An atlas of regulatory elements in chicken: A resource for chicken genetics and genomics.

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.

The Gallus gallus RJF reference genome reveals an MHCY haplotype organized in gene blocks that contain 107 loci including 45 specialized, polymorphic MHC class I loci, 41 C-type lectin-like loci, and other loci amid hundreds of transposable elements.

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 IIß, 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.

Marek's Disease Virus Telomeric Integration Profiles of Neoplastic Host Tissues Reveal Unbiased Chromosomal Selection and Loss of Cellular Diversity during Tumorigenesis.

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.

Functional annotations of three domestic animal genomes provide vital resources for comparative and agricultural research.

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.

One-step generation of a targeted knock-in calf using the CRISPR-Cas9 system in bovine zygotes.

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.

A Premature Stop Codon in RAF1 Is the Priority Candidate Causative Mutation of the Inherited Chicken Wingless-2 Developmental Syndrome.

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.

Two Proximally Close Priority Candidate Genes for diplopodia-1, an Autosomal Inherited Craniofacial-Limb Syndrome in the Chicken: MRE11 and GPR83.

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.

Vaccination and Host Marek's Disease-Resistance Genotype Significantly Reduce Oncogenic Gallid alphaherpesvirus 2 Telomere Integration in Host Birds.

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.

Marek's disease herpesvirus vaccines integrate into chicken host chromosomes yet lack a virus-host phenotype associated with oncogenic transformation.

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.

GO-FAANG meeting: a Gathering On Functional Annotation of Animal Genomes.

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.

Utilizing the chicken as an animal model for human craniofacial ciliopathies.

The chicken has been a particularly useful model for the study of craniofacial development and disease for over a century due to their relatively large size, accessibility, and amenability for classical bead implantation and transplant experiments. Several naturally occurring mutant lines with craniofacial anomalies also exist and have been heavily utilized by developmental biologist for several decades. Two of the most well known lines, talpid(2) (ta(2)) and talpid(3) (ta(3)), represent the first spontaneous mutants to have the causative genes identified. Despite having distinct genetic causes, both mutants have recently been identified as ciliopathic. Excitingly, both of these mutants have been classified as models for human craniofacial ciliopathies: Oral-facial-digital syndrome (ta(2)) and Joubert syndrome (ta(3)). Herein, we review and compare these two models of craniofacial disease and highlight what they have revealed about the molecular and cellular etiology of ciliopathies. Furthermore, we outline how applying classical avian experiments and new technological advances (transgenics and genome editing) with naturally occurring avian mutants can add a tremendous amount to what we currently know about craniofacial ciliopathies.

Using the avian mutant talpid2 as a disease model for understanding the oral-facial phenotypes of oral-facial-digital syndrome.

Oral-facial-digital syndrome (OFD) is a ciliopathy that is characterized by oral-facial abnormalities, including cleft lip and/or palate, broad nasal root, dental anomalies, micrognathia and glossal defects. In addition, these individuals have several other characteristic abnormalities that are typical of a ciliopathy, including polysyndactyly, polycystic kidneys and hypoplasia of the cerebellum. Recently, a subset of OFD cases in humans has been linked to mutations in the centriolar protein C2 Ca(2+)-dependent domain-containing 3 (C2CD3). Our previous work identified mutations in C2CD3 as the causal genetic lesion for the avian talpid(2) mutant. Based on this common genetic etiology, we re-examined the talpid(2) mutant biochemically and phenotypically for characteristics of OFD. We found that, as in OFD-affected individuals, protein-protein interactions between C2CD3 and oral-facial-digital syndrome 1 protein (OFD1) are reduced in talpid(2) cells. Furthermore, we found that all common phenotypes were conserved between OFD-affected individuals and avian talpid(2) mutants. In light of these findings, we utilized the talpid(2) model to examine the cellular basis for the oral-facial phenotypes present in OFD. Specifically, we examined the development and differentiation of cranial neural crest cells (CNCCs) when C2CD3-dependent ciliogenesis was impaired. Our studies suggest that although disruptions of C2CD3-dependent ciliogenesis do not affect CNCC specification or proliferation, CNCC migration and differentiation are disrupted. Loss of C2CD3-dependent ciliogenesis affects the dispersion and directional persistence of migratory CNCCs. Furthermore, loss of C2CD3-dependent ciliogenesis results in dysmorphic and enlarged CNCC-derived facial cartilages. Thus, these findings suggest that aberrant CNCC migration and differentiation could contribute to the pathology of oral-facial defects in OFD.

Temporal kinetics of Marek's disease herpesvirus: integration occurs early after infection in both B and T cells.

Marek's disease virus (MDV) is an oncogenic α-herpesvirus that induces Marek's disease characterized by fatal lymphomas in chickens. Here, we explored the timing during pathogenesis when the virus integrates into the host genome, the cell type involved, the role of viral integration on cellular transformation, and tumor clonality. Three immune organs of chicken (thymus, bursa, and spleen) were extracted following infection with either an oncogenic or a non-oncogenic strain of MDV. Using molecular cytogenetics, cells were investigated for viral integration at key time points throughout pathogenesis. Integration profiling of tumors (early to late stage) was conducted. Virus integration was widespread in B and T lymphocytes based on their abundance in bursa and thymus, respectively. Viral replication was detected early after infection as was viral integration into the host genome. Integration is a natural part of the MDV herpesvirus life cycle. In addition, our data using a non-oncogenic virus establish that although integration is a hallmark of tumor cell populations, integration alone is not sufficient for cellular transformation. Our results provide evidence for progression of lineage clonality within tumors. Understanding the features of integration provides insight into the mechanisms of herpesvirus pathology which could lead to disease mitigation strategies.

