Jerry B. Dodgson

Multi-platform next-generation sequencing of the domestic Turkey (Meleagris gallopavo): Genome assembly and analysis

The combined application of next-generation sequencing platforms has provided an economical approach to unlocking the potential of the turkey genome.

A physical map of the chicken genome

Strategies for assembling large, complex genomes have evolved to include a combination of whole-genome shotgun sequencing and hierarchal map-assisted sequencing. Whole-genome maps of all types can aid genome assemblies, generally starting with low-resolution cytogenetic maps and ending with the highest resolution of sequence. Fingerprint clone maps are based upon complete restriction enzyme digests of clones representative of the target genome, and ultimately comprise a near-contiguous path of clones across the genome. Such clone-based maps are used to validate sequence assembly order, supply long-range linking information for assembled sequences, anchor sequences to the genetic map and provide templates for closing gaps. Fingerprint maps are also a critical resource for subsequent functional genomic studies, because they provide a redundant and ordered sampling of the genome with clones. In an accompanying paper we describe the draft genome sequence of the chicken, Gallus gallus, the first species sequenced that is both a model organism and a global food source. Here we present a clone-based physical map of the chicken genome at 20-fold coverage, containing 260 contigs of overlapping clones. This map represents approximately 91% of the chicken genome and enables identification of chicken clones aligned to positions in other sequenced genomes.

Direct evidence of host genome acquisition by the alphaherpesvirus Marek's disease virus.

Summary.Many herpesviruses including Marek’s disease virus (MDV), a poultry alphaherpesvirus, carry homologous host genes presumably acquired during viral evolution. We have characterized one recent acquisition by MDV in considerable detail. The virulent MDV strain Md11 previously was isolated from a commercial chicken and initially propagated on duck cells. In the process of cloning the entire Md11 genome in a bacterial artificial chromosome (BAC), we obtained an infectious clone in which the entire terminal repeat short segment was replaced with a portion of the duck genome that corresponds to chicken chromosome 19. This sequence is not predicted to express any protein even though it contains one exon of the VAMP1 gene. The replacement did not affect MDV replication in vitro, despite the virus having only one copy of ICP4. Furthermore, we have shown that the variant MDV genome containing the duck genome substitution is present in the parental Md11 population and has been maintained through several subsequent propagations of the virus on chicken cells. This finding provides direct evidence that host genome acquisition by MDV actually occurs during virus replication, and that one or more such MDV genomes with host sequences may exist within MDV viral stocks which tend to be polyclonal, due to the cell-associated nature of its infection process.

Chromosomal arrangement of the chicken β-type globin genes

Abstract We have isolated the chicken β-type globin genes from a library of chicken DNA-λ Charon 4A recombinant bacteriophage. There are four β-type genes within this segment of the genome; we believe this represents all of the β-type genes of the chicken. The recombinant λCβG1 contains the embryonic ϵ- and adult β-globin genes. The hatching β H and embryonic p -globin genes are found in the recombinant λCβG2. Although λCβG1 and λCβG2 do not physically overlap, we present evidence that all four genes are closely linked and transcribed from the same DNA strand. These experiments demonstrate that the chromosomal regions represented by λCβG1 and λCβG2 lie approximately 1.6 kb apart in the chicken genome. A third recombinant λCβG3 extends the genomic locus studied in the vicinity of the β-type globin genes to approximately 39 kb. The physical order of the chicken β-type globin genes within this segment of the chromosome is 5′ … ϱ - β H - β - ϵ … 3′. This arrangement is unique among the vertebrate β-type globin gene clusters thus far examined, in that embryonic genes are located at the 5′ and 3′ ends of the cluster while the hatching and adult genes occupy central positions.

Integration of animal linkage and BAC contig maps using overgo hybridization.

The alignment of genome linkage maps, defined primarily by segregation of sequence-tagged site (STS) markers, with BAC contig physical maps and full genome sequences requires high throughput mechanisms to identify BAC clones that contain specific STS. A powerful technique for this purpose is multi-dimensional hybridization of “overgo” probes. The probes are chosen from available STS sequence data by selecting unique probe sequences that have a common melting temperature. We have hybridized sets of 216 overgo probes in subset pools of 36 overgos at a time to filter-spotted chicken BAC clone arrays. A four-dimensional pooling strategy, including one degree of redundancy, has been employed. This requires 24 hybridizations to completely assign BACs for all 216 probes. Results to date are consistent with about a 10% failure rate in overgo probe design and a 15–20% false negative detection rate within a group of 216 markers. Three complete rounds of overgo hybridization, each to sets of about 39,000 BACs (either BamHI or EcoRI partial digest inserts) generated a total of 1853 BAC alignments for 517 mapped chicken genome STS markers. These data are publicly available, and they have been used in the assembly of a first generation BAC contig map of the chicken genome.

