NC1170: Advanced Technologies for the Genetic Improvement of Poultry
(Multistate Research Project)
NC1170: Advanced Technologies for the Genetic Improvement of Poultry
Duration: 10/01/2018 to 09/30/2023
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 world’s 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 2015 was $47.9 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.
The importance of the work, and what the consequences are if it is not done
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.
The technical feasibility of the research.
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.
The advantages for doing the work as a multistate 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 organism’s 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.
What the likely impacts will be 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: Years 2013-2017
Membership and Institutional Abbreviations: Beckman Research Institute at the City of Hope (BRI: M. Miller); California State University- Fresno (CSUF: K. Tarrant); Cornell University (CU, P. Johnson); Iowa State University (IA: S. Lamont, J. Dekkers); Michigan State University (MSU: J. Dodgson, G. Strasburg); Mississippi State University (MS: M. Edelmann, B. Nanduri); North Carolina State University (NCSU: C. Ashwell, J. Petitte), Oregon State University (OSU: D. Froman); Pennsylvania State University (PSU: A. Johnson); Purdue University (PU: W. Muir); University of Arizona (AZ: F. McCarthy, S. Burgess); University of Arkansas (AR: B. Kong, D. Rhoads, W. Kuenzel); University of California, Davis (UCD: M. Delany, H. Zhou), University of Delaware (UD: B. Abasht); University of Florida (FL: M. Edelmann), University of Georgia (GA: S. Aggrey); University of Maryland (UMD: T. Porter, J. Song); University of Minnesota (MN: K. Reed); Virginia Tech (VT: Eric Wong, E. Smith); University of Tennessee (TN: B. Voy) University of Wisconsin (UW: G. Rosa); USDA-ARS-Avian Disease and Oncology Lab (ADOL: H. Cheng).
Below we describe by objective the major accomplishments achieved by the NC1170 Multistate Research Project covering 2013 to the summer of 2017. Achievements included the application of next generation sequencing technologies to improving poultry genomes and numerous contributions to advances in bioinformatics, annotation, and transcriptomics as applied to fundamental disciplines in poultry biology. Our work is conducted under the auspices of many collaborative arrangements and with stakeholders in the allied poultry industries for the purpose of improving poultry production. 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 among members of this Project as well as international researchers.
Objective 1: Create and share data and technology to enhance the development and application of genomics and systems biology in poultry.
ADOL performed targeted mapping to aid the Gallus_gallus-5.0 assembly. Specifically, 9,585 unassigned sequence contigs 2 Kb or larger in size were identified and aligned to existing Illumina reads, revealing 4,160 scaffolds that cover ~39.6 Mb with at least 1 SNP of reasonable confidence. Further filtering based on Affymetrix design scores gave 5,907 SNPs on 547 contigs that could be potentially genotyped. However, in the end, 3,440 SNPs and 510 contigs could be assayed and successfully scored. Following genetic mapping, 3,437 SNPs were assigned to 29 new linkage groups (designated E101 to E129), none of which were linked to existing markers; three SNPs were unlinked and assigned to E00.
MI conducted next generation sequence analysis of several chicken lines, particularly for developmental mutants. Several candidate genes and SNPs were identified.
MN collaborated with VT on the refinement of the turkey genome sequence, RNAseq-based support for genome annotation and characterization of the developmental transcriptome. MN expanded the core MHC (B and Y) sequences of the turkey and measured MHC diversity in the species through SNP-based genotyping. Capture-array NGS sequencing of the MHC identified over 5000 potential turkey SNVs, over 400 SNVs in the chicken (RJF-256) MHC, and support for an expanded BG gene cluster in the turkey.
TX generated whole genome sequences from two wild Gallus species, and from three domestic breeds of Gallus to characterize structural variation in chicken genomes. Comparison against the Gallus reference genome V4 identified small and large structural variants. Further analysis of inversions and large deletions helped identify variants that were unique or shared among compared varieties. Over 200 previously unreported variants have been identified and are being verified by standard laboratory techniques. Furthermore, a new software was developed to classify and compare variants and catalog into a new database.
