NC_old1170: Advanced Technologies for the Genetic Improvement of Poultry

(Multistate Research Project)

Status: Inactive/Terminating

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Annual/Termination Reports:

[03/12/2019] [03/08/2020] [04/23/2021] [02/16/2022] [03/20/2023]

Date of Annual Report: 03/12/2019

Report Information

Annual Meeting Dates: 01/12/2019 - 01/13/2019
Period the Report Covers: 01/01/2018 - 12/31/2018

Participants

Participants Attachment:

1_CoverPage.pdf

Brief Summary of Minutes

NC1170 Business Meeting

January 13, 2019 San Diego NC1170/NRSP8

Attendees: (AR) Douglas Rhoads, (DE) Carl Schmidt, (AR) Adnan Alrubay, (IA) Susan J. Lamont, (WUHS) Yvonne Drechler, (MS) Bindu Nanduri, (AZ) Fiona McCarthy, (DE) Behnam Abasht, (AR) Wayne Kuenzel, (CA) Huaijun Zhou, (VA) Eric Wong, (CSU Fresno) Katy Tarrant, (WI) Roger Sunde, (MN) Kent Reed, (MI) Gale Strasburg.

Agenda approved unanimously

Sue Lamont USDA Administrative Advisor

New project was approved and implemented for a five-year term Oct 1, 2018 – Sept 30, 2023

Gale was thanked for his leadership on assembling and submitting the proposal.

Publication lists annual reports need to report categories, but only report for this project, what is relevant to the project.

All publications should acknowledge USDA support from the Hatch program; wording: USDA/NIFA funding through Hatch program (program #)

Comments from NIFA Representative: Dr. Matukumalli was not present due to government shut-down

Comments from NRSP8 Representative Huaijun Zhou

NRSP8 renewed but significant reduction in NRSP8 because has been successful. Need to think about new directions. NRSP8 being sunset?

Coordination funding -faculty need to be a member of NRSP8 to get travel support etc. for students.

Support essential small projects especially junior faculty

New farm bill support $40,000,000 for high throughput phenotyping and genotyping of plants and animals. Still need to get appropriation (money) to actually fund this effort. Need to think about priorities of species that might be funded for this effort.

ADOL genetic lines: USDA indicates ADOL will move to Georgia by 2022 but facility not sufficient until 2022, but facility not sufficient to all of the lines. Michigan ADOL location will be shut down.Are there any stations that could adopt one or more lines as there may not be spaces?

Discussion of workshop external speakers

Purpose of Poultry workshop

Lamont: multistate meeting at PAG. Need to do annual written reporting,

No requirement for every member to speak every year.

Invite relevant outside scientists Wes Warren, Wageningen(?).

Who do we invite to speak?

Comments fromcommunity:
- Wayne Kuenzel - liked concept of minisymposium 2,3 speakers per year in such a relevant target (microbiome, gene knockout).
- Support for Wayne’s idea: Eric Wong
- Susan Lamont: value of meeting is learning about people outside of group and what they are doing. Should we shorten technical talks?
- Vet school poultry also supports idea of bringing in outside talks. There was a general consensus of support for bringing outside speakers.
- Perhaps 15 minutes as a limit for the members?

Suggestion shorten the members talk to 15 minutes.

Doug Rhoads: travel funds for outside speakers an issue

What is the next minisymposium?

Chicken genome improvement: Fiona -suggestions for improving chicken genome

Wes-sequencing the Trio of parents/offspring

May help with MHC, minichromosome.

There was support for the idea.

Red Jungle fowl x parent with very small variability

Perhaps back to UCD red jungle fowl line x something as different as possible.

Fiona will contact Wes with discussion of email

Doug Rhoads moved have meeting on weekend prior to 2020 PAG meted

Unanimously supported.

