Poultry genome sequences: progress and outstanding challenges.

The first build of the chicken genome sequence appeared in March, 2004 - the first genome sequence of any animal agriculture species. That sequence was done primarily by whole genome shotgun Sanger sequencing, along with the use of an extensive BAC contig-based physical map to assemble the sequence contigs and scaffolds and align them to the known chicken chromosomes and linkage groups. Subsequent sequencing and mapping efforts have improved upon that first build, and efforts continue in search of missing and/or unassembled sequence, primarily on the smaller microchromosomes and the sex chromosomes. In the past year, a draft turkey genome sequence of similar quality has been obtained at a much lower cost primarily due to the development of 'next-generation' sequencing techniques. However, assembly and alignment of the sequence contigs and scaffolds still depended on a detailed BAC contig map of the turkey genome that also utilized comparison to the existing chicken sequence. These 2 land fowl (Galliformes) genomes show a remarkable level of similarity, despite an estimated 30-40 million years of separate evolution since their last common ancestor. Among the advantages offered by these sequences are routine re-sequencing of commercial and research lines to identify the genetic correlates of phenotypic change (for example, selective sweeps), a much improved understanding of poultry diversity and linkage disequilibrium, and access to high-density SNP typing and association analysis, detailed transcriptomic and proteomic studies, and the use of genome-wide marker- assisted selection to enhance genetic gain in commercial stocks.

Allele-specific expression analysis reveals CD79B has a cis-acting regulatory element that responds to Marek's disease virus infection in chickens.

Marek's disease (MD) is a T cell lymphoma disease of domestic chickens induced by the Marek's disease virus (MDV), a highly infectious and naturally oncogenic alphaherpesvirus. Enhancing genetic resistance to MD in poultry is an attractive method to augment MD vaccines, which protect against MD but do not prevent MDV replication and horizontal spread. Previous work integrating QTL scans, transcript profiling, and MDV-chicken protein-protein interaction screens revealed 3 MD resistance genes; however, a major challenge continues to be the identification of the other contributing genes. To aid in this search, we screened for allele-specific expression (ASE) in response to MDV infection, a simple and novel method for identifying polymorphic cis-acting regulatory elements, which may contain strong candidate genes with specific alleles that confer MD genetic resistance. In this initial study, we focused on immunoglobulin ß (CD79B) because it plays a critical role in the immune response and, more important, is transcriptionally coupled with growth hormone (GH1), one of the previously identified MD resistance genes. Using a coding SNP in CD79B and pyrosequencing to track the relative expression of each allele, we monitored ASE in uninfected and MDV-infected F(1) progeny from reciprocal intermatings of highly inbred chicken lines 6(3) (MD resistant) and 7(2) (MD susceptible). Upon screening 3 tissues (bursa, thymus, and spleen) at 5 time points (1, 4, 7, 11, and 15 d postinfection), we observed that MDV infection alters the CD79B allelic ratios in bursa and thymus tissues at 4 and 15 d postinfection in both mating directions. Our results suggest that CD79B has a cis-acting regulatory element that responds to MDV infection and probably cooperates with GH1 in conferring genetic resistance to MD. This result helps validates the use of ASE screens to identify specific candidate genes for complex traits such as genetic resistance to MD.

Retroviral delivery of RNA interference against Marek's disease virus in vivo.

The process of RNA interference (RNAi) has been exploited in cultured chicken cells and in chick embryos to assess the effect of specific gene inhibition on phenotypes related to development and disease. We previously demonstrated that avian leukosis virus-based retroviral vectors are capable of delivering effective RNAi against Marek's disease virus (MDV) in cell culture. In this study, similar RNAi vectors are shown to reduce the replication of MDV in live chickens. Retroviral vectors were introduced into d 0 chick embryos, followed by incubation until hatching. Chicks were challenged with 500 pfu of strain 648A MDV at day of hatch, followed by assays for viremia at 14 d postinfection. Birds were monitored for signs of Marek's disease for 8 wk. A stem-loop PCR assay was developed to measure siRNA expression levels in birds. Delivery of RNAi co-targeting the MDV gB glycoprotein gene and ICP4 transcriptional regulatory gene significantly reduced MDV viremia in vivo, although to lesser extents than were observed in cell culture. Concomitant reductions in disease incidence also were observed, and the extent of this effect depended on the potency of the MDV challenge virus inoculum. Successful modification of phenotypic traits in live birds with retroviral RNAi vectors opens up the possibility that such approaches could be used to alter the expression of candidate genes hypothesized to influence a variety of quantitative traits including disease susceptibility.