BRI concentrated on defining the gene content of chicken chromosome 16 (GGA16). Even though GGA16 is of particular interest for its contribution to the immune responses of chickens, genomic organization and gene content of GGA16 remains poorly defined. GGA16, a microchromosome, has been particularly difficult to sequence. In one project with NC1170 collaborators, BRI used trisomy mapping to find and assign additional genes to GGA16. Of interest for their potential to contribute to immunity in chickens are genes for scavenger receptors and olfactory receptors that were identified and mapped proximal to the nucleolar organizer region on the q-arm of GGA16. In a second, ongoing study also carried out with NC1170 collaborators and which is nearing completion, BRI isolated clones corresponding to the MHC-Y region in the red jungle fowl (RJF) reference genome. The BAC sequences were successfully determined with SMRT sequencing made possible with a small award provided from NC1170 funds. The MHC-Y genomic sequence revealed the presence of more than 80 genes within the RJF MHC-Y haplotype. These are located within dense arrays of repetitive sequences. Members of two gene families predominate. In a third project, BRI collaborated in the development of a SNP panel highly useful in defining haplotypes in the MHC-B region of GGA16.
AZ has continued to develop tools and resources to support the application of genomic technologies to improve poultry production and health. Standardized gene nomenclature is available for chicken genes, including manually curated gene symbols and names for >1,600 genes and Gene Ontology (GO) annotations for more than 45,00 chicken and turkey gene products (proteins and transcripts). In addition, there is a comprehensive Chicken Expression resource (Chickspress). VERVE Net is an online forum to support collaboration among virology researchers, and includes help with protocols and methods. A collaborative project with MS developed host-pathogen resources for chicken pathogens, and now provides more than 12,000 interactions (both predicted and experimentally confirmed) for Marek’s Disease Virus and Salmonella species.
MS and AZ continued to develop AgBase, a curated, open-source web-accessible resource that provides Gene Ontology data, tools, training and support for understanding functional genomics data for agricultural species including chicken. AgBase provide 1,735,018 GO annotation for 341,526 proteins for 528 species, including more than 40 agriculturally important species and their pathogens (as of 15 February 2017). AgBase database also provides support for Chickspress, chicken expression atlas (transcripts, miRNA, and protein expressed in different chicken tissues). The Chicken Gene Nomenclature Consortium (CGNC) is the sole source globally for chicken gene nomenclature. AgBase provides standardized nomenclature for 29,308 chicken genes disseminated to international resources such as NLM/NIH National Center for Biotechnology Information (NCBI) Entrez database, and the European Bioinformatics Institute (EBI) Universal Protein Resource (UniProt) and Ensembl genomes browser. A host-pathogen interaction database (HPIDB) was developed containing 55,505 unique protein interactions between 55 host and 523 pathogen species (as of March 2017). HPIDB is one of 12 databases that are partners in the International Molecular Exchange Consortium (IMEX) and the only database that provides detailed, manual curation for livestock pathogens.
UCD developed advanced epigenetic and epigenomic assays for various tissues. The bioinformatics pipeline in analyzing omic data were developed to identify peaks for DNase-seq, ChIP-seq and regulatory elements in the genome.
FL in collaboration with MS has created a map of approximately 200 kinases in spleen and liver tissues in chicken based on their reactivity with the ATP and ADP desthiobiotin acyl phosphate probe. The assumed functions of these kinases were analyzed by comparing functional pathways and disease involvement of human, murine and rat orthologs of these kinases. We have also screened active deubiquitinases in chicken tissues including spleen, liver, and cecum by using chemical proteomics, which was done to obtain a map of active ubiquitin processing enzymes in these tissues.
IA has RNAseq data sets from several tissues plus one cell line of chickens under various treatments (heat stress, pathogen challenges, lipopolysaccharide exposure) and control conditions were deposited in public databases.
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.
UCD maintained developmental mutant and immunogenetic congenic inbred lines of chicken along with other genetics stocks important to the community (e.g., UCD 001, line that the genome builds are based upon; GGA16 aneuploid and deletion lines). Genotyping, phenotyping and annual breeding were undertaken; on a request basis lines were made available on a collaborative or recharge basis to poultry investigators. UCD identified hundreds of genes and signaling pathways associated with Newcastle disease virus and avian influence virus infection and disease resistance. Key microorganisms in the cecum associated with Salmonella infection were identified and provided great insights to develop better strategy in improving poultry gut health.
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.
GA maintains the Athens Canadian and Athens Randombred populations. These two populations were established in the 1950s and serve a resource to other academic institutions in the US, and also as control for industry lines.
