Duration: 10/01/2013 to 09/30/2018

Administrative Advisor(s):

NIFA Reps:

Non-Technical Summary

Statement of Issues and Justification

How the proposed research addresses regional and/or national priorities and in context of stakeholder input:


The poultry industry in the United States underpins the global food system providing an efficient, economical and nutritious source of animal-based protein (meat and eggs). The industry foundation consists of breeding companies and growers/producers distributed throughout the nation (and the world) with a heavy concentration in states from which the members of Multistate Research Project NC1170 are drawn (e.g., Arkansas, California, Delaware, Georgia, Iowa, Minnesota, North Carolina, Virginia). The USDA Economic Research Service reports that the U.S. poultry industry is the worlds largest producer and second-largest exporter of poultry meat and a major egg producer. The USDA National Agricultural Statistics Service reported that the combined value of production from broilers, eggs, and turkeys in 2011 was $35.6 billion. Many allied industries support and are impacted by the poultry industry, e.g., from grain producers and distributors to housing/cage manufacturers. Thus, the economic impact of the poultry industry to the financial health of the U.S. is enormous. The interactions of the poultry researchers involved in NC1170 with their stakeholders takes place via a variety of formats and opportunities so as to integrate our expertise with their needs and goals. These formats include, but are not limited to, organized informal meetings, workshops, and conferences (e.g., Poultry workshop at annual International Plant and Animal Genome meetings, annual National Breeders Roundtable, annual Poultry Science Association meeting), listserves, web based tools/utilities useful to the industry, as well as collaborative ventures among research institutions and poultry industry businesses. Many NC1170 members have leveraged USDA-AFRI or other federal grants and have substantive collaborations with poultry breeders and producers. It is noteworthy that many NC1170 researchers not only utilize specialized poultry genetic lines for experimental analysis but also routinely employ commercially-selected foundational lines and crosses in their work.


Importance of work and consequences if not conducted:


The work of NC1170 explores fundamental biological mechanisms using the most advanced technologies available that contribute new knowledge for application to improve the genetics, breeding and production of the poultry industry. These tools and our continued emphasis on their development and application are essential for improving efficiency of the birds directly (genotype selection) or indirectly (the management system, e.g., nutrition). Most important is improvement in the sustainability of poultry production. We operate in a world wherein it is imperative that researchers and stakeholders together consider the role and impact of our industries on the animal systems and the environment  thus there is an essential need for continued creation of novel opportunities made possible by new technologies and knowledge to assist the industry in its continued positive evolution. It can be easily argued that significant advancements will not occur or at least at the same rate without the engine of research and development at both the fundamental and applied levels. Key is the fact that the members of the project are often involved in the development stages of the technologies (e.g., genomics tools) and engage the community of project members in employment of the technologies. Other important facets of the NC1170 project are the educational opportunities provided for the next generation of researchers at the undergraduate and graduate levels, and the interactions with visiting scholars from numerous international locations and including commercial industry collaborators.


Technical feasibility of the work:


The methods employed by the NC1170 investigators are state of the art techniques for genomics, transcriptomics, proteomics, and bioinformatics, for the purpose of exploring the fundamental biological mechanisms underpinning growth, development, physiology, nutrition, immunity and animal health. Each of the investigators has extensive expertise in poultry and avian biology  to manage the wealth of opportunities and enormous volume of data. Efforts range among all scales, from fundamental to translational to applied. The investigators are the leaders in the various fields, involved in development and application of the tools and resources (e.g., sequencing and analysis of both the chicken and turkey genomes was accomplished with the direction and collaboration of NC1170 researchers). Further, one can easily have confidence that the investigators involved in the NC1170 project will be at the forefront of development and application of new technologies during the next five year project.