The chicken HMG-17 gene is dispensable for cell growth in vitro.

HMG-17 is a highly conserved and ubiquitous nonhistone chromosomal protein that binds to nucleosome core particles. HMG-17 and HMG-14 form a family of chromosomal proteins that have been reported to bind preferentially to regions of active chromatin structure. To study the functional role of the single-copy chicken HMG-17 gene, null mutants were generated by targeted gene disruption in a chicken lymphoid cell line, DT40. Heterozygous and homozygous null mutant cell lines were generated by two independent selection strategies. Heterozygous null mutant lines produced about half the normal level of HMG-17 protein, and homozygous null lines produced no detectable HMG-17. No significant changes in cell phenotype were observed in cells harboring either singly or doubly disrupted HMG-17 genes, and no compensatory changes in HMG-14 or histone protein levels were observed. It is concluded that HMG-17 protein is not required for normal growth of avian cell lines in vitro, nor does the absence of HMG-17 protein lead to any major changes in cellular phenotype, at least in lymphoid cells.

Structure and expression of the chicken ferritin H-subunit gene.

Although the genomes of many species contain multiple copies of ferritin heavy (H)- and light (L)-chain sequences, the chicken genome contains only a single copy of the H-subunit gene. The primary transcription unit of this gene is 4.6 kilobase pairs and contains four exons which are posttranscriptionally spliced to generate a mature transcript of 869 nucleotides. Chicken and human ferritin H-subunit genomic loci are organized with similar exon-intron boundaries. They exhibit approximately 85% nucleotide identity in coding regions, which yield proteins 93% identical in amino acid sequence. We have identified a sequence of 22 highly conserved nucleotides in the 5' untranslated sequences of chicken, human, and tadpole ferritin H-subunit genes and propose that this conserved sequence may regulate iron-modulated translation of ferritin H-subunit mRNAs.

Isolation and characterization of six different chicken actin genes.

Genes representing six different actin isoforms were isolated from a chicken genomic library. Cloned actin cDNAs as well as tissue-specific mRNAs enriched in different actin species were used as hybridization probes to group individual actin genomic clones by their relative thermal stability. Restriction maps showed that these actin genes were derived from separate and nonoverlapping regions of genomic DNA. Of the six isolated genes, five included sequences from both the 5' and 3' ends of the actin-coding area. Amino acid sequence analysis from both the NH2- and COOH-terminal regions provided for the unequivocal identification of these genes. The striated isoforms were represented by the isolated alpha-skeletal, alpha-cardiac, and alpha-smooth muscle actin genes. The nonmuscle isoforms included the beta-cytoplasmic actin gene and an actin gene fragment which lacked the 5' coding and flanking sequence; presumably, this region of DNA was removed from this gene during construction of the genomic library. Unexpectedly, a third nonmuscle chicken actin gene was found which resembled the amphibian type 5 actin isoform (J. Vandekerckhove, W. W. Franke, and K. Weber, J. Mol. Biol., 152:413-426). This nonmuscle actin type has not been previously detected in warm-blooded vertebrates. We showed that interspersed, repeated DNA sequences closely flanked the alpha-skeletal, alpha-cardiac, beta-, and type 5-like actin genes. The repeated DNA sequences which surround the alpha-skeletal actin-coding regions were not related to repetitious DNA located on the other actin genes. Analysis of genomic DNA blots showed that the chicken actin multigene family was represented by 8 to 10 separate coding loci. The six isolated actin genes corresponded to 7 of 11 genomic EcoRI fragments. Only the alpha-smooth muscle actin gene was shown to be split by an EcoRI site. Thus, in the chicken genome each actin isoform appeared to be encoded by a single gene.