IA maintains chicken genetics research lines (including highly inbred, MHC-congenic, and closed populations; and advanced intercross lines) which are used and distributed as the biological discovery platform for research at IA, by collaborators in NC-1170 and others. Studies of these lines identified, duplication of defensin genes in a relatively disease-resistant line, wide-spread allele-specific expression, and genomic regions and pathways associated with response to various pathogens and high temperature.
TX is maintaining a breeding population or Red Jungle fowl (Richardson strain). Active management of breeding individuals is being undertaken based on molecular marker-based parentage analyses. Blood and/or DNA samples from these birds are available for academic institutions or companies for comparative studies.
AZ has developed more than 1,700 standardized anatomy terms to add to add to the Uberon ontology. This will support comparative studies among vertebrate species and provide a resource than will enable more refined searching and accessibility to large-scale poultry data sets.
NCSU has established several cell lines of chicken primordial germ cells (PGCs) for use in the study of germ cell development and the development of transgenic chickens. Cultures of PGCs were adapted to serum-free or low serum culture. Using PGCs expressing GFP, it was established that that Z-bearing female germ cells undergo normal spermatogenesis in genetic males, but are incapable of fertilization. Gene editing of NOS2 and NOX2 using CRISPR/CAS9 was accomplished in two macrophage cells lines, Q-NCSU and HD11 cells.
Objective 3: Elucidate genetic mechanisms that underlie economic traits and develop new methods to apply that knowledge to poultry breeding practices.
ADOL, hypothesizing that variation in gene expression is the major mechanism underlying complex traits, utilized allele-specific expression (ASE) in response to Marek’s disease virus (MDV) infection to identify genes associated with genetic resistance to Marek’s disease (MD). Specifically, a ~1,000 6x7 F6 (advanced intercross) MD resource population was genotyped with a custom SNP array. Analysis yielded an h2 of 0.53 for MD genetic resistance, and SNPs accounted for >83% of the genetic variance. Furthermore, the resulting information was successfully used to select 30 MD resistant and 30 MD susceptible sires in the next generation; the accuracy of selection was 125% higher for selection based on ASE SNPs compared to that of pedigree evaluation. UCD collaborating with ADOL elucidated the genome interactions between Marek’s disease virus, vaccines and the chicken host genome establishing viral genome integrations are a key feature of infection, pathogenesis and tumorigenesis and that vaccines integrate as well as the disease-causing virus. UCD, through fine mapping and sequencing, continued to narrow the causative region of interest for several developmental mutations and possible candidate genes.
AR used whole genome resequencing to identify 31 potential QTLs for ascites phenotype in their experimental ascites lines. Three of these regions have been further validated in additional samples and commercial lines. AR identified probiotics and prebiotics effective in reducing bacterial chondronecrosis with osteomyelitis (BCO lameness) in broilers using two different models (wire flooring vs litter with and without bacterial challenge). In addition, they have sequenced bacterial isolates to identify pathogenicity markers for BCO lameness. AR and DE collaborated on RNAseq analyses of primordial germ cell cultures from layers and broilers and both genders. This work is aimed understanding gender differences in germ line cells and to identify germ cell specific expression in broilers and layers.
IA utilized high-density SNP genotyping to reveal selection signatures in local populations of chickens from Africa and northern Europe. Genome-wide association studies using a 600K SNP chip identified quantitative trait loci associated with phenotypic response to heat stress in a broiler by Fayoumi advanced intercross. Differential expression of genes from RNA-sequencing of various tissues of chickens challenged with E. coli, with lipopolysaccharide, or with high ambient temperature revealed putative genes and genetic pathways important in the chicken’s response to biotic and abiotic stressors. Statistical methodologies for application of low and high-density SNP genotypes in commercial genetic improvement were developed. Success of implementing genomic selection in commercial egg-layers was demonstrated.
PSU focused on characterizing the most proximal cellular mechanisms regulating the process by which a single follicle is recruited into the preovulatory hierarchy together with causes of misregulation in broiler breeder hens. One outcome was to identify abnormal glucose and lipid metabolism as a prominent cause of ovarian dysfunction. Results to date have demonstrated that the anti-diabetic drug, metformin, can prevent dysfunctional follicle metabolism, in vitro, and enhance the rate of egg production, in vivo, in broiler breeders. A second outcome has been to identify a cell signaling event believed to be the most proximal cellular event that results in the daily recruitment of a single follicle into the preovulatory hierarchy. Each of these outcomes will contribute to developing strategies to further enhance reproductive efficiency in poultry.