Advantages for conducting the work as Multistate Research effort:


Improvements in knowledge generation that are applicable for poultry production are not generated by single disciplinary efforts. Significant improvement in poultry production traits is the result of genotype by environment interactions. With the tools of advanced technologies we can now better dissect the phenotype which is the sum of the organisms genome (sequence), development, management, physiology, nutrition, immune status, etc. The NC1170 multistate research effort brings together principal investigators with their local priorities, a range of tool/technology backgrounds and interests (e.g., genome biologists interact with bioinformaticians and physiologists and immunologists) and employing a multidisciplinary approach (e.g., understanding the immune system in the context of genomics). The NC1170 project is in fact a sophisticated tool to bring together expertise and responsibilities (researchers and stakeholders) to advance mission-oriented knowledge with an emphasis on the fundamentals for eventual use to translate and apply that knowledge towards a meaningful goal, such as food production to feed the growing world population and meaningful contributions to the economic development of regions, states and the U.S. New goals of the industry can be more effectively addressed by the combination of resources available by the NC1170 team approach. The NC1170 researchers possess an exciting combination of expertise to create and contribute new tools and knowledge to apply to poultry improvement. And the ability to meet, interact, and share information and resources fosters an environment for multidisciplinary interactions needed to address the complex biological questions facing researchers today.


Likely impacts from successfully completing the work:


The NC1170 project impacts will include the development of new experimental tools and knowledge to opportunistically apply new breeding strategies and management systems to improve growth, development, immunity, and physiology for a stronger poultry industry which is sustainable (economically viable) and produces a safe, nutritious protein source with reduced environmental impacts.

Related, Current and Previous Work

Previous Work and Accomplishments NC1170 2008-2012


Membership and Institutional Abbreviations: Beckman Research Institute at the City of Hope (BRI: M. Miller); Iowa State University (IA: S. Lamont, J. Dekkers); Michigan State University (MSU: J. Dodgson); Mississippi State University (MS: M. Edelmann); North Carolina State University (NCSU: C. Ashwell, J. Petitte), Oregon State University (OSU: D. Froman); Purdue University (PU: W. Muir); University of Arizona (UAZ: F. McCarthy, S. Burgess); University of Arkansas (UA: B. Kong, D. Rhoads, W. Kuenzel); University of California, Davis (UCD: M. Delany, H. Zhou), University of Delaware (UD: B. Abasht); University of Georgia (GA: S. Aggrey); University of Maryland (UMD: T. Porter, J. Song); University of Minnesota (MN: K. Reed, D. Foster, F. A. Ponce de Leon); Virginia Tech (VT: Eric Wong); University of Wisconsin (UW: G. Guilherme); USDA-ARS-Avian Disease and Oncology Lab (ADOL: H. Cheng, H. Zhang).


Below we describe by objective the major accomplishments achieved by the NC1170 Multistate Research Project covering 2008 to the summer of 2012. Achievements included the application of next generation technologies in sequencing to poultry genomes and numerous contribution to advances in bioinformatics, annotation, and transcriptomics as applied to a variety of fundamental disciplines in poultry biology. Our work is conducted under the auspices of many collaborative arrangements with stakeholders involved in the allied poultry industries for the purpose of improving poultry production. Our mission-oriented research also included a large educational component wherein approximately 97 undergraduate, 29 M.S., and 57 Ph.D. students and 33 postdoctoral scholars were trained. Approximately 50 visiting scholars were hosted by our membership. The scholarly publications resulting from this project (see appendix) includes over 300 articles in peer-reviewed journals, books and proceedings. Special note should be made of the large number of publications involving collaboration between members of this Project as well as international scholars located around the world.


Objective 1: Create and share data and technology to enhance the development and application of genomics and systems biology in poultry.