Replacement variant histone genes contain intervening sequences.

The nucleotide sequences of two chicken histone genes encoding replacement variant H3.3 polypeptides are described. Unlike the replication variant genes of chickens (and almost all other organisms), these genes contain intervening sequences; introns are present in both genes in the 5' noncoding and coding sequences. Furthermore, the replacement variant histone mRNAs are post-transcriptionally polyadenylated. The locations, but not the sizes, of the two introns within the coding segments of the two genes have been exactly conserved, whereas the intron positions in their respective 5' flanking regions differ. Although both H3.3 genes predict the identical histone polypeptide sequence, they are as different from one another as each of them is from a more common replication variant H3.2 gene in silent base substitutions within the coding sequences. Thus, the H3.3 polypeptide sequence has been precisely maintained over a great evolutionary period, suggesting that this class of histones performs a strongly selected biological function. Although replacement variant histones can account for more than 50% of the total H3 protein in the nuclei of specific chicken tissues, the steady-state level of H3.3 mRNA is nearly the same (and is quite low) in all tissues and ages of animals examined. These properties suggest novel mechanisms for the control of the basal histone biosynthesis which takes place outside of the S phase of the cell cycle.

The chicken genome: some good news and some bad news.

The sequencing of the chicken genome has generated a wealth of good news for poultry science. It allows the chicken to be a major player in 21st century biology by providing an entrée into an arsenal of new technologies that can be used to explore virtually any chicken phenotype of interest. The initial technological onslaught has been described in this symposium. The wealth of data available now or soon to be available cannot be explained by simplistic models and will force us to treat the inherent complexity of the chicken in ways that are more realistic but at the same time more difficult to comprehend. Initial single nucleotide polymorphism analyses suggest that broilers retain a remarkable amount of the genetic diversity of predomesticated Jungle Fowl, whereas commercial layer genomes display less diversity and broader linkage disequilibrium. Thus, intensive commercial selection has not fixed a genome rich in wide selective sweeps, at least within the broiler population. Rather, a complex assortment of combinations of ancient allelic diversity survives. Low levels of linkage disequilibrium will make association analysis in broilers more difficult. The wider disequilibrium observed in layers should facilitate the mapping of quantitative trait loci, and at the same time make it more difficult to identify the causative nucleotide change(s). In addition, many quantitative traits may be specific to the genetic background in which they arose and not readily transferable to, or detectable in, other line backgrounds. Despite the obstacles it presents, the genetic complexity of the chicken may also be viewed as good news because it insures that long-term genetic progress will continue via breeding using quantitative genetics, and it surely will keep poultry scientists busy for decades to come. It is now time to move from an emphasis on obtaining "THE" chicken genome sequence to obtaining multiple sequences, especially of foundation stocks, and a broader understanding of the full genetic and phenotypic diversity of the domesticated chicken.

Functional genomics of the chicken--a model organism.

Since the sequencing of the genome and the development of high-throughput tools for the exploration of functional elements of the genome, the chicken has reached model organism status. Functional genomics focuses on understanding the function and regulation of genes and gene products on a global or genome-wide scale. Systems biology attempts to integrate functional information derived from multiple high-content data sets into a holistic view of all biological processes within a cell or organism. Generation of a large collection ( approximately 600K) of chicken expressed sequence tags, representing most tissues and developmental stages, has enabled the construction of high-density microarrays for transcriptional profiling. Comprehensive analysis of this large expressed sequence tag collection and a set of approximately 20K full-length cDNA sequences indicate that the transcriptome of the chicken represents approximately 20,000 genes. Furthermore, comparative analyses of these sequences have facilitated functional annotation of the genome and the creation of several bioinformatic resources for the chicken. Recently, about 20 papers have been published on transcriptional profiling with DNA microarrays in chicken tissues under various conditions. Proteomics is another powerful high-throughput tool currently used for examining the dynamics of protein expression in chicken tissues and fluids. Computational analyses of the chicken genome are providing new insight into the evolution of gene families in birds and other organisms. Abundant functional genomic resources now support large-scale analyses in the chicken and will facilitate identification of transcriptional mechanisms, gene networks, and metabolic or regulatory pathways that will ultimately determine the phenotype of the bird. New technologies such as marker-assisted selection, transgenics, and RNA interference offer the opportunity to modify the phenotype of the chicken to fit defined production goals. This review focuses on functional genomics in the chicken and provides a road map for large-scale exploration of the chicken genome.

