Duration: 10/01/2008 to 09/30/2013

Administrative Advisor(s):

NIFA Reps:

Non-Technical Summary

Statement of Issues and Justification

Poultry meat and eggs are major protein sources in the American diet, with steady per capita consumption increases yearly to current levels of broiler meat and eggs at 87 pounds and 256 eggs, respectively in 2006 and turkey per capita consumption at 17 pounds. The U.S. consumption of chicken meat is higher than either beef or pork, which are estimated at 66 and 49 pounds, respectively for the same year (USDA Economic Research Service). The U.S. also continues to be a leading exporter of poultry (broiler) meat with 5.3 billion pounds exported in 2006. To meet domestic and international demands for chicken meat, the U.S. boiler chicken industry produced 35.8 billion pounds in 2006. Turkey production is estimated at 5.8 billion pounds for 2007, up 2.4 percent from 2006. The U.S. egg industry reached a production level of 6,500 million dozen for 2006.

The unprecedented growth of the poultry industry (five-fold increase in past 40 years; three-fold increase in the U.S. in the past 25 years) is partly due to the applications of modern scientific principles in genetic selection, disease control, nutrition, and management programs. To support and sustain the poultry sector, continuous improvements in science and technology must be made, tested, and transferred to the poultry industry which must then apply these new technologies to improve the production efficiency and performance of their populations. This is particularly important today due to increased international competition from Brazil and China.

Primary breeders are responsible for the genetic improvements in poultry, and the improved genetic products are multiplied through the hatchery system. The hatcheries, in turn, supply these more efficient birds to producers and growers in nearby states. The result is greater efficiency at all levels, with gains in production efficiency being passed to the consumer, as lower commodity prices.

Very few commercial poultry breeding operations remain in the world, and operate on a narrow profit margin. As a result any technologies that are developed by one company are kept for proprietary use. At the same time, they are heavily dependent upon public information from academia and other institutions that provide experimental results associated with new technologies. The important interactions of NC-1008 researchers with poultry industry representatives allow communication through a public knowledgebase and help to avoid many of the potentially serious problems in the industry associated with production and disease. Our efforts in advanced genetic and genomic technologies will be most useful to the primary breeders where the technologies can be applied to their breeding stocks. Because of the concentrated structure of the poultry breeding industry, this segment of animal agriculture is one of the best suited for applications of new and profitable technologies.

Complementary to the highly structured industry is the information that is readily available about the biology of the chicken including its short generation interval, large family size, well-documented biochemical and morphological mutations and clearly identified embryonic stages. All contribute to making the chicken an ideal model for development and application of biotechnological discoveries from emerging fields of scientific investigations. The advances in molecular biology and genetics point the way to new technologies that can be coupled with established breeding programs to provide the tools needed by the US. Poultry industry to maintain its competitive edge in the future.

Animal agriculture, to date, has depended upon effecting genetic improvement by estimating genotypic value by measuring phenotypic traits. This practice has severe limitations, especially in traits that are sex-limited (egg production) or require expensive testing (measurement of carcass traits or disease resistance) or testing late in life (reproductive traits). Progress made in molecular genetics and genomics will allow evaluation of some traits at the gene level, opening the possibility of early classification of live birds and use of marker-assisted selection in breeding programs. In addition to the identification of genes, understanding their action within regulatory pathways and the interactions across systems even when gene transfer is utilized will be important in the application of new discoveries.

The first genetic maps for the chicken were developed early in the 20th century and have expanded into a whole genome sequence and genomewide inquiry of gene expression and trait-associations. The chicken has also been an important model organism in development and immunology providing many discoveries including: the discovery of B cells, the isolation of the first oncogenes, and the model for molecular patterning in vertebrates, with the discovery of the apical ectodermal ridge and the polarizing region for limb development. Modern chicken genomics was born with the development of the first genetic-linkage maps based on molecular markers in the 1990s which were developed for mapping QTL for economically important traits. The creation of comparative maps between the gene-poor maps of the chicken with the gene-rich map of human was at first seen as the only realistic way of predicting the gene content of QTL. Complete physical maps of the chicken genome are now available based on large BAC clone assemblies.. Expressed sequence information for the chicken has reached approximately 600,000 ESTs, along with the full-length sequencing of 17,000 cDNAs. These resources have been used to create cDNA and oligo microarrays for high throughput gene expression studies.

The first draft sequence of the chicken genome, made public in 2004 (WASHUC1), was assembled using a whole genome shotgun sequencing strategy. This 6.6-fold coverage of the genome was produced using the DNA from a single inbred female Jungle Fowl. In 2006, additional data from 250,000 targeted sequencing reads were integrated with more extensive genetic and physical maps to produce a second assembly WASHU2. The new version has resolved many errors, in particular the assembly of the sex chromosome Z, which has increased from 34 to 75 Mb with the assembly now containing 1,030 Mb or 98% of the total genome. The microchromosomes are still to be resolved.