TN focused on understanding the molecular pathways that control adipose tissue development and deposition in chicks, and on how diet can be used to manipulate these pathways and reduce fatness. Microarrays and RNAseq have been used to identify biochemical pathways that are coordinately regulated with adipose mass and adipocyte size. Methods for applying metabolomics and lipidomics to chicken tissues have been developed, as have approaches to integrate transcriptomic and metabolomic data.
VT has examined the mRNA expression profiles of nutrient transporters in the yolk sac and intestine during embryogenesis and post-hatch using qPCR and in situ hybridization. These transporters show tissue- and age-specific expression patterns. Challenge of broilers or layers with different Eimeria species revealed differential expression of intestinal nutrient transporters.
MN conducted research directed to expand the turkey MHC gene cluster(s) and quantified MHC gene expression in immune system tissues. In collaboration with MI analyzed differential expression of skeletal muscle genes in cultured muscle satellite cells and in genetically selected turkey poults in the context of a thermal challenge model. MN used applied genomics in collaboration with Utah State in work aimed at understanding the mechanisms of aflatoxin susceptibility/resistance in turkeys through expression analyses of liver and spleen trancriptomes in embryonic and juvenile birds in challenge models. Experiments were also conducted to examine variation in and activity of the Mx protein and investigate the genetics of round heart disease, including expression analysis of cardiac genes.
MI conducted studies in collaboration with Ohio State University and MN demonstrating that posthatch thermal challenge of turkey alters breast muscle ultrastructure.
TX carried out studies to investigate functional hallmarks of the broiler meat quality condition called wooden breast syndrome. RNAseq and SNP datasets were generated to elucidate the mechanisms underlying the economically important conditions. RNAseq analyses has identified genes and pathways that are affected in woody breast tissue, especially when comparing against breeds with differing growth profiles. SNP data from the 650K Affymetrix chip is being analyzed to provide further context to gene expression data.
UD combined transcriptomics (RNA-seq), metabolomics, histology, electron microscopy and bioinformatics approaches to investigate the onset and pathogenesis of wooden breast disease in broilers. Also, UD collaborated with IA to investigate genomic imprinting, allele-specific expression and linkage disequilibrium in experimental and commercial chicken lines.
AR and collaborators conducted shotgun proteomics on feed efficiency phenotypes of breast muscle and woody breast muscle myopathies for broilers, resulting in differentially expressed proteins. AR used RNAseq to analyze differentially expressed genes in breast muscle in relation to feed efficiency. AR searched genome wide genetic markers for Smyth line chicken model for autoimmune vitiligo pigment disorder and divergently selected muscle color line.
GA focused on developing new models for feed utilization efficiency. Using microarrays and next generation sequencing, GA delineated the molecular basis of feed efficiency, and developed the ANT colony algorithm for SNP association with traits.
CU focused on the hormonal mechanisms underlying the differential effect of feeding level on follicle development in broiler chickens. The relationship between the metabolic and reproductive axis in broiler breeder hens was examined by investigating the reproductive parameters and the liver transcriptome of broiler breeders maintained on a feed-restricted diet (RF) or an ad libitum (FF) diet. 120 genes were differentially expressed with a > 2 fold change (p<0.05; FDR< 0.05); 51 genes were up-regulated and 69 genes were down-regulated in FF compared to RF.
UW and collaborators focused research on genome-enabled prediction of complex traits. They investigated differential contribution of genomic regions to marked genetic variation and prediction of quantitative traits in broiler chickens. In addition, they developed models to incorporate parent-of-origin effects in prediction models. Another central area of research from the group referred to the use of graphical models such structural equation models to investigate gene-phenotypic networks.
VT focused on genotype:phenotype relationships in turkeys and chickens. Additionally, we continued our efforts to improve graduate training, especially those from underrepresented groups by testing out the effect of cohorts and “reflections by scientists that we called scientific journeys” on retention and completion of PhD and competitiveness for prestigious fellowships and awards. Among awards received by our trainees were NIH’s F31, Goldwater Fellowship, and NSF’s GRFP. Using this approach, a total of 48 PhDs have been produced: and our retention rate of 85% in the PhD program far exceeds the average of 51% reported by NSF. Since graduate students contribute significantly to research productivity we propose that animal genome scientists use a cohort approach (and to move away from the one-student-PI-driven to recruiting grad students for their research to ensure a community that can be used to support each other.