Progress from chicken genetics to the chicken genome.

The chicken has a proud history, both in genetic research and as a source of food. Here we attempt to provide an overview of past contributions of the chicken in both arenas and to link those contributions to the near future from a genetic perspective. Companion articles will discuss current poultry genetics research in greater detail. The chicken was the first animal species in which Mendelian inheritance was demonstrated. A century later, the chicken was the first among farm animals to have its genome sequenced. Between these firsts, the chicken remained a key organism used in genetic research. Breeding programs, based on sound genetic principles, facilitated the global emergence of the chicken meat and egg industries. Concomitantly, the chicken served as a model whose experimental populations and mutant stocks were used in basic and applied studies with broad application to other species, including humans. In this paper, we review some of these contributions, trace the path from the origin of molecular genetics to the sequence of the chicken genome, and discuss the merits of the chicken as a model organism for furthering our understanding of biology.

The gene encoding chicken chromosomal protein HMG-14a is transcribed into multiple mRNAs.

The sequence and structure of the chicken HMG14a gene encoding HMG-14a non-histone chromosomal protein suggest that it may be a unique member of the HMG (high mobility group) gene family with properties intermediate to those of the typical HMG-14 and HMG-17 genes. Genomic clones were isolated which together contain the complete chicken HMG-14a gene. The gene covers about 10 kb while coding for an mRNA of about 1000 nt in size. Primer extension, S1 mapping and further cDNA clone analysis suggest that HMG-14a codes for multiple mRNAs arising from two or more transcription start points with alternative splicing and utilization of two or more polyadenylation sites. However, no variation in the coding portion of the mRNA has been observed. The sequence of the promoter region of HMG-14a is similar to that of chicken HMG-14b and human HMG-14 in that it is very G+C rich, contains several putative Sp1-binding sequences and has an unusually high density of CpG dinucleotides. Expression studies confirm earlier results suggesting that the gene is expressed at low levels in most tissue types.

Chicken chromosomal protein HMG-14 and HMG-17 cDNA clones: isolation, characterization and sequence comparison.

A cDNA clone coding for the chicken high-mobility group 14 (HMG-14) mRNA has been isolated from a chicken-liver cDNA library by screening with two synthetic oligodeoxynucleotide pools whose sequences were derived from the partial amino acid sequence of the HMG-14 protein. A chicken HMG-17 cDNA clone was also isolated in a similar fashion. Comparison of the two chicken HMG cDNA clones to the corresponding human cDNA sequences shows that chicken and human HMG-14 mRNAs and polypeptides are considerably less similar than are the corresponding HMG-17 sequences. In fact, the chicken HMG-14 is almost as similar to the chicken HMG-17 in amino acid sequence as it is to mammalian HMG-14 polypeptides. HMG-14 and HMG-17 mRNAs seem to contain a conserved sequence element in their 3'-untranslated regions whose function is at present unknown. The chicken HMG-14 and HMG-17 genes, in contrast to their mammalian counterparts, appear to exist as single-copy sequences in the chicken genome, although there appear to exist one or more additional sequences which partially hybridize to HMG-14 cDNA. Chicken HMG-14 mRNA, about 950 nucleotides in length, was detected in chicken liver RNA but was below our detection limits in reticulocyte RNA.