A significant contribution of the genome sequence has been the ability to predict gene sequences and compare them to mammalian genomes. Gene detection and comparisons for rapidly evolving genes such as those involved in the immune system remain unresolved. Information on gene function from model organisms and human to orthologs in the chicken has provided important clues to their role in birds. The availability of the chicken genome and a predicted genome-wide set of genes provides new opportunities for whole genome-based gene association and gene expression-based investigations. The chicken genome project has generated many new resources and experimental tools such as whole genome gene expression arrays, full-length cDNA clones, etc. for studies of gene function.

In the dbSNP, there are more than 2.9 million entries for chicken single nucleotide polymorphisms, most generated by a consortium led by the Beijing Genome Institute from a comparison of shotgun sequences from Silkie, broiler, and layer chicken lines with the Red Jungle Fowl as reference. Surprisingly, the segregation of large numbers of common SNP between and within broiler and layer populations provides information on a large number of potential genetic markers for QTL mapping in poultry. Recently, low-cost methods for typing 10,000 or more SNP have become available and with the ability to produce resource populations QTL detection will identify new trait-associations. QTL were first detected for susceptibility to Mareks disease and growth traits and since have expanded to include over 1,000 QTL for a wide range of traits which can be found in databases recording information on more than 200 traits, for almost 1,200 QTL loci, in addition to 400 gene associations. The chicken genome sequence has reinforced the importance of the chicken as a model avian species. Clearly, much has been achieved in our understanding of the chicken genome, but it has also highlighted how much is still to be learned about avian genomes as a whole.

Many of the Advanced Technologies that we are proposing in this new project derive from the remarkable progress that has been made in very recent years. The completion of the chicken genome sequence will clearly revolutionize the poultry industry and the future will be in the post-genome utilization of a systems biology approach to understand better the mechanisms of gene function to improve economics traits of poultry. Poultry researchers are now poised to apply these methods (genomics and transgenesis) to critical problems in the poultry industry, including viral and bacterial disease resistance, fertility, nutrient utilization, reproductive efficiency, and carcass yield.

Related, Current and Previous Work

Participants in the current Regional Project NC-1008 have been instrumental in bringing poultry genomics to its present location both in the U.S. and internationally. Long standing collaborations with the major poultry genome research centers in the UK, France, Netherlands, Sweden, and Israel has resulted in joint projects with NC-1008 members and contributed to a coordinated global approach to poultry genome research. Additional support from the Poultry committees of NRSP-8 and the International Society for Animal Genetics (ISAG) has been instrumental in fostering these international relationships. Cooperation with NRSP-8 provides much constructive interaction with researchers trying to solve common problems across a wide diversity of agricultural species and is of particular scientific value.

A CSREES-CRIS search was performed to identify research projects similar to the proposed project described here. Keywords used in the search were: poultry, livestock, genomics, gene expression, breeding, and quantitative trait loci. Alone and in combination these keywords resulted in hundreds apparently related CRIS projects. A review of active individual projects identified research that complement this project and for the most part build off of foundations provided by NC-1008, its participants, or their collaborators. Complementary multi-state CRIS projects include the ongoing National Research Support Project -8 (NRSP-8) as well as current regional Research Projects (S-1020, NE-60, and S-1037) which has resulted in coordinated efforts and remarkable progress in the areas of domestic species genomics. These projects have provided the scientific community with comprehensive linkage and physical maps, genetic markers for traits involved in health, production, and product quality, as well as genome and transcript sequence information.

Particular Regional Projects with close ties to NC-1008 are NE-60, "Genetic Basis for Resistance and Immunity in Avian Diseases" and S-1037, "Integrative Functional and Physiological Genomics of Poultry". As one of the closest related projects to NC-1008, the NE-60 project has focused primarily on a single family of genes, the major histocompatability complex (MHC) and related Rfp-Y genes for evaluating resistance to disease. This chromosomal regions represented by these genes are also of interest in NC-168 in a more global sense. Many participating stations in NE-60 also participate in NC-1008 and clearly joint efforts across the two projects are likely. Information exchange is critical between these two projects for technologies developed by NC-1008 may be of significant importance to NE-60 efforts for identifying disease resistance genes and genes identified by NE-60 could be verified by introduction into new populations by NC-1008.

The relationship between S-1037 and NC-1008 is that S-1037 is dedicated to evaluating existing populations for genetic relationships to production traits whereas NC-1008 is dedicated to developing new technologies for genetically improving poultry populations for the future. The primary goal of the S-1037 project is to use an integrative systems approach to determine cellular, physiological and biochemical mechanisms as impacted by traditional methods of selection. Current advances in science, especially molecular biology and proteomics have provided unique opportunities for further elucidation of biochemical, physiological and molecular genetic mechanisms as impacted by selection.