MSU, UCD and MN, with collaborators, generated a detailed turkey BAC contig-based physical map, one of the most complete for any vertebrate genome. This turkey BAC contig map provided the primary platform for assembly of the first draft sequence of the turkey genome using next-generation sequencing (NGS) as part of the Turkey Genome Sequencing Consortium. Comparison to the chicken genome confirmed the high evolutionary stability of avian chromosomes and elucidated 20-27 major rearrangements between turkey and chicken genomes over 40 million years of separate evolution.
ADOL contributed the (1) generation of a consensus genetic map that consolidated the East Lansing, Wageningen, and Uppsala maps and contained 9,285 genetic markers, (2) generation of a 60K chicken SNP chip that was distributed worldwide, and (3) development and field evaluation of genomic selection in commercial broilers and layers in collaboration with PU.
MS contributed (1) 180,345 functional annotations for 47,969 chicken genes, subsequently expanded to include turkey annotation, (2) new tools for functional modeling of high-throughput data now freely available on AgBase or via iAnimal, (3) to the Chicken Gene Nomenclature Committee (CGNC) which manually approved 1,300 chicken genes, 4) a tissue expression atlas for chicken (Chickspress) that currently displays data from 11 different tissues with more being added, and 5) the development of a comparative genome browser for birds that contains the three public bird genomes (chicken, turkey, zebra finch) and is being expanded to include another 50 bird genomes sequenced by the BGI and Genome 10K projects.
UCD contributed to the comparative analysis of poultry genomes providing cytogenetic evidence for the chromosome structural changes between chicken and turkey, in collaboration with MN and MSU. UCD contributed to the molecular analysis of genomic diversity within and among chicken breeds and strains using the 60K SNP array, collaborating with PU and ADOL. The same array was used to establish the chromosomal regions of interest responsible for a series of chicken developmental mutations and determination of priority candidate genes for continued study. UCD developed a chicken whole genome 44K gene expression array that has been widely used by the poultry community.
BRI, in collaboration with other members, contributed detailed binding motifs for two MHC-B class I molecules, extensive sequence data for the MHC-B region, identification of BG1 as a Mareks tumor suppressor gene, revelation of the MHC-YF class I protein as a new type antigen presentation molecule, and characterization of chicken natural killer cells. BRI collaborated with UCD to find the correct order of MHC genes on GGA-16 revealing the presence of a heretofore unknown gene segment within the MHC.
MN collaborated on construction of genetic linkage maps and sequencing of the turkey genome (the latter with VT, MSU and UCD); investigated immune system genomics to enhance disease resistance in turkeys, including sequencing the core turkey MHC gene cluster, estimation of MHC haplotype diversity within the species through SNP analysis and quantification of MHC gene expression in immune system tissues; identified, mapped and analyzed differential expression of skeletal muscle genes in genetically selected turkeys; applied genomics to increase aflatoxin resistance in turkeys, including sequencing key genes; and investigated the genetics of round heart disease, including expression analysis of cardiac genes.
UA investigated neuroendocrine regulation of stress in chickens using the vasotocin receptor family, with a focus on the vasotocin two and vasotocin four receptors. Both receptors are highly expressed in the anterior pituitary specifically in corticotropes that contain adrenocorticotropic hormone. Gene expression of these receptors and two corticotropin releasing hormone receptors were shown to be significantly altered following several different stress models. The data suggest that these four receptors play critical roles in the neuroendocrine regulation of stress in birds.
MS used chemical proteomics to identify chicken deubiquitinating enzymes (DUBs), especially ones regulated during Salmonella infection, and to identify ubiquitinated proteins in chicken. They established an enrichment technique for ubiquitinated proteins in chicken macrophages and optimized ubiquitin-specific probe labeling to identify new chicken DUBs or confirm their annotation. Further new chicken DUBs were identified with consistent differences in abundance during infection with Salmonella.
UMD used genome-wide histone methylation analysis and pathway predictions to identify virus-induced DNA methylation changes during Mareks disease virus (MDV) infections, and identified associations between lipoprotein metabolism and MDV infection. Computational epigenetics methods were developed for evaluating the similarity between epigenetics patterns.


Objective 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.


IA maintained unique chicken research lines [including highly inbred; MHC-congenic; closed populations; and advanced intercross lines (AIL)] as resources for identifying genes and QTL of economic importance. Genetic material (chicks, fertile eggs, blood, tissue, DNA or RNA) was shared with cooperating investigators to expand studies on the chicken genome. Financial constraints resulted in the termination of 11 of the 24 lines in 2009-2010.
MSU, in collaboration with ADOL, demonstrated that retroviral short hairpin microRNA vectors were capable of generating antiviral RNA interference against Mareks disease virus, both in cell culture and in vivo.
ADOL maintains a large number of chicken lines that are characterized for a number of traits, especially those associated with viral diseases, including maintenance under specific pathogen free (SPF) conditions. Besides providing unique genetic resources to our location, ~1500 embryos or chicks are supplied yearly to academic institutions or companies in the United States.
MS supported avian phenotype analysis by developing chicken anatomy ontology. The ontology currently contains 1,829 adult chicken anatomical terms from 11 anatomical systems.
MN developed the SC-2 immortal chicken embryo fibroblast cell line; analyzed gene expression in senescent and immortal chicken CEF cells; performed SAGE analysis of chicken primordial germ cells; used reverse genetics to generating a biomarker avian metapneumovirus vaccine; investigated development of chicken inducible pluripotent stem cells; developed immunogens to protect against turkey cellulitis; and induced chicken embryonic stem-like cells using recombinant SV40 large T antigen.
NCSU established lines of chicken primordial germ cells (PGCs) from transgenic chickens expressing green fluorescent protein. Retinoid acid (RA) was found to induce chicken PGCs of both sexes to enter meiosis, act directly on the PGCs, and up-regulate Stra8 expression in PGCs with similar increases in the expression of Dmc1 and Sycp3. In collaboration with Zhejiang University, Epidermal Growth Factor (EGF) was found to stimulate proliferation of chicken PGCs via activation of Ca(2+)/PKC involving the NFKB1 signaling pathway.