Chicken genome sequence: a centennial gift to poultry genetics.

A draft sequence of the chicken genome will be available by early 2004. This event conveniently marks the start of the second century of poultry genetics, coming 100 years after the use of the chicken to demonstrate Mendelian inheritance in animals by William Bateson. How will the second, post-genomic century of poultry genetics differ from the first? A whole genome shotgun (WGS) approach is being used to obtain the chicken sequence, with the goal of generating approximately six-fold coverage of the genome. Bacterial artificial chromosome (BAC) and fosmid clone end sequences, along with a BAC contig map integrated with genetic linkage and radiation hybrid maps, will form the platform for assembly of the WGS data. Rapid progress in global analysis of chicken gene expression patterns is also being made. Comparative genomics will link these new discoveries to the knowledge base for all other animal species. It's hoped that the genome sequence will also provide common ground on which to unite studies of the chicken as a model species with those aimed at agriculturally-relevant applications. The current status of chicken genomics will be assessed with projections for its near and long term future.

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.

Elements regulating differential activity of chicken histone H1 gene promoters.

The chicken genome contains six closely related histone H1 genes, each of which encodes a different H1 protein. The four common regulatory elements previously identified in H1 histone promoters are very similar in sequence and location in all chicken H1 genes, which gives rise to the question of how the six H1 variants are expressed at significantly different levels. Transient transfections of reporter gene transcriptional fusions indicate that approximately 200 bp of each promoter is sufficient to generate the observed spectrum of H1 promoter activity. The differences in H1 promoter-driven expression are shown to be explained by the relative activity of the previously characterized G box region and that of a novel element found between CCAAT and TATA that we have termed differential upstream sequence (Dus). Gel shift analysis indicated that the primary nuclear binding protein to the G box is one or more avian homologues of the Sp1 transcription factor. The Dus region binds multiple nuclear proteins, one of which is the recently described IBR/IBF factor. The differential affinities of the G box and Dus sequences of the H1 promoters for their respective nuclear binding factors correlate well with their relative promoter activities.

DNA marker technology: a revolution in animal genetics.

The development of DNA-based markers has had a revolutionary impact on gene mapping and, more generally, on all of animal and plant genetics. With DNA-based markers, it is theoretically possible to exploit the entire diversity in DNA sequence that exists in any cross. For this reason, high resolution genetic maps are being developed at an unprecedented speed. The most commonly used DNA-based markers include those based on a cloned and (usually) sequenced DNA fragment and other, more random, assays for genetic polymorphism that can be grouped under the heading of fingerprint markers. The advantages and disadvantages of the various marker types are discussed, along with their application to the reference chicken genetic linkage maps and to the search for quantitative trait loci (QTL). The prospects for the use of DNA-based markers in marker-assisted selection are considered, along with likely future trends in poultry gene mapping. Further development of both physical and linkage genome maps of the chicken will allow animal scientists to more efficiently detect and characterize QTL and will provide them access to the wealth of genetic information that is being generated about the human genome and the genomes of model species, such as the mouse and Drosophila.

A comprehensive screen for chicken proteins that interact with proteins unique to virulent strains of Marek's disease virus.

Genetic resistance to Marek's disease (MD) has been proposed as a method to augment current vaccinal control of MD. Although it is possible to identify QTL and candidate genes that are associated with MD resistance, it is necessary to integrate functional screens with linkage analysis to confirm the identity of true MD resistance genes. To help achieve this objective, a comprehensive 2-hybrid screen was conducted using genes unique to virulent Marek's disease virus (MDV) strains. Potential MDV-host protein interactions were tested by an in vitro binding assay to confirm the initial two-hybrid results. As a result, 7 new MDV-chicken protein interactions were identified and included the chicken proteins MHC class II beta (BLB) and invariant (Ii) chain (CD74), growth-related translationally controlled tumor protein (TPT1), complement component Clq-binding protein (C1QBP), retinoblastoma-binding protein 4 (RBBP4), and alpha-enolase (ENO1). Mapping of the encoding chicken genes suggests that BLB, the gene for MHC class II beta chain, is a positional candidate gene. In addition, the known functions of the chicken proteins suggest mechanisms that MDV might use to evade the chicken immune system and alter host gene regulation. Taken together, our results indicate that integrated genomic methods provide a powerful strategy to gain insights on complex biological processes and yield a manageable number of genes and pathways for further characterization.