With the current sequence of the chicken genome and the along with more extensive genetic and physical maps have produced an assembly containing 1,030 Mb or 98% of the total genome. A significant contribution of the chicken genome sequence has been the ability to predict gene sequences and compare them to mammalian genomes. This comparative approach has been extremely useful and resulted in many recent accomplishments by members of the technical committee for NC-1008. Many of the Advanced Technologies that we are being proposing in this new project are derived from this marked progress. The completion of the chicken genome sequence will clearly revolutionize the poultry industry and the future will be in the post-genome utilization of a systems biology approach to understand better the mechanisms of gene function to improve economics traits of poultry. Poultry researchers are now poised to apply these methods (genomics and transgenesis) to critical problems in the poultry industry, including viral and bacterial disease resistance, fertility, nutrient utilization, reproductive efficiency, and carcass yield.

Past Accomplishments

Past work to develop high resolution integrated maps to facilitate the identification of poultry genes and other DNA sequences of economic importance has resulted in significant progress made in both chicken and turkey genome resources. A clearly noticeable trend has been the utilization of the available chicken tools, the sequence and EST s, to develop resources for turkey and for usage in poultry biological research to understand gene and sequence organization as well as function. Research has also continued on the development and alignment of genetic, physical and cytogenetic maps. Advances towards this objective have involved considerable collaborative research among technical committee members and included sharing of information, reagents, tools and genetic resources including cell lines and specialized stocks of chicken.

This work has included the development of resources for: mapping specific neurons in the avian brain, telomere organization and function as well as on telomerase gene expression, population studies on the genetic basis of reproductive problems in broiler breeders, characterization of the genes within the major histocompatibility complex (MHC), population studies using unique well defined genetic lines, integrated maps combining linkage and BAC contig maps in the chicken, comparative mapping across many avian species using overgo hybridizations, SNP genotyping in diverse populations, increased density of markers within the turkey linkage map, nutritional effects on gene expression, and characterizing the innate immune response to pathogens.
Previous work has also developed methods for creating new genetic variation in poultry by gene transfer and chromosome alteration. This work has included the development of technologies for: liposome delivery of sperm mediated gene transfer, novel delivery methods for the use of RNAi in poultry, the immortalization of avian cell lines, and the identification of the chromosomal location of transgenic events in poultry.

The development, comparison, and integration of emerging technologies with classical quantitative genetics for the improvement of economic traits in poultry has also been furthered. Prior work by NC-1008 participants include the development of resources for: the identification of genes involved in resistance to Mareks disease, use of dense SNP marker screens and linkage disequilibrium mapping to identify QTL in commercial populations, the identification of associations between immune-related genes and mortality and performance traits in commercial populations, the molecular characterization of the MHC-B locus, expression profiling of genes and their relationship to performance traits.

ADOL continues to curate the East Lansing genetic map. In the past year, 710 SNPs were added, which brings the total number of genetic markers to 3209. The East Lansing and Wageningen maps were combined into a consensus map that contained 3850 genetic markers, which was used in the second genome sequence assembly. Of the 3072 SNPs screened in our Illumina panel generated in 2005, 233 changed chromosomal positions from the first to the second genome assemblies.

At AR, the promoter regions from the chicken DAZL gene, a PGC specific promoter, has been cloned. The promoter is being fused to a reporter gene to allow us to determine time of induction and extent of expression (collaborator: NCSU).

To protect scientifically valuable research genetic lines of highly inbred chickens, semen samples were collected again this year from all adult males of the 21 highly and partially inbred lines held at Iowa State University (IA). Semen samples were placed into the long-term cryopreservation bank in the National Animal Germplasm program in Ft. Collins, CO.

At CA (City of Hope) work continues on construction of a gene map for the chicken MHC B and Y regions. The B map for Red Jungle Fowl now spans 242 kb and encompasses 46 genes including TRIM, C-type lectin and Ig superfamily type genes.

At CA (UC Davis), research continued to explore variation for telomere length within genomes (among chromosomes) and among different genotypes. Differences among genotypes are evident as telomere profiles differ (slightly) between inbred lines, although locations for some mega-telomere loci are held in common. Work was also initiated to examine integration of MDV into the chicken genome using MDV-BACs. We are studying the hypothesis that there is targeted integration into the mega-telomeres. This work will assist in determining clonality of tumors within individuals and will establish commonalities (and/or preferences) of integration sites among individuals.

