A comprehensive characterization of regulatory elements in the chicken genome across tissues will have substantial impacts on both fundamental and applied research. Here, we systematically identified and characterized regulatory elements in the chicken genome by integrating 377 genome-wide sequencing datasets from 23 adult tissues. In total, we annotated 1.57 million regulatory elements, representing 15 distinct chromatin states, and predicted about 1.2 million enhancer-gene pairs and 7662 super-enhancers. This functional annotation of the chicken genome should have wide utility on identifying regulatory elements accounting for gene regulation underlying domestication, selection, and complex trait regulation, which we explored. In short, this comprehensive atlas of regulatory elements provides the scientific community with a valuable resource for chicken genetics and genomics.

MHCY is a second major histocompatibility complex-like gene region in chickens originally identified by the presence of major histocompatibility complex class I-like and class II-like gene sequences. Up to now, the MHCY gene region has been poorly represented in genomic sequence data. A high density of repetitive sequence and multiple members of several gene families prevented the accurate assembly of short-read sequence data for MHCY. Identified here by single-molecule real-time sequencing sequencing of BAC clones for the Gallus gallus Red Jungle Fowl reference genome are 107 MHCY region genes (45 major histocompatibility complex class I-like, 41 c-type-lectin-like, 8 major histocompatibility complex class II beta, 8 LENG9-like, 4 zinc finger protein loci, and a single only zinc finger-like locus) located amid hundreds of retroelements within 4 contigs representing the region. Sequences obtained for nearby ribosomal RNA genes have allowed MHCY to be precisely mapped with respect to the nucleolar organizer region. Gene sequences provide insights into the unusual structure of the MHCY class I molecules. The MHCY class I loci are polymorphic and group into 22 types based on predicted amino acid sequences. Some MHCY class I loci are full-length major histocompatibility complex class I genes. Others with altered gene structure are considered gene candidates. The amino acid side chains at many of the polymorphic positions in MHCY class I are directed away rather than into the antigen-binding groove as is typical of peptide-binding major histocompatibility complex class I molecules. Identical and nearly identical blocks of genomic sequence contribute to the observed multiplicity of identical MHCY genes and the large size (>639 kb) of the Red Jungle Fowl MHCY haplotype. Multiple points of hybridization observed in fluorescence in situ hybridization suggest that the Red Jungle Fowl MHCY haplotype is made up of linked, but physically separated genomic segments. The unusual gene content, the evidence of highly similar duplicated segments, and additional evidence of variation in haplotype size distinguish polymorphic MHCY from classical polymorphic major histocompatibility complex regions.

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.

The avian α-herpesvirus known as Marek’s disease virus (MDV) linearly integrates its genomic DNA into host telomeres during infection. The resulting disease, Marek’s disease (MD), is characterized by virally-induced lymphomas with high mortality. The temporal dynamics of MDV-positive (MDV+) transformed cells and expansion of MD lymphomas remain targets for further understanding. It also remains to be determined whether specific host chromosomal sites of MDV telomere integration confer an advantage to MDV-transformed cells during tumorigenesis. We applied MDV-specific fluorescence in situ hybridization (MDV FISH) to investigate virus-host cytogenomic interactions within and among a total of 37 gonad lymphomas and neoplastic splenic samples in birds infected with virulent MDV. We also determined single-cell, chromosome-specific MDV integration profiles within and among transformed tissue samples, including multiple samples from the same bird. Most mitotically-dividing cells within neoplastic samples had the cytogenomic phenotype of ‘MDV telomere-integrated only’, and tissue-specific, temporal changes in phenotype frequencies were detected. Transformed cell populations composing gonad lymphomas exhibited significantly lower diversity, in terms of heterogeneity of MDV integration profiles, at the latest stages of tumorigenesis (>50 days post-infection (dpi)). We further report high interindividual and lower intraindividual variation in MDV integration profiles of lymphoma cells. There was no evidence of integration hotspots into a specific host chromosome(s). Collectively, our data suggests that very few transformed MDV+ T cell populations present earlier in MDV-induced lymphomas (32–50 dpi), survive, and expand to become the dominant clonal population in more advanced MD lymphomas (51–62 dpi) and establish metastatic lymphomas.