Objective 3: Elucidate genetic mechanisms that underlie economic traits and develop new methods to apply that knowledge to poultry breeding practices.


OSU and UA identified genetic regions contributing to low sperm mobility in roosters, a major determinant of male fertility. Proteomic analysis with MS and UAZ indicated reduced energy metabolism in low mobility sperm. A model was developed to explain mobility through stochastic processes arising from reduced energy reserves and transit time in the excurrent ducts.
UA studied the basis of ascites in broilers. Experimental lines were generated and used to map some of the genes involved in susceptibility and to study production traits associated with ascites susceptibility. UA performed microarray analyses to find differentially expressed host genes in cultured chicken cells infected by wild type and vaccine strain infectious laryngotracheitis virus (ILTV) to identify gene networks and functional roles of candidate genes in host-ILTV interactions. ILTV encoding microRNAs were identified using next generation sequencing (NGS). Microarray analyses were used to identify differentially expressed genes in DF-1 chicken embryo fibroblast (CEF) cells and in senescent CEF cells compared to primary CEF cells. Hundreds of differentially expressed genes were identified and bioinformatics was used to interpret gene network and functional roles of candidate genes in cellular proliferation and lifespan in avian cells. UA examined genetic mechanisms for neuroendocrine regulation of testes development to identify the distribution of specialized neurons in the brain that respond to changes in photoperiod and their neuroanatomical pathway responsible for activating the reproductive system for the production of semen. Identification of key genes and specific neurons comprising the neural pathway could aid in the future sustainability of fertility over a typical reproductive cycle thereby assisting in elucidating markers for the selection of poultry male breeders.
IA focused on the host response to bacterial infections, including Salmonella, for which many new candidate genes and quantitative trait loci associated with resistance to bacterial colonization were identified. Research in resistance to pathology resulting from infection with avian pathogenic E. coli (APEC) using microarrays identified many gene networks that are important in host genetic resistance. A new area of focus  on resistance to heat stress  was recently initiated, and large-scale animal trials generated samples and data for future analysis by transcriptomic and bioinformatics study. Several statistical methods for the analysis of high-density SNP genotype data were implemented and evaluated, using both simulated and real data. This included whole-genome association analyses and whole-genome prediction of breeding values. Further strategies to implement whole-genome selection methods based on the use of less-costly low-density SNP panels combined with genotype imputation were developed and evaluated. The latter strategies have now become the norm for most applications of whole-genome selection for production animals.
ADOL focused on genes and biological pathways that confer genetic resistance to Mareks disease (MD). Using a combination of (1) allele-specific expression (ASE) in response to viral challenge, (2) ChIP seq for Meq, the viral oncogene and a bZIP transcription factor, and (3) selective sweeps using next generation sequencing, we identified 5,360 SNPs in 3,773 genes that are involved in the transcriptional response to viral infection and are overrepresented in selective sweeps. Efforts are underway to validate these SNPs and other SNPs using a custom 15K SNP chip and an ~1,000 6x7 F6 MD resource population. ADOL identified a set of 172 SNPs highly associated with Mareks disease (MD), on chromosomes 1, 3, 5, and Z; (2) a series of challenge trials provided experimental evidence that host genetics, in addition to the major histocompatibility complex, plays an important role in modulating vaccinal protective efficacy; (3) challenge trials also suggested HVT, a low efficacy MD vaccine, conveys comparable protection against very virulent MD virus challenge as CVI988/Rispens (gold standard); (4) differential DNA methylation patterns and microRNA profiles were found between MD resistant and susceptible lines of chickens in collaboration with UMD.