Over 700 genetic markers (primarily microsatellites) have been genotyped by researchers at MN on the UMN/NTBF and Nte mapping families. Combined analysis of both mapping families found 684 markers to be significantly linked to at least one other marker in the UMN database; 41 linkage groups have been identified. Two MHC-B BAC clones were identified and sequenced. Gene annotation indicates three class IIb genes in the sequenced turkey haplotype, one more than in the sequenced chicken haplotype. DNA sequence polymorphisms (SNPs) identified in the turkey MHC were used to develop genotyping assays for genetic mapping in the UMN/NTBF mapping families. Segregation analysis found two turkey MHC-B SNPs (BTN2 and C4) were genetically linked. Genetic linkage was not observed between the MHC-B and MHC-Y SNPs.

NC has been using microarray analysis to identify differential chicken gene expression in response to dietary nutrient restriction. Adaptation to P and Ca restricted diets has been previously reported in chickens. Animals respond to nutrient restriction by increasing absorption rates and utilization efficiency, which decreases excretion of the restricted nutrients. The outcome of this research will provide a means to improve the innate ability of poultry to utilize environmentally important nutrients such as N and P, therefore reducing their excretion, and therefore greatly aid in reducing the cost of poultry litter disposal and in maintaining the productivity of poultry industry.

CA completed and published research in collaboration with Origen Therapeutics (Burlingame, CA) on long term culture procedures of stem cells (embryonic stem cells, primordial germs cells) and their utilization/utility for making transgenics (Origen) and analysis of features related to genome stability and differentiation status of these cell systems (UCD). Long term cultures were typically found to be normal in terms of karyotype to the level of analysis conducted, although occasionally cell lines were found with macrochromosome aberrations (in one case involving a deletion of a large portion of GGA 2) and of course these would not be suitable for use. Thus, the analysis of cells to be used for transgenic purposes as to their chromosomal status is an important parameter which needs consideration.

MI generated a Gateway-compatible entry plasmid containing the micro RNA (miRNA) sequence of chicken miR-30a that allows RNAi cassettes to be inserted into an ALV subgroup(A) retroviral destination vector. RNAi targeted either against the viral envB gene or host receptor tvb gene has been shown to be an effective antiviral strategy against subgroup(B) ALV. Similar reductions in plaque number and size of Marek's disease virus (serotype III) have been achieved by constructs that target the essential gB glycoprotein gene.
Work conducted at MN indicated that the phenotype of the spontaneously immortalized chicken cell line SC-2, changed dramatically at about passage 80, appearing smaller and more compact than at earlier passages. Passage 43 SC-2 cells expressed undetectable levels of p53 mRNA, but the elevated levels detected by passage 95 did not correlated to functional protein activity. The altered expression of genes involved in the p53 and Rb pathways, specifically, p53 and p21WAF1, may have contributed to the immortalization of the SC-2 CEF cell line. The regulation of chicken p15INK4b was shown to increase substantially at senescence and was transcriptionally silenced in two immortalized chicken cell lines. Short-hairpin RNA (shRNA)-mediated knockdown of chicken INK4b provided only modest lifespan extension, suggesting that other factors contribute to senescence in CEFs.

The transgenic chicken line developed in NC, now designated NCSU-Blue1, is a useful tool for several areas of research, and they have been used for studies of early embryonic development (Stem Cells Dev. (2006) 15:17-28). Functional beta-galactosidase was expressed in all tissues of the digestive tract, particularly in the small intestine. This should give the birds the ability to hydrolyze lactose, which normally cannot be utilized as a source of energy in birds. NC examined this possibility through a feeding trial in which isocaloric diets containing 0, 5, 7.5 and 10% dietary lactose were fed to wild type and transgenic birds from 10-24 days of age. Transgenic birds were observed to have a greater ability to digest lactose through the hydrolysis of lactose to galactose and glucose than those of nontransgenic wild-type chicks at least by 10%.. The culture of PGCs from male and female embryos at the NC station will have significant applications in reproductive biology, developmental biology and transgenics. Work with the NCSU-Blue1 line of transgenic chickens suggests that the nutritional requirements of transgenic poultry may need to be evaluated, particularly in high expressing lines.

ADOL continues to work on genetic resistance to Mareks disease. Evaluation of the line 6 x 7 F2 resource population for two-epistatic interactions identified a large number (239) of highly significant interactions involving loci located throughout the genome that account for MDV viremia titers in infected birds. Based on prior two-hybrid results, the MDV protein R-LORF10 may be the responsible protein in the novel up-regulation of MHC class II cell surface expression based upon defined MDV recombinants via MDV-BAC clones. In addition, a worldwide and genome-wide assessment was made for commercial poultry by genotyping 2551 informative SNPs spaced throughout the chicken genome on 2580 unique individuals including 1440 commercial birds. Results from several analytical methods combined with theory indicate that individual commercial breeding stocks have lost 70% or more genetic diversity of which no more than 10% can be recovered by combining all breeds from commercial poultry. These results emphasize a need for concerted national and international efforts to preserve chicken biodiversity.