Background The homologous recombination (HR) pathway is largely inactive in early embryos prior to the first cell division, making it difficult to achieve targeted gene knock-ins. The homology-mediated end joining (HMEJ)-based strategy has been shown to increase knock-in efficiency relative to HR, non-homologous end joining (NHEJ), and microhomology-mediated end joining (MMEJ) strategies in non-dividing cells. Results By introducing gRNA/Cas9 ribonucleoprotein complex and a HMEJ-based donor template with 1 kb homology arms flanked by the H11 safe harbor locus gRNA target site, knock-in rates of 40% of a 5.1 kb bovine sex-determining region Y ( SRY )-green fluorescent protein ( GFP ) template were achieved in Bos taurus zygotes. Embryos that developed to the blastocyst stage were screened for GFP, and nine were transferred to recipient cows resulting in a live phenotypically normal bull calf. Genomic analyses revealed no wildtype sequence at the H11 target site, but rather a 26 bp insertion allele, and a complex 38 kb knock-in allele with seven copies of the SRY-GFP template and a single copy of the donor plasmid backbone. An additional minor 18 kb allele was detected that looks to be a derivative of the 38 kb allele resulting from the deletion of an inverted repeat of four copies of the SRY-GFP template. Conclusion The allelic heterogeneity in this biallelic knock-in calf appears to have resulted from a combination of homology directed repair, homology independent targeted insertion by blunt-end ligation, NHEJ, and rearrangement following editing of the gRNA target site in the donor template. This study illustrates the potential to produce targeted gene knock-in animals by direct cytoplasmic injection of bovine embryos with gRNA/Cas9, although further optimization is required to ensure a precise single-copy gene integration event.

SummaryAn embryonic lethal mutation in chicken named cleft primary palate (cpp) is inherited in an autosomal recessive mode and results in a severely truncated upper beak. In this study, genotyping and sequencing techniques were employed to advance our genetic and genomic knowledge of the mutation’s chromosomal location, candidate region and possible causative element using a congenic inbred line. Herein, the candidate region for the cpp developmental mutation was established as a ca. 5.1 Mb region of chicken chromosome 11 (GGA 11) through the use of a 600K Affymetrix SNP array. The SNPs identified from this array linked to cpp were used to genotype individuals from the congenic inbred line over several generations and thereby fine‐map the causative region resulting in an approximately 200 kb size reduction. This candidate region (4.9 Mb) was sequenced via capture array in a cohort of 24 individuals, including carriers, mutants and their wild type (wt) siblings. Interestingly, the GGA 11 region for cpp encompasses the predicted centromere location and is thus unlikely to be highly disrupted by further recombination. Here we report on the variation unique to the cpp mutation, i.e. single‐nucleotide variants and insertions or deletions. Although the candidate region contains several genes of interest with regard to the cpp phenotype, only one cpp‐linked variant was predicted to have a significant physiological effect by causing a frameshift mutation in ESRP2, which has a role in tissue‐specific splicing during development.

The use of Assay for Transposase-Accessible Chromatin (ATAC-seq) to profile chromatin accessibility has surged over the past years, but its applicability to tissues has been very limited. With the intent of preserving nuclear architecture during long-term storage, cryopreserved nuclei preparations from chicken lung were used to optimize ATAC-seq. Sequencing data were compared with existing DNase-seq, ChIP-seq, and RNA-seq data to evaluate library quality, ultimately resulting in a modified ATAC-seq method capable of generating high quality chromatin accessibility data from cryopreserved nuclei preparations. Using this method, nucleosome-free regions (NFR) identified in chicken lung overlapped half of DNase-I hypersensitive sites, coincided with active histone modifications, and specifically marked actively expressed genes. Notably, sequencing only the subnucleosomal fraction dramatically improved signal, while separation of subnucleosomal reads post-sequencing did not improve signal or peak calling. The broader applicability of this modified ATAC-seq technique was tested using cryopreserved nuclei preparations from pig tissues, resulting in NFR that were highly consistent among biological replicates. Furthermore, tissue-specific NFR were enriched for binding motifs of transcription factors related to tissue-specific functions, and marked genes functionally enriched for tissue-specific processes. Overall, these results provide insights into the optimization of ATAC-seq and a platform for profiling open chromatin in animal tissues.

