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Gene regulatory elements are central drivers of phenotypic variation and thus of critical importance towards understanding the genetics of complex traits. The Functional Annotation of Animal Genomes consortium was formed to collaboratively annotate the functional elements in animal genomes, starting with domesticated animals. Here we present an expansive collection of datasets from eight diverse tissues in three important agricultural species: chicken ( Gallus gallus ), pig ( Sus scrofa ), and cattle ( Bos taurus ). Comparative analysis of these datasets and those from the human and mouse Encyclopedia of DNA Elements projects reveal that a core set of regulatory elements are functionally conserved independent of divergence between species, and that tissue-specific transcription factor occupancy at regulatory elements and their predicted target genes are also conserved. These datasets represent a unique opportunity for the emerging field of comparative epigenomics, as well as the agricultural research community, including species that are globally important food resources. In order to interpret non-coding variants, information about regulatory elements in the genome is essential. Here, the authors annotate regulatory elements in chicken, pig and cattle, and characterize conservation of these elements between species.

Many livestock and human vaccines are leaky because they block symptoms but do not prevent infection or onward transmission. This leakiness is concerning because it increases vaccination coverage required to prevent disease spread and can promote evolution of increased pathogen virulence. Despite leakiness, vaccination may reduce pathogen load, affecting disease transmission dynamics. However, the impacts on post-transmission disease development and infectiousness in contact individuals are unknown. Here, we use transmission experiments involving Marek disease virus (MDV) in chickens to show that vaccination with a leaky vaccine substantially reduces viral load in both vaccinated individuals and unvaccinated contact individuals they infect. Consequently, contact birds are less likely to develop disease symptoms or die, show less severe symptoms, and shed less infectious virus themselves, when infected by vaccinated birds. These results highlight that even partial vaccination with a leaky vaccine can have unforeseen positive consequences in controlling the spread and symptoms of disease.

Background: In livestock species like the chicken, high throughput single nucleotide polymorphism (SNP) genotyping assays are increasingly being used for whole genome association studies and as a tool in breeding (referred to as genomic selection). To be of value in a wide variety of breeds and populations, the success rate of the SNP genotyping assay, the distribution of the SNP across the genome and the minor allele frequencies (MAF) of the SNPs used are extremely important. Results: We describe the design of a moderate density (60k) Illumina SNP BeadChip in chicken consisting of SNPs known to be segregating at high to medium minor allele frequencies (MAF) in the two major types of commercial chicken (broilers and layers). This was achieved by the identification of 352,303 SNPs with moderate to high MAF in 2 broilers and 2 layer lines using Illumina sequencing on reduced representation libraries. To further increase the utility of the chip, we also identified SNPs on sequences currently not covered by the chicken genome assembly (Gallus_gallus-2.1). This was achieved by 454 sequencing of the chicken genome at a depth of 12x and the identification of SNPs on 454-derived contigs not covered by the current chicken genome assembly. In total we added 790 SNPs that mapped to 454-derived contigs as well as 421 SNPs with a position on Chr_random of the current assembly. The SNP chip contains 57,636 SNPs of which 54,293 could be genotyped and were shown to be segregating in chicken populations. Our SNP identification procedure appeared to be highly reliable and the overall validation rate of the SNPs on the chip was 94%. We were able to map 328 SNPs derived from the 454 sequence contigs on the chicken genome. The majority of these SNPs map to chromosomes that are already represented in genome build Gallus_gallus-2.1.0. Twenty-eight SNPs were used to construct two new linkage groups most likely representing two micro-chromosomes not covered by the current genome assembly. Conclusions: The high success rate of the SNPs on the Illumina chicken 60K Beadchip emphasizes the power of Next generation sequence (NGS) technology for the SNP identification and selection step. The identification of SNPs from sequence contigs derived from NGS sequencing resulted in improved coverage of the chicken genome and the construction of two new linkage groups most likely representing two chicken micro-chromosomes.