AR initiated a SNPlotype mapping project for Sperm Degeneration and Sperm Mobility, with partial support from Species Coordinator funds. A detailed full-length cDNA sequencing project to characterize over 450 novel transcripts expressed in the chicken reproductive tract was begun.

In work conducted at IA, Fatness QTL were mapped in two F2 resource populations that were established by crossing one broiler sire with dams from two unrelated highly inbred lines (Fayoumi and Leghorn). Thirty-three markers in 8 regions on chromosomes 1 to 4 showed significant association (1% FDR) with AF. IA also evaluated the influence of heritability used in analysis of SNP data on the significance and magnitude of SNP effects and their standard errors (SE) and developed an approximation that would allow results for alternate levels of heritability to be obtained without reanalysis, using actual data from a broiler breeder population. Use of SNP-trait associations detected in one population for use in other populations requires LD between loci to be consistent across populations. The correlation between LD measured by r2 between lines for SNP at short distances is a good predictor of line relationships, although somewhat less so than the typical allele frequency-based distance.

At MD, analysis of global gene expression in the neuroendocrine system of chickens genetically selected for high and low body weight or body fat was accomplished using custom cDNA microarrays produced in collaboration with DE. Hundreds of genes were identified that are expressed at different levels in the pituitary gland or hypothalamus in either fat versus lean chickens or high growth versus low growth chickens. These are excellent candidate genes for controlling body fat and body growth in chickens. Single nucleotide polymorphisms (SNPs) have been identified in many of these genes and a study has been initiated at MD to assess these SNPs for their utility as genetic markers in marker assisted selection programs. mRNA splicing variants have been identified in BDNF, which is known to control body fat accumulation. In collaboration with ADOL, it was determined that these BDNF splice variants are associated with chicken lines which accumulate different amounts of body fat.

MS has developed and demonstrated computational tools, as well as proteomics techniques, to improve the structural annotation of the chicken genome. This is especially important for identifying those genes unique to birds for which obvious mammalian homologs do not exist. Resistance to MD is the result of complex interactions between chicken and MDV genes and current research is aimed at defining one molecular genetic mechanism that may be a critical determinant of this host-pathogen relationship. Results demonstrate the applicability of high throughput proteomics followed by computational modeling to understand biological function in the chicken. Databases are being maintained and computational tools developed that facilitate chicken researchers ability to derive biological meaning from their functional genomics datasets. This is broadly applicable throughout the chicken research community regardless of the field of study.

NC has focused on the identification of QTL for immune response and disease resistance in lines differentially selected for antibody production. NC has developed a new resource population which consists of reciprocal crosses of lines divergently selected for antibody response to sheep red blood cells. Selection for immune response parameters may lead to improved general disease resistance in part due to they are difficult to measure and have low to moderate heritability. The nature of selection appears to favor the contribution of a large number of loci with relatively small effects as opposed to single loci with large effects. Further characterization of these selected lines and their intercross population will provide additional information on the complexity of antibody response in the chicken as well as the genetic basis of selection in general.

In work at TX, the chicken 44K Agilent array was used to analyze RNA of heterophils from SE-resistant (line A) and SE susceptible chickens (line B) with SE (I) or without SE infection (N). The results indicated that: for the comparisons of SE infection with non-infection, 3096 genes in line A and 3312 genes in line B were differentially expressed (P<0.05). In the comparison of linage (line A and line B) difference, 4377 genes in the non-infected and 4333 genes in the infected groups have shown differential expression (P<0.05). The results discovered in the present study have laid a solid foundation to elucidate cellular and molecular mechanisms of SE infection in chickens.

VA has examined the spatial and temporal expression of nutrient (amino acid, peptide, and monosaccharide) transporters in the small intestine of late embryonic and early posthatch chicks. Expression of these transporters showed different developmental profiles and different levels in the intestinal segments. The peptide transporter PepT1 is expressed highest in the duodenum, the monosaccharide transporters are expressed highest in the jejunum and the amino acid transporters are expressed predominantly in the ileum. Because early nutrition plays an important role in overall growth performance, optimizing diets to match the absorptive capacity of the posthatch chick may result in increased growth performance.

Objectives

1. Create and share data and technology to enhance the development and application of genomics and systems biology in poultry.
   
2. Facilitate the creation and sharing of poultry research populations and the collection and analysis of relevant new phenotypes including those produced by gene transfer.
   
3. Elucidate genetic mechanisms that underlie economic traits and develop new methods to apply that knowledge to poultry breeding practices.
   

