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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.

BackgroundThe 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.ResultsBy 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.ConclusionThe 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.

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 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.

The Functional Annotation of Animal Genomes (FAANG) Consortium recently held a Gathering On FAANG (GO-FAANG) Workshop in Washington, DC on October 7-8, 2015. This consortium is a grass-roots organization formed to advance the annotation of newly assembled genomes of domesticated and non-model organisms (www.faang.org). The workshop gathered together from around the world a group of 100+ genome scientists, administrators, representatives of funding agencies and commodity groups to discuss the latest advancements of the consortium, new perspectives, next steps and implementation plans. The workshop was streamed live and recorded, and all talks, along with speaker slide presentations, are available at www.faang.org. In this report, we describe the major activities and outcomes of this meeting. We also provide updates on ongoing efforts to implement discussions and decisions taken at GO-FAANG to guide future FAANG activities. In summary, reference datasets are being established under pilot projects; plans for tissue sets, morphological classification and methods of sample collection for different tissues were organized; and core assays and data and meta-data analysis standards were established.

Chicken developmental mutants are valuable for discovering sequences and pathways controlling amniote development. Herein we applied the advanced technologies of targeted sequence genomic capture enrichment and next-generation sequencing to discover the causative element for three inherited mutations affecting craniofacial, limb and/or organ development. Since the mutations (coloboma, diplopodia-1 and wingless-2) were bred into a congenic line series and previously mapped to different chromosomes, each targeted mutant causative region could be compared to that of the other two congenic partners, thereby providing internal controls on a single array. Of the ~73 million 50-bp sequence reads, ~76% were specific to the enriched targeted regions with an average target coverage of 132-fold. Analysis of the three targeted regions (2.06 Mb combined) identified line-specific single nucleotide polymorphism (SNPs) and micro (1-3 nt) indels. Sequence content for regions indicated as gaps in the reference genome was generated, thus contributing to its refinement. Additionally, Mauve alignments were constructed and indicated putative chromosomal rearrangements. This is the first report of targeted capture array technology in an avian species, the chicken, an important vertebrate model; the work highlights the utility of employing advanced technologies in an organism with only a "draft stage" reference genome sequence.

Abstract Background A robust bacterial artificial chromosome (BAC)-based physical map is essential for many aspects of genomics research, including an understanding of chromosome evolution, high-resolution genome mapping, marker-assisted breeding, positional cloning of genes, and quantitative trait analysis. To facilitate turkey genetics research and better understand avian genome evolution, a BAC-based integrated physical, genetic, and comparative map was developed for this important agricultural species. Results The turkey genome physical map was constructed based on 74,013 BAC fingerprints (11.9 × coverage) from two independent libraries, and it was integrated with the turkey genetic map and chicken genome sequence using over 41,400 BAC assignments identified by 3,499 overgo hybridization probes along with > 43,000 BAC end sequences. The physical-comparative map consists of 74 BAC contigs, with an average contig size of 13.6 Mb. All but four of the turkey chromosomes were spanned on this map by three or fewer contigs, with 14 chromosomes spanned by a single contig and nine chromosomes spanned by two contigs. This map predicts 20 to 27 major rearrangements distinguishing turkey and chicken chromosomes, despite up to 40 million years of separate evolution between the two species. These data elucidate the chromosomal evolutionary pattern within the Phasianidae that led to the modern turkey and chicken karyotypes. The predominant rearrangement mode involves intra-chromosomal inversions, and there is a clear bias for these to result in centromere locations at or near telomeres in turkey chromosomes, in comparison to interstitial centromeres in the orthologous chicken chromosomes. Conclusion The BAC-based turkey-chicken comparative map provides novel insights into the evolution of avian genomes, a framework for assembly of turkey whole genome shotgun sequencing data, and tools for enhanced genetic improvement of these important agricultural and model species.