Create and share data and technology to enhance the development and application of genomics, epigenomics, and systems biology in poultry.
Facilitate the creation and sharing of poultry research populations and the collection and analysis of relevant new phenotypes including those produced by gene editing.
Elucidate genetic mechanisms that underlie economically important traits, including genetic variants and functional regulatory elements within the genomes of poultry species, and develop new methods to apply that knowledge to poultry breeding practices.
Project Participants and Institutional Abbreviations for the years 2018-2023: Beckman Research Institute at the City of Hope (BRI: M. Miller); California State University- Fresno (CSUF: K. Tarrant); Iowa State University (IA: S. Lamont, J. Dekkers); Michigan State University (MSU: G. Strasburg); Mississippi State University (MS: B. Nanduri); North Carolina State University (NCSU: J. Petitte); University of Arizona (AZ: F. McCarthy, S. Burgess); University of Arkansas (AR: B. Kong, D. Rhoads, W. Kuenzel); University of California, Davis (UCD: M. Delany, H. Zhou), University of Delaware (UD: B. Abasht); University of Florida (FL: M. Edelmann), University of Georgia (GA: S. Aggrey); University of Maryland (UMD: T. Porter, J. Song); University of Minnesota (MN: K. Reed); Virginia Tech (VT: E. Wong, E. Smith); University of Tennessee (TN: B. Voy) University of Wisconsin (UW: G. Rosa, R. Sunde); USDA-ARS-Avian Disease and Oncology Lab (ADOL: H. Cheng).
Objective 1. Create and share data and technology to enhance the development and application of genomics, epigenomics, and systems biology in poultry.
Objective 1 will collect and disseminate both data and methods to improve our understanding of the interaction between the genome and the environment. Environment encompasses all levels including the nucleus, cell, tissue, organism and external environment. Ultimately, it is the combination of the genome and environment that gives rise to the phenotypes important to poultry production. Systems biology approaches will be applied to model and integrate data across these various levels to improve our ability to apply genomic understanding to improving poultry production. This will be achieved by employing genomic sequencing and Genome Wide Association Studies (GWAS) to characterize additional poultry lines that have distinct traits, defining regulatory elements in the genome (ChIP-seq, ATAC-seq, Hi-C), further defining expressed genes (RNA-seq) and their products (Proteomics).
BRI, TAMU, ADOL will all be applying genomic sequencing to improve poultry genomics annotation and define additional structural variants in both production and heritage lines. BRI will focus on providing a complete annotated genomic sequence of the MHC-Y region, which has been problematic. Further, BRI will develop new methods to type chicken MHC-Y sequences to better our understanding of the relationship between this important genetic region and immune traits. TX will exploit genomic sequencing to identify structural variants between domestic and wild chickens. This will provide a baseline for associating traits with genetic variants across these distinct chickens. TX will also provide bioinformatic tools to improve variant calling and a publically accessible resource to provide community access to the data. ADOL will apply sequencing along with other approaches to associate genetic and epigenetic variants with resistance to Marek’s disease (MD), improving MD vaccine efficiency and identifying mutations that are associated with distinct MD tumors. VT and UD will be employing GWAS to explore the genotype-phenotype relations affecting inflammatory response in the turkey and to identify alleles involved in woody breast disease in the chicken, respectively.
UCD, AR and UD will be applying RNA-sequencing techniques to improve our annotation of the chicken transcriptome and provide better resolution of alternative transcripts. FL will be using both chemical and proteomic approaches to annotate kinases, serine hydrolases and deubiquitinases encoded in the chicken genome. UC-Davis and UD will be employing genomic and epigenetic technologies including ChiP-seq, ATAC-seq and Hi-C to identify regulatory elements and epigenetic changes in the tissues of multiple chicken lines. WI is utilizing genomic approaches including RNA-seq in collaboration with MN to examine Se requirements and selenoprotein enzyme and transcript expression in the turkey, to better substantiate what the turkey dietary Se requirement should be, and to better understand why turkey Se requirements and metabolism are different from that of mammals. We anticipate that the groups will be sharing and integrating the genomic, transcriptomic and proteomic data generated by the NC1170 group. This data will provide an important foundation for continued systems biology approaches to allow for continued improvement to the poultry industry.