UCD created new knowledge regarding host  virus genome interactions involved in Mareks disease by establishing the integration profiles of the virus into chicken telomeres and determined that tumors within birds are largely clonally-derived. UCD mapped the location and variation of mega-telomere arrays in well-utilized cell line systems and UCD 001 (Red Jungle fowl sequenced genome). UCD discovered evidence for the alternate lengthening of telomeres in chicken in vitro cell systems. Genes and signaling pathways associated with host response to Campylobacter and Salmonella infection in the chicken and genes, microRNAs and signaling pathways related to avian influenza virus infection in chickens were identified using high-throughput technology including microarray and next-generation sequencing.
PU, in collaboration with ADOL, used NGS to identify genetic adaptations associated with chicken domestication and trait selection, including aggression and MD resistance, and developed software to call SNPs. We explored the use of genomic selection (GWMAS) to address social and ethical concerns while at the same time demonstrating the power and limitations of GWMAS in a multi-generational selection experiment. We found that accuracy was up to 50% greater with ssGBLUP than BLUP. Genetic trends mirrored the increase in accuracy with near 50% increase in response of the index over the 3 traits.
GA developed miRNA target programs based on specific features of a particular miRNA class. Selection studies for residual feed intake were undertaken which will lead to reduced feed intake and no changes in weight gain. High feed efficiency birds are characterized by rapid conversion of carbohydrates to ATP, high fat oxidation, increased de novo amino acid synthesis, cell division and proliferation, and efficient nitrogen recycling. Low feed efficiency birds are characterized by low ATP production, increased apoptosis, increased lipogenesis and increased ammonium production and excretion.
BRI, in collaboration with other members helped to elucidate genetic mechanisms controlling immune responses that underlie disease resistance traits in poultry including mechanisms underlying the activation of natural killer cells and of T lymphocyte responses.
UW focused on four main areas: (1) Statistical modeling approaches and optimal experimental design strategies for gene expression assays, including genomics experiments; (2) Genomic selection models, especially on kernel models, genotype x environment interaction, and non-additive genetic effects; (3) Computational strategies for implementation of genomic selection models, with focus on R software and high throughput computing using clusters; and (4) Inferring causal phenotypic networks.
VT focused on uptake of nutrients (amino acid, peptide, sugar) mediated by transporter proteins and digestive enzymes located in or at the cell membrane. Expression of these genes in the intestine during pre- and post-hatch showed differential expression in the small intestinal segments and throughout development. Expression profiles of some of these genes changed in response to changes in dietary protein quality and composition. In addition, the yolk sac membrane expresses many of the digestive enzyme and transporter genes normally associated with the intestine.
UMD used microarrays to identify differentially expressed genes in the anterior pituitary glands and hypothalami of fat line and lean line chickens. SNPs in the promoters of candidate genes were associated with fat yield and breast yield. Microarrays were also used to analyze gene expression in hypothalamus of newly hatched chicks following fasting and re-feeding and in the cecae of neonatal chicks following Salmonella and probiotic treatment to determine gene networks involved in feed intake and Salmonella reduction by probiotics. Promoter and chromatin precipitation analysis of the growth hormone gene identified transcription factors that regulate growth hormone production.
UD conducted whole genome sequencing of four experimental lines, to search for potential genomic targets of divergent selection underlying multiple fold differences in abdominal fatness, which differs by 3-fold between fat (FL) and lean (LL) lines or 12-fold between high growth (HG) and low growth (LG) lines. Several potential genomic targets of divergent selection and putative candidate genes subjected to divergent selection in experimental chicken lines were identified.