Methods

Objective 1. Create and share data and technology to enhance the development and application of genomics and systems biology in poultry. The methods employed to achieve Objective 1 are both broad in scope in general application as well as explicit for specific focus areas of investigation by particular project participants. The participants in Objective 1 will advance the development and sharing of information regarding the application of genomics and a systems approach to understanding problems in poultry species. ADOL will continue to enhance the accuracy, saturation, and completeness of the East Lansing genetic map. This will be conducted by increasing the marker density through the addition of SNPs and other informative genetic markers. By integrating the East Lansing map with other linkage maps (Wag) and physical maps the genome sequence will have a stronger foundation from which the final sequence can be completed. MI will continue to develop resources to further characterize the turkey genome physical map. Efforts will include the refined turkey BAC-contig map that will be aligned with the chicken genome sequence. Assembly of the 40,000 turkey BAC fingerprints and 20,000 turkey BAC end sequences with additional shotgun sequencing of the turkey genome is planned. This will build on the nearly 8000 turkey BAC-marker assignments to position the turkey for potential complete genome sequencing in the future. A coordinated project in CA will involve the development of cytogenetically-verified BACs that identify turkey chromosomes for further exploration of karyotype variation and evolution among poultry species. NC will utilize gene expression profiling to look for eQTL (expression QTL) for association with immune response phenotypes in chicken lines divergently selected for antibody response. The development of the current intercross resource population at NC consisting of reciprocal crosses of chicken lines developed in VA is in the F3 generation and will at the F4 generation be subject to microarray analysis to detect differences in gene expression during antibody response. NC will also utilize SNP genotyping to identify gene(s) involved in pigmentation in a novel resource population designed to map the genes for the Mendelian traits Id (Inhibitor of dermal melanin) and Fm (fibromelanosis). CA will use molecular technologies to explore the underlying genes responsible for a series of developmental mutations affecting chicken embryonic and post natal growth and development. SNP technologies have been used to identify chromosomes and regions for potential candidate genes responsible for the mutant phenotypes which affect limb development and craniofacial defects. These regions will be narrowed by further analysis and candidate genes will be studied by sequence and transcript analysis. AR will utilize immunocytochemistry, in situ hybridization histochemistry and real time, quantitative RT-PCR to map the location of sets of neurons in the chick brain where gene products, synthesis and changes in expression occur. The specific, neuroendocrine changes that are addressed include those associated with development of the male reproductive system and the impact of the stress response on specific neurons in the brain and cells in the pituitary gland. AR will collaborate with DE and MN on characterizing viral, virulent genes with the objective of developing recombinant vaccine production in poultry. Avian viral disease, such as avian influenza, Newcastle disease, infectious bronchitis, pneumovirus, Mareks disease, or laryngotracheitis cause economic losses in the poultry industry by decreasing productivity. A functional genomic approaches will be utilized to screen a variety of poultry genes to identify host regulatory factors responding to a virus infection. Specialized arrays focusing upon specific cellular mechanisms will be used for screening purposes and identification of potential host factors responsible for either defense or progression will be determined. Our goal is to find effective genes that contribute to disease resistance in poultry. Methods utilized include cell culture, recombinant DNA techniques, molecular and immunological hybridization, serological detection, ectopic expression and gene knock-down procedures. AR will also be sequencing novel transcripts from the chicken reproductive tract. Many of these appear to be non- coding transcripts however, some represent new protein coding genes. The transcripts are characterized for expression patterns. Many of the transcripts are developmentally regulated and restricted to particular tissues. This work will lead to a more complete annotation of the chicken genome, and identify developmentally regulated promoter regions. Objective 2. Facilitate the development and sharing of animal populations and the collection and analysis of new, unique and interesting phenotypes including those produced by gene transfer. A comprehensive approach is being taken to develop new lines and phenotypes using gene transfer. MI is investigating the application of RNAi technology against two important viral pathogens of chickens, avian leukosis virus (ALV) and Marek's Disease virus (MDV). A series of RNAi retroviral delivery vectors that inhibit ALV subgroup (B) (ALV(B)) replication will be developed along with a panel of entry plasmids containing the micro RNA (miRNA) sequence of chicken miR-30a that allow RNAi cassettes to be easily moved into the retroviral delivery vector, entry vectors based on a miRNA cluster on chicken chromosome 4 that can carry up to three target sequences simultaneously, and a vector employing a tet-responsive promoter. These RNAi vectorswill be used to inhibit replication of MDV, both serotypes I and III, using the viral glycoprotein gB gene as the target. Inhibition of MDV replication has also been achieved with RNAi vectors containing the viral ICP-4 gene as a target. Both reductions in MDV titer and smaller plaque size occur when successful target sequences are chosen. Other viral genes will be tested as targets. In addition,antiviral RNAi will be tested in birds into which the retroviral vectors have been introduced in the newly laid embryo, followed by incubation, hatching and challenge with virulent MDV. NC will continue the evaluation of new transgenic reporter lines of chicken expressing LacZ and eGFP. Lines will be established to provide eggs to the wider research community through the appropriate Material Transfer Agreements. NC will continue the work on the culture of primordial germ cells (PGCs) for the production of transgenic chickens and expand the culture system to other species of birds. AR is developing reporter gene constructs for expression restricted to the germline. AR will collaborate with NC for the generation of transgenic chicken lines expressing the constructs. These analyses will be crucial for mapping the development of the germline and contributions of the germline to somatic development. MN will continue to develop immortalized cell lines that can be of use for gene transfer and host cell substrates for molecular virology studies. In collaboration with NC to elucidate gene expression and functionality in PGCs. Objective 3. Elucidate genetic mechanisms that underlie economic traits and develop new methods to apply that knowledge to poultry breeding practices.