Meeting adjourned at 11:55 January 13, 2019

Accomplishments

Accomplishments by Objective This document is a compilation of annual reports submitted by members of NC1170. Below is a summary of achievements, by objective, reported by the NC1170 membership for the calendar year 2018. One hundred and seven peer reviewed publications were reported for this period. This number improved over the previous report year; furthermore the number of the publications with collaborations among NC1170 member institutions is notable. Over $25 Million in funding was reported; this funding is enabling ever greater advances into avian genomics – from investigations of fundamental biology to development of novel tools and databases. The funding also supported training of the next generation of talent – eight Ph.D dissertations or M.S. theses were completed in this project year. The current membership of NC1170 stands at 34 members spread across 23 institutions. Objective 1: Create and share data and technology to enhance the development and application of genomics and systems biology in poultry. AZ AgBase: supporting functional modeling in agricultural organisms. AgBase (<a href="http://www.agbase.msstate.edu/">http://www.agbase.msstate.edu</a>) provides resources to facilitate modeling of functional genomics data and structural and functional annotation of agriculturally important animal, plant, microbe and parasite genomes. We provide 2,069,320 Gene Ontology (GO) annotations for 394,599           gene products from more than 50 agriculturally important species and their pathogens. During 2018 AgBase was moved to a new server system which will provide enhanced performance. We also partnered with Phoenix Bioinformatics, a non-profit organization, to implement a subscription-based funding model for AgBase tools and large-scale analyses, and this will go into effect during 2019.  Standardized Gene Nomenclature. The Chicken Gene Nomenclature Consortium (CGNC; <a href="http://birdgenenames.org/cgnc/">http://birdgenenames.org/cgnc</a>) is the sole source globally for chicken gene nomenclature. During 2018 we mapped and updated gene names for the new Galgal5 release. This resulted in 817 obsolete records, 2,693 records with duplicated gene symbols and 1,111 records for manual review. Chickspress – developing a tissue specific compendium of gene expression for chicken gene products. The Chickspress resource (<a href="http://geneatlas.arl.arizona.edu/">http://geneatlas.arl.arizona.edu</a>) provides a detailed “atlas” of chicken gene expression, collating experimental information from Red Jungle Fowl and chicken gene expression studies. During 2018 we have completed remapping all expression data to the new Galgal5 annotations and the publication describing this resources have recently been provisionally accepted (pending minor revisions).  CA Identification of Regulatory Elements in Livestock Species The recent international FAANG (Functional Annotation of ANimal Genomes) initiative has stimulated efforts to functionally annotated important livestock species, which will ultimately be leveraged to improve production efficiency, animal welfare, and food safety. As one of the FAANG pilot projects coordinated by UC Davis, we present the current progress in generating and analyzing data from chicken, cattle, and pig. Samples were collected from adipose, cerebellum, cortex, hypothalamus, liver, lung, muscle, and spleen in two male biological replicates from each species, allowing the identification of both universal and tissue-specific functional elements. We aims to perform RNA-seq, DNase-seq/ATAC-seq, and five ChIP-seq assays on eight tissues in three farm animal species. Currently, we have completed data collection for eight tissues in cattle, three tissues in pig, and five tissues in chicken. Three ChromHMM models, one for each species, were trained to predict genome-wide chromatin states specific to each tissue. To facilitate comparison of models between species, all models were created with 14 states. Among the three species, 11 states were consistent between models while the remaining three states in each model were either unique to that species or appear in the model for only two species. The states common to all species were four promoter-associated states, four enhancer states, a repressive state, an insulator state, and a low-signal state with no strong prevalence of any ChIP-seq marks. The predicted states of all transcription start sites (TSS) were compared in liver, lung, and spleen across three species. The number of TSS with active promoter states was generally higher in pig with 49%, 45%, and 46% in liver, lung and spleen, respectively, compared to 38%, 34%, and 37% in chicken and 39%, 40%, and 38% in cattle. Less than 10% of TSS were predicted in repressed and bivalent states in all species, with the majority of the remaining TSS having a low-signal state. A small portion of TSS were predicted in an enhancer state, which is likely due to missing H3K4me3 signal. A more robust comparison between tissues and species could provide novel insights of evolutionary divergence of regulatory elements when all data collection is completed.  