The cellular and molecular etiology of the craniofacial defects in the avian ciliopathic mutant talpid2.

talpid(2) is an avian autosomal recessive mutant with a myriad of congenital malformations, including polydactyly and facial clefting. Although phenotypically similar to talpid(3), talpid(2) has a distinct facial phenotype and an unknown cellular, molecular and genetic basis. We set out to determine the etiology of the craniofacial phenotype of this mutant. We confirmed that primary cilia were disrupted in talpid(2) mutants. Molecularly, we found disruptions in Hedgehog signaling. Post-translational processing of GLI2 and GLI3 was aberrant in the developing facial prominences. Although both GLI2 and GLI3 processing were disrupted in talpid(2) mutants, only GLI3 activator levels were significantly altered in the nucleus. Through additional fine mapping and whole-genome sequencing, we determined that the talpid(2) phenotype was linked to a 1.4 Mb region on GGA1q that contained the gene encoding the ciliary protein C2CD3. We cloned the avian ortholog of C2CD3 and found its expression was ubiquitous, but most robust in the developing limbs and facial prominences. Furthermore, we found that C2CD3 is localized proximal to the ciliary axoneme and is important for docking the mother centriole to the ciliary vesicle and cell membrane. Finally, we identified a 19 bp deletion in talpid(2) C2CD3 that produces a premature stop codon, and thus a truncated protein, as the likely causal allele for the phenotype. Together, these data provide insight into the cellular, molecular and genetic etiology of the talpid(2) phenotype. Our data suggest that, although the talpid(2) and talpid(3) mutations affect a common ciliogenesis pathway, they are caused by mutations in different ciliary proteins that result in differences in craniofacial phenotype.

Comparative cytogenomics of poultry: mapping of single gene and repeat loci in the Japanese quail (Coturnix japonica).

Well-characterized molecular and cytogenetic maps are yet to be established in Japanese quail (Coturnix japonica). The aim of the current study was to cytogenetically map and determine linkage of specific genes and gene complexes in Japanese quail through the use of chicken (Gallus gallus) and turkey (Meleagris gallopavo) genomic DNA probes and conduct a comparative study among the three genomes. Chicken and turkey clones were used as probes on mitotic metaphase and meiotic pachytene stage chromosomes of the three species for the purpose of high-resolution fluorescence in situ hybridization (FISH). The genes and complexes studied included telomerase RNA (TR), telomerase reverse transcriptase (TERT), 5S rDNA, 18S-5.8S-28S rDNA (i.e., nucleolus organizer region (NOR)), and the major histocompatibility complex (MHC). The telomeric profile of Japanese quail was investigated through the use of FISH with a TTAGGG-PNA probe. A range of telomeric array sizes were confirmed as found for the other poultry species. Three NOR loci were identified in Japanese quail, and single loci each for TR, TERT, 5S rDNA and the MHC-B. The MHC-B and one NOR locus were linked on a microchromosome in Japanese quail. We confirmed physical linkage of 5S rDNA and the TR gene on an intermediate-sized chromosome in quail, similar to both chicken and turkey. TERT localized to CJA 2 in quail and the orthologous chromosome region in chicken (GGA 2) and in turkey (MGA 3). The cytogenetic profile of Japanese quail was further developed by this study and synteny was identified among the three poultry species.

Mapping genes to chicken microchromosome 16 and discovery of olfactory and scavenger receptor genes near the major histocompatibility complex.

Trisomy mapping is a powerful method for assigning genes to chicken microchromosome 16 (GGA 16). The single chicken nucleolar organizer region (NOR), the 2 major histocompatibility complex regions (MHC-Y and MHC-B), and CD1 genes were all previously assigned to GGA 16 using trisomy mapping. Here, we combined array comparative genomic hybridization with trisomy mapping to screen unassigned genomic scaffolds (consigned temporarily to chrUn_random) for sequences originating from GGA 16. A number of scaffolds mapped to GGA 16. Among these were scaffolds that contain genes for olfactory (OR) and cysteine-rich domain scavenger (SRCR) receptors, along with a number of genes that encode putative immunoglobulin-like receptors and other molecules. We used high-resolution cytogenomic analyses to confirm assignment of OR and SRCR genes to GGA 16 and to pinpoint members of these gene families to the q-arm in partially overlapping regions between the centromere and the NOR. Southern blots revealed sequence polymorphism within the OR/SRCR region and linkage with the MHC-Y region, thereby providing evidence for conserved linkage between OR genes and the MHC within birds. This work localizes OR genes to the vicinity of the chicken MHC and assigns additional genes, including immune defense genes, to GGA 16.

Case study of sequence capture enrichment technology: identification of variation underpinning developmental syndromes in an amniote model.

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.

A comparative physical map reveals the pattern of chromosomal evolution between the turkey (Meleagris gallopavo) and chicken (Gallus gallus) genomes.

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.