The chicken wingless-2 (wg-2) mutation is inherited in an autosomal recessive fashion, and the resulting phenotype in mutant (wg-2/wg-2) individuals is a developmental syndrome characterized by absent wings, truncated legs, craniofacial as well as skin and feather defects, and kidney malformations. Mapping and genotyping established that the mutation resides within 227 kilobases (kb) of chromosome 12 in a wg-2 congenic inbred line. A capture array was designed to target and sequence the candidate region along with flanking DNA in 24 birds from the line. Many point mutations and insertions or deletions were identified, and analysis of the linked variants indicated a point mutation predicted to cause a premature stop codon in the RAF1 gene. Expression studies were conducted inclusive of all genes in the candidate region. Interestingly, RAF1 transcription was elevated, yet the protein was absent in the mutants relative to normal individuals. RAF1 encodes a protein integral to the Ras/Raf/MAPK signaling pathway controlling cellular proliferation, and notably, human RASopathies are developmental syndromes caused by germline mutations in genes of this pathway. Our work indicates RAF1 as the priority candidate causative gene for wg-2 and provides a new animal model to study an important signaling pathway implicated in limb development, as well as RASopathies.

Next-generation sequencing (NGS) and expression technologies were utilized to investigate the genes and sequence elements in a 586 kb region of chicken chromosome 1 associated with the autosomal recessive diplopodia-1 (dp-1) mutation. This mutation shows a syndromic phenotype similar to known human developmental abnormalities (e.g., cleft palate, polydactyly, omphalocele [exposed viscera]). Toward our goal to ascertain the variant responsible, the entire 586 kb region was sequenced following utilization of a specifically designed capture array and to confirm/validate fine-mapping results. Bioinformatic analyses identified a total of 6142 sequence variants, which included SNPs, indels, and gaps. Of these, 778 SNPs, 146 micro-indels, and 581 gaps were unique to the UCD-Dp-1.003 inbred congenic line; those found within exons and splice sites were studied for contribution to the mutant phenotype. Upon further validation with additional mutant samples, a smaller subset (of variants [51]) remains linked to the mutation. Additionally, utilization of specific samples in the NGS technology was advantageous in that fine-mapping methodologies eliminated an additional 326 kb of sequence information on chromosome 1. Predicted and confirmed protein-coding genes within the smaller 260 kb region were assessed for their developmental expression patterns over several stages of early embryogenesis in regions/tissues of interest (e.g., digits, craniofacial region). Based on these results and known function in other vertebrates, 2 genes within 5 kb of each other, MRE11 and GPR83, are proposed as high-priority candidates for the dp-1 mutation.