COH MHC-Y region genomic sequence data and annotation (Goto, Warden, Zhang, Wu, Kang, McPherson, Delany, Stadtmueller, Bjorkman, Shiina, Hosomichi, Inoko, & Miller). MHC-Y does not fit readily within expectations for a region containing MHC genes. It is a second region of polymorphic MHC genes in chickens. The genes have distinctive features. No homologous region has been identified in mammals. MHC-Y is located on the same chromosome as the classical chicken MHC (MHC-B) and CD1 region, but haplotypes at MHC-Y are inherited independently as if located on another chromosome. MHC-Y is enigmatic with its function remaining mostly undefined. In the past year we completed annotation of the total MHC-Y region sequence determined (830,931 bp). As we reported last year, 649 kbp is the MHC-Y region and contains 115 genes. The remaining 137 kbp contains four rRNA genes and intervening sequences representing the closely adjacent NOR. Soon we will be submitting a manuscript describing MHC-Y in the Red Jungle Fowl (RJF) reference genome. All that remains is completion of an analysis of the distribution of polymorphic residues in the MHC-Y encoded MHC class I-like molecules. Data on MHC-Y class I polymorphism (Miller, Goto, Zhang, Stadtmueller, Bjorkman). Genetic/structural polymorphism is a major feature distinguishing MHC class I molecules. The binding and presentation of peptide antigens by classical MHC class I molecules is well understood. Polymorphic residues in and around the antigen binding groove, also called “the MHC-fold”, of the highly polymorphic classical MHC class I molecules determine which peptides are held within the groove of different isoforms. Other molecules also bear the MHC-fold but nearly all of these are monomorphic and are described as non-classical. The highly polymorphic class I-like molecules encoded by MHC-Y have properties that are both classical and non-classical, making them unusual and difficult to classify. They display considerable polymorphism (Afanassieff et al., 2001; Hunt et al., 2006; Thoraval et al., 2003), but do not bind peptides (Hee et al., 2010). We are now completing structural analyses defining position and side-chain orientation of the polymorphic residues found in the MHC-Y class I molecules encoded in the RJF haplotype. The distribution of the polymorphic residues shows interesting patterns. IA Transcriptome data made public. Several data sets from RNAseq experiments on chickens were deposited in public databases upon submission for journal publication of the manuscripts describing those studies. Copy number variations. Copy number variations (CNV) are an important source of genetic variation that has gained increasing attention over the last couple of years. In this study, we performed CNV detection and functional analysis for 18,719 individuals from four pure lines and one commercial cross of layer chickens. Samples were genotyped on four single nucleotide polymorphism (SNP) genotyping platforms, i.e. the Illumina 42K, Affymetrix 600K, and two different customized Affymetrix 50K chips. CNV recovered from the Affymetrix chips were identified by using the Axiom® CNV Summary Tools and PennCNV software and those from the Illumina chip were identified by using the cnvPartition in the Genome Studio software. The mean number of CNV per individual varied from 0.50 to 4.87 according to line or cross and size of the SNP genotyping set. The length of the detected CNV across all datasets ranged from 1.2 kb to 3.2 Mb. The number of duplications exceeded the number of deletions for most lines. Between the lines, there were considerable differences in the number of detected CNV and their distribution. Most of the detected CNV had a low frequency, but 19 CNV were identified with a frequency higher than 5% in birds that were genotyped on the 600K panel, with the most common CNV being detected in 734 birds from three lines. Commonly used SNP genotyping platforms can be used to detect segregating CNV in chicken layer lines. The sample sizes for this study enabled a