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 editing
We will achieve this objective by maintenance and development of specialized poultry populations, cell lines and resources for researchers to effectively investigate key production traits. During 2009-2010 financial constraints resulted in the termination of 11 poultry lines, and other populations are also becoming economically burdensome for individual research groups that support them. While we support the continued support for these valuable poultry population, we also acknowledge that the development of genetic resources for in vitro and in ovo functional analysis may help mitigate costs associated with poultry populations. We support the creation, sharing and phenotyping of genetic material (e.g., chicks, fertile eggs, blood, tissue, DNA or RNA) for collaborative studies. This includes the maintenance or development of avian cell lines to support in vitro studies, as well as resources for in vivo research.
Our approach is to identify both in vivo and in vitro resources that serve as tools for identifying the causal agents for economically important traits. Poultry lines and populations include (but are not limited to) inbred lines, congenic lines and advanced intercross lines that are genotyped for relevant traits and phenotypes, as well as pathogen-free populations. We also support the development and maintenance of avian resources for furthering in vitro studies. These studies may include (but are not limited to) investigating molecular mechanisms underlying important production traits, understanding how gene expression is regulated to produce observed phenotypes and determining function of genetic elements that contribute to traits and phenotypes. Resources to support mechanistic and functional in vitro or in ovo studies include the development of cell lines, and technologies for creating transgenics and mutants (e.g., gene delivery, over-expression, knock downs and targeted mutagenesis). With the collection of poultry samples in support of the Functional Annotation of Animal Genomes (FAANG) Project, we endorse FAANG sample metadata specifications, and characterization of poultry research resources to meet this standard.
NC will examine the characteristics of CRISPR/CAS9 gene editing in various avian cell lines and in primordial germ cells with a specific reference to off-target phenomenon. The understanding of CAS9 binding and subsequent repair associated with gene editing in avian cells will provide new information on the best approaches to develop strategies for precise manipulation of the avian genome that would avoid off-target effects.
TX will maintain and share genetic material from Red Junglefowl population (Richardson Strain). Samples from birds with pedigree data will be maintained and shared to aid in the identification of loci important for health, welfare, and performance traits.
ADOL will maintain and share chicken genetic stocks (inbred and congenic lines), most of which are genetically characterized for resistance or susceptibility to Marek’s disease or avian retroviruses.
IA will maintain and share genetic material from unique chicken research lines (including diverse, highly inbred lines; MHC-congenic lines; and highly advanced intercross lines of more than 30 generations of intercrossing) as resources for identifying genes, genomic regions and genetic networks of economic importance.
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. Highly inbred lines that differ in disease response to viral infection will be evaluated using next generation sequencing to understand molecular mechanism of genetic resistance to viral infection. Regulatory elements such as enhancer, insulator, promoter in chicken genome will be functionally annotated and functional elements related to immune function will be identified. Genetic variants associated with disease resistance to virus infection will be used to genetically enhance broad immunity and resistance to specific pathogens in poultry. Through SNP genotyping to identify carriers or cellular and organismal phenotyping, UCD will continue to maintain, breed, and make available genetic resources (lines of chickens) that are important for research of the poultry community.
Objective 3. Elucidate genetic mechanisms that underlie economically important traits (genotype to phenotype) including genetic variants and functional regulatory elements within the genomes of poultry species, and develop new methods to apply that knowledge to poultry breeding practices.
Objective 3 will identify and further investigate molecular and genetic mechanisms underlying the basic biology of growth, reproduction, and immunology using both established and emerging research approaches. We will achieve this objective by using genomic (DNA markers, SNPs) and cell biology technologies (in situ hybridization, histochemistry), expression profiles (microarray, RNAseq, proteiomics) and bioinformatic analysis (QTLs, GWAS, metagenomics) in combination with key animal resources (stem cells, inbred research lines, commercial lines, and exotic ecotypes).
Basic Biology: UMD will use cell and molecular technologies to define the mechanisms underlying pituitary development and cell differentiation and to identify genes and gene networks involved in neuroendocrine (hypothalamic and pituitary) control of growth, development, and body composition in broiler chickens. UA will investigate the effect of stressors on growth and reproductive function and develop neural pathways associated with gonadal development based upon tract-tracing methods. BRI in collaboration with UCD will work to characterize genes that regulate innate and adaptive immunity that have been newly mapped to the chicken MHC chromosome. UCD will collaborate with (ADOL) to investigate chromosomal aberrations in MDV-inducted transformation and oncogenesis in resistant and susceptible chicken lines.