Marek’s disease (MD) is a lymphoproliferative disease of chickens induced by Marek’s disease virus (MDV), an oncogenic α-herpesvirus. MDV has increased in virulence, prompting continued efforts in both improved vaccines and enhanced genetic resistance. Model pairs of genetically MD-resistant and MD-susceptible chickens that were either MHC-matched or MHC-congenic allowed characterization of T cell receptor (TCR) repertoires associated with MDV infection. MD-resistant chickens showed higher usage of Vβ-1 TCRs than susceptible chickens in both the CD8 and CD4 subsets in the MHC-matched model, and in the CD8 subset only in the MHC-congenic model, with a shift towards Vβ-1+ CD8 cells during MDV infection. Long and short read sequencing identified divergent TCRβ loci between MHC-matched MD-resistant and MD-susceptible chickens, with MD-resistant chickens having more TCR Vβ1 genes. TCR Vβ1 CDR1 haplotype usage in MD-resistant x MD-susceptible F1 birds by RNAseq indicated that the most commonly used CDR1 variant was unique to the MD-susceptible line, suggesting that selection for MD resistance in the MHC-matched model optimized the TCR repertoire away from dominant recognition of one or more B2 haplotype MHC molecules. Finally, TCR downregulation during MDV infection in the MHC-matched model was strongest in the MD-susceptible line, and MDV reactivation downregulated TCR expression in a tumor cell line.

Genetically resistant or susceptible chickens to Marek’s disease (MD) have been widely used models to identify the molecular determinants of these phenotypes. However, these prior studies lacked the basic identification and understanding of immune cell types that could be translated toward improved MD control. To gain insights into specific immune cell types and their responses to Marek’s disease virus (MDV) infection, we used single-cell RNA sequencing (scRNAseq) on splenic cells from MD resistant and susceptible birds. In total, 14,378 cells formed clusters that identified various immune cell types. Lymphocytes, specifically T cell subtypes, were the most abundant with significant proportional changes in some subtypes upon infection. The largest number of differentially expressed genes (DEG) response was seen in granulocytes, while macrophage DEGs differed in directionality by subtype and line. Among the most DEG in almost all immune cell types were granzyme and granulysin, both associated with cell-perforating processes. Protein interactive network analyses revealed multiple overlapping canonical pathways within both lymphoid and myeloid cell lineages. This initial estimation of the chicken immune cell type landscape and its accompanying response will greatly aid efforts in identifying specific cell types and improving our knowledge of host response to viral infection.

Marek’s disease (MD) is a lymphoproliferative disease of chickens caused by Marek’s disease virus (MDV), an oncogenic α-herpesvirus. Since 1970, MD has been controlled by widespread vaccination; however, more effective MD vaccines are needed to counter more virulent MDV strains. The bivalent vaccine combination of SB-1 and herpesvirus of turkey (HVT) strain FC126 has been widely used. Nonetheless, the mechanism(s) underlying this synergistic effect has not been investigated. Three experiments were conducted where SB-1 or HVT were administered as monovalent or bivalent vaccines to newly hatched chickens, then challenged five days later with MDV. In Experiment 1, levels of MDV replication in PBMCs were measured over time, and tumor incidence and vaccinal protection determined. In Experiment 2, MDV and vaccine strains replication levels in lymphoid organs were measured at 1, 5, 10, and 14 days post-challenge (DPC). In Experiment 3, to verify that the bursa was necessary for HVT protection, a subset of chicks were bursectomized and these birds plus controls were similarly vaccinated and challenged, and the levels of vaccinal protection determined. The efficacy of bivalent SB-1 + HVT surpasses that of either SB-1 or HVT monovalent vaccines in controlling the level of pathogenic MDV in PBMCs until the end of the study, and this correlated with the ability to inhibit tumor formation. SB-1 replication in the spleen increased from 1 to 14 DPC, while HVT replicated only in the bursa at 1 DPC. The bursa was necessary for immune protection induced by HVT vaccine. Synergy of SB-1 and HVT vaccines is due to additive influences of the individual vaccines acting at different times and target organs. And the bursa is vital for HVT to replicate and induce immune protection.

Necrotic enteritis (NE) is an important intestinal infectious disease of commercial poultry flocks caused by Clostridium perfringens. Using an experimental model of NE involving co-infection with C. perfringens and Eimeria maxima, transcriptome profiling and functional genomics approaches were applied to identify the genetic mechanisms that might regulate the host response to this disease. Microarray hybridization identified 1,049 transcripts whose levels were altered (601 increased, 448 decreased) in intestinal lymphocytes from C. perfringens/E. maxima co-infected Ross chickens compared with uninfected controls. Five biological functions, all related to host immunity and inflammation, and 11 pathways were identified from this dataset. To further elucidate the role of host genetics in NE susceptibility, two inbred chicken lines, ADOL line 6 and line 7 which share an identical B2 major histocompatibility complex haplotype but differ in their susceptibility to virus infection, were compared for clinical symptoms and the expression levels of a panel of immune-related genes during experimental NE. Line 6 chickens were more susceptible to development of experimental NE compared with line 7, as revealed by decreased body weight gain and increased E. maxima oocyst shedding. Of 21 immune-related genes examined, 15 were increased in C. perfringens/E. maxima co-infected line 6 vs. line 7 chickens. These results suggest that immune pathways are activated in response to experimental NE infection and that genetic determinants outside of the chicken B complex influence resistance to this disease.