A New Chicken Genome Assembly Provides Insight into Avian Genome Structure

The importance of the Gallus gallus (chicken) as a model organism and agricultural animal merits a continuation of sequence assembly improvement efforts. We present a new version of the chicken genome assembly (Gallus_gallus-5.0; GCA_000002315.3), built from combined long single molecule sequencing technology, finished BACs, and improved physical maps. In overall assembled bases, we see a gain of 183 Mb, including 16.4 Mb in placed chromosomes with a corresponding gain in the percentage of intact repeat elements characterized. Of the 1.21 Gb genome, we include three previously missing autosomes, GGA30, 31, and 33, and improve sequence contig length 10-fold over the previous Gallus_gallus-4.0. Despite the significant base representation improvements made, 138 Mb of sequence is not yet located to chromosomes. When annotated for gene content, Gallus_gallus-5.0 shows an increase of 4679 annotated genes (2768 noncoding and 1911 protein-coding) over those in Gallus_gallus-4.0. We also revisited the question of what genes are missing in the avian lineage, as assessed by the highest quality avian genome assembly to date, and found that a large fraction of the original set of missing genes are still absent in sequenced bird species. Finally, our new data support a detailed map of MHC-B, encompassing two segments: one with a highly stable gene copy number and another in which the gene copy number is highly variable. The chicken model has been a critical resource for many other fields of study, and this new reference assembly will substantially further these efforts.

Inhibition of Marek's disease virus replication by retroviral vector-based RNA interference.

RNA interference (RNAi) is a promising antiviral methodology. We recently demonstrated that retroviral vectors expressing short-hairpin RNAs (shRNA-mirs) in the context of a modified endogenous micro-RNA (miRNA) can be effective in reducing replication of other retroviruses in chicken cells. In this study, similar RNAi vectors are shown to inhibit replication of the avian herpesvirus, Mareks disease virus (MDV, also known as gallid herpesvirus type 2), and its close relative, herpesvirus of turkeys (HVT). Cells expressing shRNA-mirs targeting the MDV or HVT gB glycoprotein gene or the ICP4 transcriptional regulatory gene show significant inhibition of viral replication. Not only are viral titers reduced, but observed plaque sizes are significantly smaller when the virus is grown on cells in which RNAi is effective. We also describe a modified retroviral delivery vector that expresses a shRNA-mir containing up to three RNAi target sequences and employ this vector with multiple targets within the MDV gB gene or within both the gB and ICP4 genes. The use of targets within multiple genes potentially can provide a larger antiviral effect and/or make it more difficult for viral escape mutations to evolve.

Class II MHC cDNAs in 15I5 B-congenic chickens.

Abstract cDNA was obtained from the bursae of Fabricius of chickens from six B-congenic lines developed at this laboratory and studied for expression of class II B-LB genes. Following cDNA amplification, cloning and sequencing, genes were assigned to B-LB loci based on characteristic DNA sequences, amino acid relatedness to characterized genes, and level of expression. Genes from the B-LBI, B-LBII, and B-LBVI loci were differentially expressed in chickens with the B2, B5, B13, B15, or B21haplotypes. Chickens of all haplotypes expressed a B-LBII gene. Additional B-LB genes expressed included: B-LBI genes in the B5 and B19 haplotypes; a B-LBI/VI recombinant gene in the B2 haplotype; and a B-LBVI gene in the B13 haplotype. The B-congenic lines have demonstrable differences in resistance to Marek’s disease (MD), and in responses to MD viral vaccines. This variability in disease resistance may be correlated with polymorphisms in the expressed B-LB genes, or with differential expression of genes at different loci.

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

talpid2 is an avian autosomal recessive mutant with a myriad of congenital malformations, including polydactyly and facial clefting. Although phenotypically similar to talpid3, talpid2 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 talpid2 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 talpid2 mutants, only GLI3 activator levels were significantly altered in the nucleus. Through additional fine mapping and whole-genome sequencing, we determined that the talpid2 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 talpid2 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 talpid2 phenotype. Our data suggest that, although the talpid2 and talpid3 mutations affect a common ciliogenesis pathway, they are caused by mutations in different ciliary proteins that result in differences in craniofacial phenotype.

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

BackgroundA 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.ResultsThe 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.ConclusionThe 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.