The methods of Objective 3 appropriately span the range from techniques used in fundamental studies of molecular genetic mechanisms through the applied methodologies required to verify the effectiveness of application of genetic information to improve practical breeding programs of poultry. The expertise represented by the scientists in this project enables the effective integration of live-animal, wet-laboratory, simulation, bioinformatics, mathematical-modeling and statistical techniques. Use of experimental and commercial populations with accurately recorded phenotypes enables high-risk discovery research as well as research on the breeding populations in which the results will ultimately be applied to improve US agriculture.

The studied economic traits will encompass those of importance to the industry, including growth, feed efficiency, nutritional physiology, egg production, body composition, integrity of skeletal, cardiovascular and respiratory systems, as well as the high-priority traits of resistance to Mareks and food-safety pathogens. Developmental and dietary changes in the RNA and protein expression profiles of intestinal nutrient (amino acid, peptide, and sugar) transporters from the late embryonic stage to early posthatch will be investigated at VA using real time PCR, DNA microarrays, western blotting and in situ hybridization. Analysis of the promoter regions for a selected number of these transporter genes will be examined by transfection studies with various promoter-reporter gene constructs as well as electrophoretic mobility shift assays. MD will evaluate candidate genes by computational analysis of microarray results and genome sequence, identify SNPs unevenly distributed between experimental lines through sequencing of genomic DNA from the grandparent lines, determine association of SNPs in candidate genes with phenotypic production traits; and then test a selected panel of genetic markers in genomic DNA samples of elite broiler breeders submitted by commercial companies. AR, in collaboration with OR have identified 13 chromosomal regions that harbor probable QTLs for male fertility (sperm mobility and sperm degeneration), and those regions will be refined and the contribution of each region to male fertility determined, leading to genetic tests to predict male fertility for use in the industry.

Several stations will study genetic mechanisms of host defense against viral transformation or bacterial pathogenesis. USDA-ADOL will characterize genes and biological pathways that confer genetic resistance to Mareks disease; IA will characterize genes and biological pathways that confer genetic resistance to bacterial disease caused by Salmonella and E. coli. Techniques will include pathogen-challenge and vaccine studies, use of unique experimental chicken lines, QTL mapping, microarrays, assays of DNA-protein interaction and comparative genomics. USDA-ADOL will evaluate genomic variations among the recombinant congenic strains in relation to phenotypes of interest. IN will examine importance of epistatic interactions in disease resistance in poultry. City of Hope will apply methods of molecular and cellular biology to elucidate mechanisms of immune responses. With the long-term objective to elucidate how integration contributes to the host disease state and/or viral evolution, CA will use molecular and cellular technologies to explore the regulatory mechanisms controlling genome stability in normal and transformed cells. Studies will continue on the telomere-telomerase pathway controlling cellular lifespan in vitro and in vivo. Normal and transformed cells will be used to study cells that age as expected and cells that have been immortalized and transformed; in particular, cells from different stages of Mareks disease viral infection. The modalities the virus may use to disrupt normal cellular activities, such as viral TR expression and development of a specialized telomerase utilizing vTR will be investigated, as well as the nature of integration of the virus into the host genome. Assays will include qPCR transcript analysis and telomerase activity assays. A major research emphasis will be determining whether there are random, preferred or targeted integration sites for the virus into the host genome. The latter effort will employ molecular cytogenetics and chromosome-specific BACs.

The development, testing and refinement of new statistical and machine-learning methods, as applied to the data derived from microarrays, high-density SNP panels, and other contemporary molecular data, are essential for the effective integration of knowledge from molecular studies into poultry genetic improvement by selective breeding. WI will develop biologically-informed statistical methods, which incorporate information on prior genomic or biological knowledge (such as common regulatory DNA sequence elements, gene function, or pathway membership) into the analysis of microarray data. MD will use methods which include DNA methylation analysis, identification of biomarker and genetic regulatory networks, and other computational biology methods from machine learning. These methods will be adapted for whole genome research and candidate gene analysis. Unique machine learning algorithms for marker association studies will be applied by GA. The machine learning approach will be used to analyze association of SNP to traits of economic importance. The ant colony optimization algorithm (ACA) coupled with logistic regression on haplotypes will be adapted for association studies involving large numbers of SNP markers. The ACA uses artificial ants that communicate through a probability density function (PDF) that is updated at each iteration with weights or pheromone levels, which are analogous to the chemical pheromones used by real ants. In the case of SNP association studies, the weights can be ascertained by the strength of the association between selected haplotypes and the traits of interest. IA will develop and implement statistical methods for the use of high-density SNP genotyping data to detect and map QTL affecting economic traits using linkage disequilibrium mapping. Methods for the use of this information in poultry breeding programs will also be developed and evaluated by simulation at IA. IN will examine efficiency and methods to implement genomic selection in poultry through simulations and biological testing using dense SNP genotyping. IN will develop methods of traceability of poultry products through dense SNP genotyping.