Marek’s disease (MD) is a highly infectious lymphoproliferative disease in chickens with a significant economic impact. Mardivirus gallidalpha 2, also known as Marek’s disease virus (MDV), is the causative pathogen and has been categorized based on its virulence rank into four pathotypes: mild (m), virulent (v), very virulent (vv), and very virulent plus (vv+). A prior comparative genomics study suggested that several single-nucleotide polymorphisms (SNPs) and genes in the MDV genome are associated with virulence, including nonsynonymous (ns) SNPs in eight open reading frames (ORF): UL22, UL36, UL37, UL41, UL43, R-LORF8, R-LORF7, and ICP4. To validate the contribution of these nsSNPs to virulence, the vv+MDV strain 686 genome was modified by replacing nucleotides with those observed in the vMDV strains. Pathogenicity studies indicated that these substitutions reduced the MD incidence and increased the survival of challenged birds. Furthermore, using the best-fit pathotyping method to rank the virulence, the modified vv+MDV 686 viruses resulted in a pathotype similar to the vvMDV Md5 strain. Thus, these results support our hypothesis that SNPs in one or more of these ORFs are associated with virulence but, as a group, are not sufficient to result in a vMDV pathotype, suggesting that there are additional variants in the MDV genome associated with virulence, which is not surprising given this complex phenotype and our previous finding of additional variants and SNPs associated with virulence.

Breed utilization, genetic improvement, and industry consolidation are predicted to have major impacts on the genetic composition of commercial chickens. Consequently, the question arises as to whether sufficient genetic diversity remains within industry stocks to address future needs. With the chicken genome sequence and more than 2.8 million single-nucleotide polymorphisms (SNPs), it is now possible to address biodiversity using a previously unattainable metric: missing alleles. To achieve this assessment, 2551 informative SNPs were genotyped on 2580 individuals, including 1440 commercial birds. The proportion of alleles lacking in commercial populations was assessed by (1) estimating the global SNP allele frequency distribution from a hypothetical ancestral population as a reference, then determining the portion of the distribution lost, and then (2) determining the relationship between allele loss and the inbreeding coefficient. The results indicate that 50% or more of the genetic diversity in ancestral breeds is absent in commercial pure lines. The missing genetic diversity resulted from the limited number of incorporated breeds. As such, hypothetically combining stocks within a company could recover only preexisting within-breed variability, but not more rare ancestral alleles. We establish that SNP weights act as sentinels of biodiversity and provide an objective assessment of the strains that are most valuable for preserving genetic diversity. This is the first experimental analysis investigating the extant genetic diversity of virtually an entire agricultural commodity. The methods presented are the first to characterize biodiversity in terms of allelic diversity and to objectively link rate of allele loss with the inbreeding coefficient.

Marek’s disease (MD) is an economically significant disease in chickens caused by the highly oncogenic Marek’s disease virus (MDV). Understanding the genes and biological pathways that confer MD genetic resistance should lead towards the development of more disease resistant commercial poultry flocks or improved MD vaccines. MDV mEq, a bZIP transcription factor, is largely attributed to viral oncogenicity though only a few host target genes have been described, which has impeded our understanding of MDV-induced tumorigenesis. Given the importance of mEq in MDV-induced pathogenesis, we explored the role of mEq in genetic resistance to MDV. Using global transcriptome analysis and cells from MD resistant or susceptible birds, we compared the response to infection with either wild type MDV or a nononcogenic recombinant lacking mEq. As a result, we identified a number of specific genes and pathways associated with either MD resistance or susceptibility. Additionally, integrating prior information from ChIP-seq, microarray analysis, and SNPs exhibiting allele-specific expression (ASE) in response to MDV infection, we were able to provide evidence for 24 genes that are polymorphic within mEq binding sites are likely to account for gene expression in an allele-specific manner and potentially for the underlying genetic differences in MD incidence.