex genomic architecture.

5350 CNVRs detected by CNVRuler based on 15,041 CNVs identified by PennCNV in 222 Friesian horses. 5350 CNVRs detected by CNVRuler based on 15,041 CNVs identified in PennCNV [33â 35] in 222 Friesian horses. Information that is presented includes CNVR identification (CNVR_ID), Equus caballus chromosome (ECA), start position (in bp), end position (in bp), size (in bp), copy number state, number of horses (N) with CNVR and whether the CNVR includes a gene(s) and is present in 1 horse (private) or more (shared). The gene(s) located within the CNVR were identified using human orthologues. Start and end position of the genes annotated in the horse genome are presented, including Ensembl IDs and whether the CNVR is upstream, inside, downstream of the gene or encompasses the gene. (XLSX 603 kb)

Autosomal dwarfism (adw) in chickens is a growth deficiency caused by a recessive mutation. Characteristic for adw is an approximately 30% growth reduction with short shank. The adw variant was first recognized in the Cornell K-strain of White Leghorns, but the genetic causal variant remained unknown. To identify the causal variant underlying the adw phenotype, fine mapping was conducted on chromosome 1, within 52–56 Mb. This region was known to harbor the causal variant from previous linkage studies. We compared whole-genome sequence data of this region from normal-sized and adw chickens in order to find the unique causal variant. We identified a novel nonsense mutation NP_001006244.1:p.(Trp59∗), in the transmembrane protein 263 gene (TMEM263), completely associated with adw. The nonsense mutation truncates the transmembrane protein within the membrane-spanning domain, expected to cause a dysfunctional protein. TMEM263 is reported to be associated with bone mineral deposition in humans, and the protein shows interaction with growth hormone 1 (GH1). Our study presents molecular genetic evidence for a novel loss-of-function variant, which likely alters body growth and development in autosomal dwarf chicken.