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

Partner Institution Abbreviations used in the NC1170 Multistate Research Project Methods Section: ADOL - USDA-ARS Avian Disease and Oncology Laboratory; AR - University of Arkansas; AZ - University of Arizona; BRI - Beckman Research Institute at the City of Hope; GA - University of Georgia; IA - Iowa State University; MS - Mississippi State University; MN - University of Minnesota; MSU - Michigan State University; NC - North Carolina State University; OSU - Oregon State University; PU - Purdue University; UCD - University of California, Davis; UD - University of Delaware; UMD - University of Maryland; VT - Virginia Tech; UW - University of Wisconsin OBJECTIVE 1: Create and share data and technology to enhance the development and application of genomics and systems biology in poultry. Objective 1 will employ (and develop further) a broad range of advanced tools to study genomes and trait specific candidate regions, the transcriptome, and proteome of unique genetic resources to better understand poultry growth, development and disease (the phenome). MSU will use next generation sequencing (NGS) technology to analyze genomes of chickens and chicken cell lines to identify trait or mutant-encoding alleles in collaboration with ADOL and UCD. AR will investigate global gene expression using microarrays and NGS. Broilers will be subjected to specific stressors to determine their neuroendocrine response and regulation. Birds will be anesthetized, perfused and organs prepared for immunohistochemistry using primary antibodies to neuropeptides, receptors, enzymes, hormones and/or transporters. Microarrays, brain regional, and laser capture microdissection, followed by real time quantitative PCR will be used to detect and quantify additional genes and their products. Gene expression will be correlated with steroid levels. AR, OSU, and AZ are contributing RNAseq data for a new assembly of the chicken transcriptome coordinated out of Roslin Institute (UK). This will lead to a better annotation for genome assembly. ADOL in collaboration with PU will continue genomic selection for Mareks disease resistance. A ~1,000 F6 bird resource population will be genotyped using a focused set of single nucleotide polymorphisms (SNPs), most of which exhibit allele-specific expression in response to Mareks disease virus challenge. Based on the association analyses, genomic estimated breeding values will be calculated for additional males and females; individuals will be inter-mated to select for birds with higher or lower incidences of Mareks disease in their progeny. UMD will use bioinformatics, statistical genomics, biopathway analysis and gene regulatory networks to develop novel computational methodologies for epigenetics and genetics. AZ will provide functional annotation, tools for functional modeling and develop a chicken expression atlas. They will provide education, training and outreach for researchers with poultry genomics data that they wish to model. They will collaborate with UD to develop additional functional and comparative genomics tools and data. MN will use NGS to refine the genome sequence and transcriptome of the turkey and closely related species. NGS data will be used to identify trait- or mutant-encoding alleles for targeted genomic regions including the major histocompatibility complex (MHC) and other immune system genes. UCD will utilize advanced sequencing technologies to study the genetic basis for inherited developmental malformations affecting craniofacial, organ and limb development in chicken. UCD will also develop a systems biology modeling pipeline to integrate omics data for comprehensive analyses. BRI will create an extensive, heavily annotated publicly available database providing detailed knowledge of the MHC in poultry. The site will provide easy access to the nomenclature of the region with connections to current understandings of the functions of polymorphic genes so that they can be more readily evaluated and drawn upon in new approaches to poultry breeding. NC will generate DNA methylation data at the whole-genome level. This data will be annotated to local gene loci and shared with the community through BirdBase. Methylation frequency at specific loci can then be correlated with other molecular data including gene expression data from microarrays and RNAseq.  OBJECTIVE 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. Objective 2 will address development and sharing of tools and resources describing genes/networks underlying phenotypes and will maintain and share unique genetic resources, i.e., living bird populations. AZ will provide tools to support the annotation of poultry phenotypic data (development of ontologies and tools for using these ontologies). UCD will maintain and share chicken genetic stocks (inbred, congenic inbred, and mutant) of specific genotypes expressing unique disease-related or developmental phenotypes. IA will maintain and share genetic material from unique chicken research lines (including highly inbred; MHC-congenic; closed populations; and advanced intercross lines) as resources for identifying genes and quantitative trait loci of economic importance. NC will continue to distribute eggs to the wider research community from the transgenic chicken lines expressing LacZ and eGFP through the appropriate Material Transfer Agreements. NC will develop additional transgenic chicken lines using transposase technology and primordial germ cell culture. In addition, the culture of primordial germ cells will be expanded to other species of poultry. NC will support the genetic stocks currently maintained, specifically those with well-defined MHC and disease susceptibility characteristics. Additional resource populations will also be developed to map and characterize Mendelian traits for unique phenotypes including conformation and feather pattern as well as disease resistance and alloantigens. ADOL will maintain and share chicken genetic stocks (inbred and congenic lines), most of which are genetically characterized for resistance or susceptibility to Mareks disease or avian retroviruses.  