Measurement of Progress and Results

Outputs

• Generation of comprehensive genetic maps for the chicken and turkey.
• Generation of comprehensive physical maps for the chicken.
• Development of novel techniques for gene transfer and transgenesis in chickens.
• A preliminary understanding of how DNA sequence variation leads to phenotypic variation in chickens and turkeys.
• Identification of QTL, genes, and/or pathways that are associated with production traits and disease resistance in chickens and turkeys.

Outcomes or Projected Impacts

• Increased scientific knowledge of the chicken and turkey genomes and their organization.
• Enhanced understanding of gene function and expression, and identification of candidate genes affecting economically important traits in poultry.
• Coordination of genome reagents and tools, databases, cell lines, and experimental poultry populations.
• Coordination of poultry research, education and extension programs.
• Enhanced collaborations and communications to the poultry breeders and associated industries.
• Outcome 6 Improved genetic selection programs at the primary poultry breeding companies by utilization of modern, cost-efficient breeding practices based on marker-assisted selection. Outcome 7 Technology transfer to the poultry breeders and associated industries.

Milestones

Projected Participation

View Appendix E: Participation

Outreach Plan

New information and techniques are promptly transferred to relevant users by public-access databases, popular press articles, presentations at scientific conferences and industry meetings, scientific journal publications and by inviting industry personnel to attend the annual Technical Committee meeting.

Interesting Web Sites and Databases: http://poultry.mph.msu.edu/ ; http://chicksnps.afs.udel.edu/; http://www.agbase.msstate.edu; http://www.animalgenome.org

The previous NC-1008 project period (2003-2006) resulted in 250 peer-reviewed manuscripts; book chapters/proceedings manuscripts, patents, and popular press articles. The previous NC-1008 project also involved the education of 30 MS and 50 Ph.D graduate students.

Organization/Governance

The planning and supervision of this new NC Regional Research Project shall be the responsibility of the Regional Technical Committee which shall consist of an Administrative Advisor, a Technical Representative of each participating agency or experiment station and a Representative of the USDA Cooperative State Research, Education and Extension Service (CSREES). Industry representatives, USDA-ARS researchers, and investigators from non-land grant institutions are welcomed as full technical members and they will be subject to the same governance as the experiment station committee members (described below). Voting membership of the committee shall consist of the Technical Representatives. Only one member representing each participating agency or experiment station shall be eligible to vote.

The Technical Committee will meet yearly. The Chair and Secretary for the technical committee, elected by the committee members, will serve two years. An Executive Committee will conduct all business of the committee between annual meetings and will consist of the current Chair, the Secretary and the immediate past Chair. The Chair will appoint a Technical Committee member to act as a Coordinator for each objective for the purpose of maintaining and organizing an approach to each objective, communicating progress to the Executive Committee and preparing necessary reports on their respective objective. Other subcommittees may be named by the Chairperson as needed to perform specific assignments. They may include subcommittees to develop procedures, manuals, and phases of the regional project, to review work assignments, to develop research methods, to prepare publications, and to write proposals.

The Technical Committee shall be responsible for review and acceptance of contributing projects, preparation of reviews, modification of the regional project proposal, and preparation of an annual report for transmittal by the Administrative Advisor upon approval to CSREES. Annual written reports will be prepared by each Technical Committee member and distributed at the annual meeting. Annual reports are to be submitted in sufficient copies to be distributed to all Technical Committee members. In the circumstance that a member is unable to attend the meeting the reports shall be mailed in advance to the local host member for distribution to meeting participants.

Minimum expectations for Technical Committee members are attendance at an annual meeting at least three years out of five and submission of a written annual report at or before the meeting. Collaborators may include emeritus members with an interest in attending annual meetings, scientists who wish to contribute and participate by virtue of having a special skill or interest, and those who participate in research with a special focus or interaction with an individual Technical Committee member.

Literature Cited

Attachments

Land Grant Participating States/Institutions

AR, AZ, CA, DE, FL, GA, IA, IN, MD, MI, MN, NC, OR, VA, WI, WV

Non Land Grant Participating States/Institutions

City of Hope Beckman Research Institute, USDA-ARS-Avian Disease & Oncology Laboratory