Production traits: IA will elucidate genetic mechanisms that underlie heat tolerance, and work to implement whole-genome selection in breeding programs associated with traits of economic importance. CSUF will create genome wide association studies, SNP genotyping, and classical genetic data. Associations will be made between gene regions and economically important broiler chickens traits. UA will identify differentially represented gene regions in divergently selected lines that contribute to muscle quality, ascites, tumor progression, autoimmunity and the cellular basis for susceptibility to bacterial chondronecrosis with osteomyelitis leading to lameness. MN will examine genomic genome responses in turkeys associated external stressors including; the genetic susceptibility/resistance to aflatoxin, thermal challenge, and immunogenetic mechanisms. MSU will work in collaboration with MN to define genetic mechanisms responsible for meat quality characteristics in inbred research lines and commercial turkeys subjected to thermal challenge. TN will use a combination of RNAseq and lipidomics/metabolomics to understand how diet regulates body composition, growth and adipose tissue development in young broiler chicks. TAMU will identify genomic regions associated with traits important for performance, health and welfare in poultry including host-genotype influence on microbiota profile and its relevance for innate immune stimulation. UD will use a variety of complementary approaches such as genome-wide association analysis, metabolomics and RNA-sequencing to gain a comprehensive understanding of the genetic basis of Wooden Breast Disease (WBD). Findings are expected to identify major genes associated with WBD and provide mechanistic insights underlying etiology of WBD. Results from metabolite profiling of plasma samples from WBD-affected and unaffected birds are expected to identify biomarkers of WBD. UD will investigate biological mechanisms leading to high or low feed efficiency phenotype in broiler chickens. UW will investigate the genetics underlying feed efficiency as well as the feeding. VaT will use genome-wide analyses to identify genes associated with economically important traits in poultry and model avian species to establish phenotype:genotype relationships of external factors such as incubation temperature, delayed access to feed and probiotics in the yolk sac and small intestine.
Disease susceptibility/resistance: ADOL will identify genes, alleles and pathways that affect genetic resistance to Marek’s disease (MD) or MD vaccinal efficiency. UCD will identify differentially expressed genes associated with Campylobacter, Salmonella, avian influenza virus infection in immune-related tissues. IA will examine response to Newcastle Disease virus in chickens, differences in resistance to highly pathogenic Avian Influenza virus infection. MS will support infectious disease research by providing host-pathogen interactions (HPI) for agricultural host-pathogen systems
Bioinformatics/metagenomics: PU will use methods to establish genotype to phenotype associations and suggest candidate genes for traits of economic importance, 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. MN will use co-expression analyses to further refine gene networks. NCSU will generate DNA methylation data at the whole-genome level for correlation with other molecular data including gene expression data from microarrays and RNAseq. UAZ will provide functional annotations, bioinformatics tools and support for functional genomics modeling. MS will work with FL to contribute to annotation of active kinases and deubiquitinases in chicken using quantitative proteomics.
Measurement of Progress and Results
- 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
- Establishment and maintenance of genetically and phenotypically characterized poultry populations, cell lines, and associated resources that support genetic and functional analysis of production traits in poultry.
- Identification of genes, pathways and mechanisms involved in generating phenotypes such as tolerance of thermal challenge (heat or cold stress) and disease resistance
- Identification of sequence variations involved in generating phenotype
Outcomes or Projected Impacts
- Identification of key production traits for poultry
- Increased access and opportunity for stakeholders to genetic and functional information that will inform breeding programs aimed at improvements in poultry production
- Development of new tools for bioinformatics and genome analysis for research community use made available in easily accessible databases
- Improved understanding of genotype x environment interactions creating phenotype
- Advancement of understanding of gene interactions in pathways
- Education of students and postdoctoral scholars and collaboration with many visiting international scholars drawing their attention to our project goals
Projected ParticipationView Appendix E: Participation
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 (e.g., Poultry Science annual meeting, National Breeders Roundtable, 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 welcome stakeholder participation and interaction. In addition, committee members frequently provide seminars on their work at other academic institutions and companies. Those conducting genome sequencing and analysis 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.
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 USDA-NIFA 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 a 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.
Please see the attachments section for a file which includes 300 peer-reviewed citations as well as over 50 book chapters, conferenc proceedings, theses and dissertations resulting from the collaborative research of this project from 2013 through the summer of 2017.