OBJECTIVE 3. Elucidate genetic mechanisms that underlie economic traits and develop new methods to apply that knowledge to poultry breeding practices. Objective 3 will identify and further investigate molecular mechanisms underlying growth, reproduction, immunology and diseases using genomics, proteomics and transcriptomics approaches. DNA markers, QTLs and SNPs associated with economic traits will be identified. UMD will use advanced cell and molecular technologies to define the genetic and molecular mechanisms underlying pituitary development and cell differentiation. NGS and microarray technologies will also be used to identify genes and gene networks involved in neuroendocrine (hypothalamic and pituitary) control of growth, development, and body composition in broiler chickens. UD will use the RNA-seq approach to understand biological mechanisms leading to high or low feed efficiency phenotype in broiler chickens. Currently, cDNA libraries are being constructed from mRNA isolated from multiple tissue types to sequence in an Illumina Hi-seq 2000. Sequence data from this study will be analyzed to identify genes differentially expressed between birds showing low and high feed efficiency phenotypes. Differentially expressed genes will then be mapped to biological pathways and physiological functions to understand the fundamental basis of differences in feed efficiency between low and high feed-efficient broilers. VT will determine the molecular mechanism that regulates expression of nutrient (amino acid, peptide sugar) transporters in the intestine following in ovo feeding and in response to infection by intestinal parasites such as Eimeria. IA will elucidate genetic mechanisms that underlie resistance to heat-stress by conducting live-bird trials with heat stress, and with combined challenges of heat stress and LPS sickness; by mapping quantitative trait loci for heat-stress response in an Advanced Intercross Line (AIL); and by transcriptomic and bioinformatic analysis of various tissues sampled in acute and chronic heat stress. AR will utilize male poultry and subject birds to different photoperiods as well as administer a compound, sulfamethazine, that interacts with photoperiod to enhance testes development. Microdissection of brain areas coupled with real time quantitative PCR will be used to quantify gene expression. To validate dissected brain areas showing significant changes in expression, in situ hybridization histochemistry will be utilized to identify specific brain nuclei. Neural pathways associated with gonadal development will be developed based upon tract-tracing methods including retrograde and anterograde procedures. AR will use classical genetics, NGS, and SNP genetics to identify gene regions differentially represented in divergently selected lines for contribution to the following traits: muscle quality, ascites, tumor progression, autoimmunity. AR, OSU and AZ will continue analyses using NGS, SNP chips, quantitative genetics, physiology, proteomics, and gene expression, to identify the genes involved in reduced sperm mobility affecting broiler breeder roosters. ADOL in collaboration with PU, UMD and UCD will identify genes, alleles and pathways that affect genetic resistance to Mareks disease. Experimental or commercial birds will be challenged with Mareks disease virus. Splenic RNA will be isolated, sequenced, and analyzed to reveal SNPs that show allele-specific expression in response to virus challenge, i.e., the allelic ratios will change between uninfected and Mareks disease virus-infected birds. ADOL will continue to explore genetic and epigenetic factors as well as biological pathways that confer genetic resistance to Mareks and vaccine protective efficacy with technologies including genome wide association analysis (GWAS), Pyrosequencing, and NGS. UMD will also focus on epigenetic alterations induced by virus exposure and explore regulatory mechanisms related to etiology of the disease. BRI in collaboration with UCD will use genomics, fluorescence in situ hybridization, and immunological methods to characterize genes newly mapped to chicken GGA16 (MHC-encoding) chromosome. The focus will be on genes that regulate innate and adaptive immunity. BRI will pursue the epigenetic regulation of BG1, a polymorphic gene that affects the incidence of Mareks disease and also develop new methods to test the role of polymorphic MHC-Y class I molecules in antigen presentation. UCD will use transcriptome, RNAome and proteome profiling utilizing high-throughput technologies such as NGS to identify differentially expressed genes associated with Campylobacter, Salmonella, avian influenza virus infection in immune-related tissues. Further, UCD will employ molecular cytogenetics techniques and collaborate with ADOL to investigate the role of chromosomal aberrations in Mareks disease virus induced transformation and oncogenesis in resistant and susceptible lines. MS will test the upregulation of ubiquitin-specific enzymes in response to Salmonella enterica infection previously detected by proteomics. RNAi will be used to knockdown expression of these enzymes in the chicken cell model and test their effect on the infection rates. Microscopy will be used to visualize localization of these proteins within the infected/uninfected chicken cell. Moreover, proteomics will be used to identify more ubiquitin-specific proteases in chicken, which have been only so far identified as possible orthologues of human enzymes. MN will identify genes, alleles and pathways that affect genetic susceptibility/resistance to aflatoxin in turkey. MN will study immunogenetic mechanisms that underlie disease resistance/susceptibility in turkey. PU will use methods to establish genotype to phenotype associations and suggest candidate genes for traits of economic importance. These methods include a combination of "hitchhiking mapping", using next generation sequencing to identify patterns associated with selection; allele specific expression (ASE) to find polymorphisms associated with differential gene expression, and genome wide association analysis GWAS using the 60K SNP chip. These will be followed up by actual selection on genotype to prove that identified genotypes will result in desired phenotype. GA will use the machine learning approach 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, 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 also continue to work on implementation of whole-genome selection in poultry breeding programs and on genome-wide association analyses to identify genetic markers or genomic regions associated with traits of economic importance in poultry. NC will generate DNA methylation data at the whole-genome level. This data will be annotated to local gene loci and shared with the community through BirdBase. Methylation frequency at specific loci can then be correlated with other molecular data including gene expression data from microarrays and RNAseq. AZ will combine the use of -omics data with the functional modeling and phenotype tools to support the identification of key genes and genetic mechanisms that can be applied to improve poultry through breeding. UW will conduct research on development and application of statistical methods for inferring functional (causal) relationships between phenotypic traits, and investigate how knowledge regarding phenotypic causal networks can be exploited in the context of genome-wide association analysis and genomic selection.