Abstract Background Herpesviruses are a major health concern for numerous organisms, including humans, causing both acute and chronic infections recurrent over an individual's lifespan. Marek's disease virus (MDV) is a highly contagious herpesvirus which causes a neoplastic condition in chicken populations. Several vertebrate-infecting herpesviruses have been shown to exist in an integrated state during latent periods of infection. However the status of MDV during latency has been a topic of debate. Results Here we employed high-resolution multi-color fluorescence in situ hybridization (FISH) to show integration of MDV at the telomeres of chicken chromosomes. Cytogenomic mapping of the chromosomal integrations allowed us to examine the clonal relationships among lymphomas within individuals, whereas analysis of tumors from multiple individuals indicated the potential for chromosomal preferences. Conclusions Our data highlight that substantive genome-level interactions between the virus and host exist, and merit consideration for their potential impact and role in key aspects of herpesvirus pathobiology including infection, latency, cellular transformation, latency-breaks and viral evolution.

This study investigated telomeric array organization of diverse chicken genotypes utilizing in vivo and in vitro cells having phenotypes with different proliferation potencies. Our experimental objective was to characterize the extent and nature of array variation present to explore the hypothesis that mega-telomeres are a universal and fixed feature of chicken genotypes. Four different genotypes were studied including normal (UCD 001, USDA-ADOL Line 0), immortalized (DF-1), and transformed (DT40) cells. Both cytogenetic and molecular approaches were utilized to develop an integrated view of telomeric array organization. It was determined that significant variation exists within and among chicken genotypes for chromosome-specific telomeric array organization and total genomic-telomeric sequence content. Although there was variation for mega-telomere number and distribution, two mega-telomere loci were in common among chicken genetic lines (GGA 9 and GGA W). The DF-1 cell line was discovered to maintain a complex derivative karyotype involving chromosome fusions in the homozygous and heterozygous condition. Also, the DF-1 cell line was found to contain the greatest amount of telomeric sequence per genome (17%) as compared to UCD 001 (5%) and DT40 (1.2%). The chicken is an excellent model for studying unique and universal features of vertebrate telomere biology, and characterization of the telomere length variation among genotypes will be useful in the exploration of mechanisms controlling telomere length maintenance in different cell types having unique phenotypes.

Author(s): Marie-Cecile van de Lavoir [1]; Jennifer H. Diamond [1]; Philip A. Leighton [1]; Christine Mather-Love [1]; Babette S. Heyer [1]; Renee Bradshaw [1]; Allyn Kerchner [1]; Lisa T. Hooi [...]

Abstract Background A robust bacterial artificial chromosome (BAC)-based physical map is essential for many aspects of genomics research, including an understanding of chromosome evolution, high-resolution genome mapping, marker-assisted breeding, positional cloning of genes, and quantitative trait analysis. To facilitate turkey genetics research and better understand avian genome evolution, a BAC-based integrated physical, genetic, and comparative map was developed for this important agricultural species. Results The turkey genome physical map was constructed based on 74,013 BAC fingerprints (11.9 × coverage) from two independent libraries, and it was integrated with the turkey genetic map and chicken genome sequence using over 41,400 BAC assignments identified by 3,499 overgo hybridization probes along with > 43,000 BAC end sequences. The physical-comparative map consists of 74 BAC contigs, with an average contig size of 13.6 Mb. All but four of the turkey chromosomes were spanned on this map by three or fewer contigs, with 14 chromosomes spanned by a single contig and nine chromosomes spanned by two contigs. This map predicts 20 to 27 major rearrangements distinguishing turkey and chicken chromosomes, despite up to 40 million years of separate evolution between the two species. These data elucidate the chromosomal evolutionary pattern within the Phasianidae that led to the modern turkey and chicken karyotypes. The predominant rearrangement mode involves intra-chromosomal inversions, and there is a clear bias for these to result in centromere locations at or near telomeres in turkey chromosomes, in comparison to interstitial centromeres in the orthologous chicken chromosomes. Conclusion The BAC-based turkey-chicken comparative map provides novel insights into the evolution of avian genomes, a framework for assembly of turkey whole genome shotgun sequencing data, and tools for enhanced genetic improvement of these important agricultural and model species.

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. [ABSTRACT FROM AUTHOR]