Background Numerous long non-coding RNAs (lncRNAs) have been identified and their roles in gene regulation in humans, mice, and other model organisms studied; however, far less research has been focused on lncRNAs in farm animal species. While previous studies in chickens, cattle, and pigs identified lncRNAs in specific developmental stages or differentially expressed under specific conditions in a limited number of tissues, more comprehensive identification of lncRNAs in these species is needed. The goal of the FAANG Consortium (Functional Annotation of Animal Genomes) is to functionally annotate animal genomes, including the annotation of lncRNAs. As one of the FAANG pilot projects, lncRNAs were identified across eight tissues in two adult male biological replicates from chickens, cattle, and pigs. Results Comprehensive lncRNA annotations for the chicken, cattle, and pig genomes were generated by utilizing RNA-seq from eight tissue types from two biological replicates per species at the adult developmental stage. A total of 9393 lncRNAs in chickens, 7235 lncRNAs in cattle, and 14,429 lncRNAs in pigs were identified. Including novel isoforms and lncRNAs from novel loci, 5288 novel lncRNAs were identified in chickens, 3732 in cattle, and 4870 in pigs. These transcripts match previously known patterns of lncRNAs, such as generally lower expression levels than mRNAs and higher tissue specificity. An analysis of lncRNA conservation across species identified a set of conserved lncRNAs with potential functions associated with chromatin structure and gene regulation. Tissue-specific lncRNAs were identified. Genes proximal to tissue-specific lncRNAs were enriched for GO terms associated with the tissue of origin, such as leukocyte activation in spleen. Conclusions LncRNAs were identified in three important farm animal species using eight tissues from adult individuals. About half of the identified lncRNAs were not previously reported in the NCBI annotations for these species. While lncRNAs are less conserved than protein-coding genes, a set of positionally conserved lncRNAs were identified among chickens, cattle, and pigs with potential functions related to chromatin structure and gene regulation. Tissue-specific lncRNAs have potential regulatory functions on genes enriched for tissue-specific GO terms. Future work will include epigenetic data from ChIP-seq experiments to further refine these annotations.

Wingless-2 (wg-2) is an autosomal recessive mutation in chicken that results in an embryonic lethal condition. Affected individuals exhibit a multisystem syndrome characterized by absent wings, truncated legs, and craniofacial, kidney, and feather malformations. Previously, work focused on phenotype description, establishing the autosomal recessive pattern of Mendelian inheritance and placing the mutation on an inbred genetic background to create the congenic line UCD Wingless-2.331. The research described in this paper employed the complementary tools of breeding, genetics, and genomics to map the chromosomal location of the mutation and successively narrow the size of the region for analysis of the causative element. Specifically, the wg-2 mutation was initially mapped to a 7 Mb region of chromosome 12 using an Illumina 3 K SNP array. Subsequent SNP genotyping and exon sequencing combined with analysis from improved genome assemblies narrowed the region of interest to a maximum size of 227 kb. Within this region, 3 validated and 3 predicted candidate genes are found, and these are described. The wg-2 mutation is a valuable resource to contribute to an improved understanding of the developmental pathways involved in chicken and avian limb development as well as serving as a model for human development, as the resulting syndrome shares features with human congenital disorders.

Marek's disease (MD) is an infectious disease characterized by lymphomas and high mortality in susceptible chickens. The causative and ubiquitous alpha-herpesvirus known as MD virus (MDV) integrates into host telomeres during early infection through latency, known to be an important phase for oncogenic transformation. Herein, we sought to determine the influence of vaccination and host genetics on the temporal dynamics of MDV-host genome interactions. We studied integration profiles using 2 MD vaccines that vary in protective efficacy in 2 genetic lines that differ in MD resistance/susceptibility. Virus integration of both oncogenic MDV and vaccine strains was observed in both MD susceptible and resistant birds, however, the lines differed in their dynamic telomere-integration profiles. Notably, the resistant host genotype exhibited a smaller percentage of replicating cells with the virus telomere-integrated only phenotype as compared to the susceptible genotype. Vaccination with Rispens, the most protective MD vaccine, also reduced the establishment of the virus telomere-integrated only phenotype, suggesting a significant role of the phenotype in MD lymphoma development. The effect of Rispens vaccination was most dramatic in the susceptible genotype. These results suggest important connections between vaccinal immunity, MDV telomere integration, virus-induced oncogenesis, and virus-host genome interactions in the context of host genetics and disease susceptibility.