Measurement of Progress and Results

Outputs

• Continued resolution and improvement of genomic sequence content and genetic maps for turkey and chicken
• Enhancement of bioinformatic methods and tools for use in genome analysis of poultry
• Maintenance of genetic resource populations for use by the scientific community and development of new lines
• Identification of genes and pathways involved in generating phenotype
• Identification of sequence variation involved in generating phenotype

Outcomes or Projected Impacts

• Increased opportunity for stakeholders to utilize genomic data in breeding programs
• New genome analysis and bioinformatics tools for community use in accessible databases
• Improved knowledge of genotype x environment interactions creating phenotype
• New knowledge regarding gene interactions in pathways
• Education of students and post doctoral scholars and collaboration with many visiting international scholars drawing their attention to our project goals

Milestones

Projected Participation

View Appendix E: Participation

Outreach Plan

Technical Committee (TC) members will publish NC1170 project generated data in the peer reviewed literature thus making content accessible to the scientific community. In addition members participate in conferences and workshops (Poultry Science annual meeting, International Plant and Animal Genome annual meeting, and many disciplinary or technology-related meetings) providing oral presentations of results and proceedings (written communications). Most conference and workshop environments include stakeholder groups and further committee members provide seminars on their work at other academic institutions and companies. Those conducting genome sequencing deposit their results in relevant federally-funded databases allowing access by the scientific community and stakeholders. Several NC1170 members also participate in other multistate research projects (e.g., NE1034) allowing for cross fertilization of techniques and relevant content. Several members are organizers of databases as part of their project efforts and ensure dissemination of NC1170 data, tools and content. The Poultry Genome Newsletter with a national and international distribution list covers NC1170 meeting highlights.

Organization/Governance

The organization and planning of the NC1170 Project is the responsibility of the Technical Committee (TC) members consisting of scientists from land-grant or non-land grant research institutions, the USDA-ARS and industry. Administrative guidance will be provided by an assigned Administrative Advisor and a CSREES Representative. Voting membership shall consist of the TC members; only one member representing each participating agency shall be eligible to vote. Our governance includes a two year term for Chair and a Secretary. An executive committee of Chair, Secretary and immediate past-Chair (if needed) coordinate business between meetings. Volunteer members of the TC organize objective content for necessary reports. Members are expected to write and distribute annual reports prior to each annual meeting. Members are expected to attend annual meetings at least three of five years. Attendees at meetings can include emeritus members, collaborating scientists, invited colleagues, scientists interested in the topical areas discussed.

Literature Cited

Please see the appendix which includes over 300 citations covering the publications (peer-reviewed journals, book chapters, proceedings) resulting from the collaborative research of this project from 2008 through the summer of 2012.

Attachments

Land Grant Participating States/Institutions

AR, AZ, CA, DE, FL, GA, IA, IN, MD, MI, MN, MS, NC, NY, OR, PA, TN, TX, VA, WI, WV

Non Land Grant Participating States/Institutions

California State University, Fresno, City of Hope Beckman Research Institute, USDA-ARS-Avian Disease & Oncology Laboratory, Washington University in St. Louis, Western University of Health Sciences