Examination of antisense RNA and oligodeoxynucleotides as potential inhibitors of avian leukosis virus replication in RP30 cells.

Avian leukosis virus (ALV) is an economically important pathogen of chickens. Both antisense RNA and antisense oligodeoxynucleotides (ODN) have been used to diminish the replication and spread of other retroviruses. The use of antisense RNA and ODN to inhibit ALV replication has been examined in cultured RP30 cells. Using an expression system that constitutively transcribes antisense ALV RNA, one transfected cell clone showed a significant reduction in virus growth. However, this effect was not reproducibly observed in other transfected cell lines or in cells in which the antisense transcript was expressed from a regulatable promoter, even though a substantial amount of antisense transcript was generated. Antisense ODN complementary to several different target sites near the 5' end of the ALV genome were also tested for antiviral activity, by comparison of antisense ODN effects to those of randomized sequence controls. An antisense ODN complementary to the ALV primer binding site demonstrated a reproducible reduction in viral replication. However, when the corresponding region was specifically employed as a target for intracellular antisense RNA expression, there again was no significant inhibition of ALV. These results suggest that in vivo expression of antisense RNA is unlikely to be an effective way to generate transgenic poultry that are resistant to field strains of ALV.

Identification of class II major histocompatibility complex polymorphisms predicted to be important in peptide antigen presentation.

Chickens of the B2, B5, B15, B19, and B21 B-congenic haplotypes differ in disease resistance. Complementary DNA from B-congenic chicken strains have been analyzed for allelic diversity of expressed Class II MHC genes. The predicted amino acid sequences of eight genes from five haplotypes were subjected to Wu-Kabat variability analysis. The B-L gene polymorphic regions and conserved regions are highly similar to the human leukocyte antigen Class II genes. Therefore, the present analysis reveals candidate polymorphisms important in determining the spectrum of antigenic peptides presented to T helper cells, and allelic differences possibly important in resistance to avian disease.

Development of a genetic map of the chicken with markers of high utility.

Microsatellites are tandem duplications with a simple motif of one to six bases as the repeat unit. Microsatellites provide an excellent opportunity for developing genetic markers of high utility because the number of repeats is highly polymorphic, and the assay to score microsatellite polymorphisms is quick and reliable because the procedure is based on the polymerase chain reaction (PCR). We have identified 404 microsatellite-containing clones of which 219 were suitable as microsatellite markers. Primers for 151 of these microsatellites were developed and used to detect polymorphisms in DNA samples extracted from the parents of two reference populations and three resource populations. Sixty, 39, 46, 49, and 61% of the microsatellites exhibited length polymorphisms in the East Lansing reference population, the Compton reference population, resource population No. 1 (developed to identify resistance genes to Marek's disease), resource population No. 2 (developed to identify genes involved in abdominal fat), and resource population No. 3 (developed to identify genes involved in production traits), respectively. The 91 microsatellites that were polymorphic in the East Lansing reference population were genotyped and 86 genetic markers were eventually mapped. In addition, 11 new random amplified polymorphic DNA (RAPD) markers and 24 new markers based on the chicken CR1 element were mapped. The addition of these markers increases the total number of markers on the East Lansing genetic map to 273, of which 243 markers are resolved into 32 linkage groups. The map coverage within linkage groups is 1,402 cM with an average spacing of 6.7 cM between loci. The utility of the genetic map is greatly enhanced by adding 86 microsatellite markers. Based on our current map, approximately 2,550 cM of the chicken genome is within 20 cM of at least one microsatellite marker.