Wingless-2 (wg-2) is an autosomal recessive mutation in chicken that results in an embryonic lethal condition. Affected individuals exhibit a multisystem syndrome characterized by absent wings, truncated legs, and craniofacial, kidney, and feather malformations. Previously, work focused on phenotype description, establishing the autosomal recessive pattern of Mendelian inheritance and placing the mutation on an inbred genetic background to create the congenic line UCD Wingless-2.331. The research described in this paper employed the complementary tools of breeding, genetics, and genomics to map the chromosomal location of the mutation and successively narrow the size of the region for analysis of the causative element. Specifically, the wg-2 mutation was initially mapped to a 7 Mb region of chromosome 12 using an Illumina 3 K SNP array. Subsequent SNP genotyping and exon sequencing combined with analysis from improved genome assemblies narrowed the region of interest to a maximum size of 227 kb. Within this region, 3 validated and 3 predicted candidate genes are found, and these are described. The wg-2 mutation is a valuable resource to contribute to an improved understanding of the developmental pathways involved in chicken and avian limb development as well as serving as a model for human development, as the resulting syndrome shares features with human congenital disorders.

Multiple Sequence Alignments for Conserved LncRNAs. A multiple sequence alignment file generated by ClustalW is included for each of the 39 groups of lncRNAs associated with orthologous genes across all five species. (ZIP 445 kb)

Marek’s disease (MD), a virally-induced lymphoproliferative disease of chickens epitomized by T cell lymphomas, maintains as a premier threat to the world’s poultry industries. Despite control measures like widespread usage of MD vaccines and biosecurity, new and more virulent strains of Marek’s disease virus (MDV), the causative agent, have repeatedly arisen resulting in disease outbreaks. With the most efficacious MD vaccine in use for 10–15 yr, there is fear that more virulent field strains will emerge soon as well as the need for improved control strategies. Therefore, there is a critical need to understand MD tumorigenesis and the genomic landscapes inside tumors to utilize genetic resistance as a sustainable control MD measure. To identify somatic mutations driving tumorigenesis, highly inbred experimental White Leghorn lines that were MD resistant or susceptible were intermated to produce a cohort of F1 chickens that were challenged with MDV, and the most homogenous tumors and matching normal tissues collected. To date, most samples have been screened to generate four primary datasets: (1) whole genome sequencing analysis, (2) whole transcriptome sequencing analysis, (3) 15K SNP microarray analysis, and (4) cytogenetic analysis to identify MDV integration sites. Collectively these data should allow for the robust characterization of somatic cancer-associated variants including single nucleotide variants (SNVs), short insertions and deletions (indels), structural variations (SVs), copy number variations (CNVs), and loss-of-heterozygosity (LOH). Thus far with DNA resequencing of 26 MD tumors (and matched normal tissues), 6 different somatic single nucleotide variant callers have repeatedly identified ∼300 SNPs per tumor sample, many of which are associated cancer-genes in humans. The low number of variants increases our chances of finding causative driver gene(s). Furthermore, the low somatic mutation rate (∼0.3 SNPs/Mb), which is similar to human juvenile cancers, also suggests a significant genetic component. Further refinements in the computational pipelines as well as integration with the other datasets should enable the identification of key driving genes for MD as well as information on somatic mutation frequency, somatic mutation signatures, and significantly mutated regions. Collectively these cancer-associated inferences can be used to understand the genetic forces and players involved in tumorigenesis and tumor progression to develop sustainable genetic resistance strategies, e.g., genomic selection. While a work in progress, the latest results and analyses will be presented as well as the challenges to implement computational tools developed primarily for the biomedical community.

The technologies and assays developed in the human and mouse ENCODE projects provide a solid foundation to functionally annotate domesticated animal genomes. Chicken, pig, and cattle are major farm animals for providing global food production. Robust functional annotations of their genomes could be leveraged to improve their production efficiency and animal and human health. A recent international FAANG (Functional Annotation of Animal Genomes) initiative has stimulated new efforts in functional annotation in these species. The overall objective of this study is to functionally annotate regulatory elements in 3 livestock species. The first key step is to identify regulatory elements in the genomes by integrating RNA-seq, DNase-seq, and ChIP-seq data from important tissues. We will present current progress in generating and analyzing data, including analysis of 48 RNA-seq libraries (16 per species) collected from 2 biological replicates across 8 tissues: adipose, cerebellum, cortex, hypothalamus, liver, lung, muscle, and spleen. For chicken, an analysis of 15 DNase-seq data from all issues with 2 replicates, except hypothalamus (one replicate), show that identified tissue-specific DNase I hypersensitivity (DHS) sites are associated with genes that relate to the unique biological functions of the organs or tissues. Using the 2 replicates to construct a set of DHS sites present in each replicate, we find 29,190 sites in cerebellum, 43,672 in cortex, 52,337 in liver, 64,149 in lung, 27,433 in muscle, and 63,605 in spleen. In addition, 24 ChIP-seq data sets from all tissues (2 replicates), except adipose and muscle (H3K4me3 and H3K27me3 histone modification marks), were generated. For H3K4me3, 19,940 peaks were shared among replicates in cerebellum, 9,979 in cortex, 6,104 in hypothalamus, 5,872 in liver, 29,891 in lung, and 20,323 in spleen. For H3K27me3, 14,000 peaks were shared in cerebellum, 7,525 in cortex, 16 in hypothalamus, 7,642 in liver, 5,807 in lung, and 4,326 in spleen. Integrative analysis of these data sets should allow the identification of genome-wide active and inactive promoter regions, enabling an in-depth comparison of the regulatory landscapes of multiple tissues within chicken and ultimately across the 3 livestock species. This work will lead to a better understanding of the role regulatory elements play on how the genotype of organisms determines the phenotype.

Marek’s disease (MD) is a lymphotropic and oncogenic disease of chickens that can lead to death in susceptible and unvaccinated host birds. The causative pathogen, MD virus (MDV), a highly oncogenic alphaherpesvirus, integrates into host genome near the telomeres. MD occurrence is controlled across the globe by biosecurity, selective breeding for enhanced MD genetic resistance, and widespread vaccination of flocks using attenuated serotype 1 MDV or other serotypes. Despite over 40 years of usage, the specific mechanism(s) of MD vaccine-related immunity and anti-tumor effects are not known. Here we investigated the cytogenetic interactions of commonly used MD vaccine strains of all three serotypes (HVT, SB-1, and Rispens) with the host to determine if all were equally capable of host genome integration. We also studied the dynamic profiles of chromosomal association and integration of the three vaccine strains, a first for MD vaccine research. Our cytogenetic data provide evidence that all three MD vaccine strains tested integrate in the chicken host genome as early as 1 day after vaccination similar to oncogenic strains. However, a specific, transformation-associated virus-host phenotype observed for oncogenic viruses is not established. Our results collectively provide an updated model of MD vaccine-host genome interaction and an improved understanding of the possible mechanisms of vaccinal immunity. Physical integration of the oncogenic MDV genome into host chromosomes along with cessation of viral replication appears to have joint signification in MDV’s ability to induce oncogenic transformation. Whereas for MD vaccine serotypes, a sustained viral replication stage and lack of the chromosome-integrated only stage were shared traits during early infection.

Marek's Disease Virus (MDV) is a chicken alphaherpesvirus that causes paralysis, chronic wasting, blindness, and fatal lymphoma development in infected, susceptible host birds. This disease and its protective vaccines are highly relevant research targets, given their enormous impact within the poultry industry. Further, Marek's disease (MD) serves as a valuable model for the investigation of oncogenic viruses and herpesvirus patterns of viral latency and persistence—as pertinent to human health as to poultry health. The objectives of this article are to review MDV interactions with its host from a variety of genomic, molecular, and cellular perspectives. In particular, we focus on cytogenetic studies, which precisely assess the physical status of the MDV genome in the context of the chicken host genome. Combined, the cytogenetic and genomic research indicates that MDV-host genome interactions, specifically integration of the virus into the host telomeres, is a key feature of the virus life cycle, contributing to the viral achievement of latency, transformation, and reactivation of lytic replication. We present a model that outlines the variety of virus-host interactions, at the multiple levels, and with regard to the disease states.