detailed characterization of the CNV landscape within commercially relevant lines. The size of the SNP panel used affected detection efficiency, with more CNV detected per individual on the higher density 600K panel. In spite of the high level of inter-individual diversity and a large number of CNV observed within individuals, we were able to detect 19 frequent CNV, of which, 57.9% overlapped with annotated genes and 89% overlapped with known quantitative trait loci. MN Additional RNAseq datasets compiled for the turkey have been accessioned in NCBI’s Gene Expression Omnibus (GEO) repository. MS MSU continues to generate data and develop bioinformatics resources to facilitate the analysis of functional genomics data in agricultural species. MSU contributed to Host-Pathogen Interaction Database (HPIDB): HPIDB provides predicted and curated host-pathogen protein-protein interaction data to support animal health/disease studies. During 2018 HPIDB provides predicted and curated host-pathogen protein-protein interaction data to support animal health/disease studies. During 2018 HPIDB continued to develop a set of predicted interologs (a total of 130,966 http://hpidb.igbb.msstate.edu/hpi30_interologs.html). Working with University of Florida, We identified active kinases in spleen and liver tissues in chicken based on their reactivity with the ATP and ADP desthiobiotin acyl phosphate probes combined with mass spectrometry. We identified 188 chicken kinases and their ATP-binding regions to create a tissue-specific atlas of active kinase expression in chicken. We also determined the possible functions of these kinases by utilizing bioinformatics approach by comparing functional pathways and disease involvement of human, murine and rat orthologs of these kinases. We also performed chemical proteomic profiling of active deubiquitinases (DUBs) by utilizing active-site directed ubiquitin (Ub)-vinyl sulfone (VS)-HA probe labeling combined with western blot-based or mass spectrometric identification of DUBs. By using these techniques we identified 29 DUBs in cecum, liver and spleen tissues. We have identified that DUBs such as USP5, USP4, UCH-6, UNP, USP7, UCH- L5, USP9x, USP10, USP19, USP47, OTUD6B, and USP8 are the top 12 DUBs identified in liver while in spleen there were USP5, USP4, UCH-6, UNP, UCH-L5, UCH-L1, USP9x, USP10, USP19, USP16, OTUD6B, and USP8. Cecum overall contained less active DUBs, and the most active DUBs were USP5, USP4, UCH-6, UNP, USP7, UCH-L5, UCH-L1, USP47, USP8, USP10, USP19 and USP9x. TX Structural variants are an important source of phenotypic variation in domestic species, including chicken, standardized databases for comparison across phenotypes are not available at present. Athrey Lab at continued development and testing of new structural variant cataloging tools, Girar.  After initial development in 2017, additional datasets downloaded from NCBI were tested using the approach. Over 400 unique structural variant variants have been identified and are being verified by standard laboratory techniques. One main approach to validate structural variants has been a partnership with Bionano, who are currently running samples on the Saphyr platform to validate structural variants identified and validated using the in-silico methods. Also towards Objective 1, Athrey lab graduate student Travis Williams continued ongoing work on in-silico approach to validate currently predicted miRNAs in the chicken genome. The results of this work were presented at the Poultry Science Association annual meeting. This work also produced a short pipeline tool that will deposited on Github. WI R Sunde RNAseq datasets for the turkey transcriptome have been collected in collaboration with K. Reed (U Minnesota); better annotation of selenoprotein transcripts for NCBI is being developed. <ol> <li> Rosa</li> </ol> Quantitative Genetic Analysis of Reproductive Traits on a Male Line of Turkey Turkey body weight and growth rate have been successfully improved with selective breeding. However, reproductive traits such as egg production, fertility, hatchability and egg weight have not been actively selected for, especially on male lines. Improving performance for these traits is crucial to further increase production and decreasing cost. In this context, understanding the genetic background of reproductive traits is important so that efficient breeding programs can be developed to meet the demand for turkey meat. The aim of this study was to estimate genetic parameters and perform a genome-wide association analyses for reproductive traits in a male turkey line. Data on 11,467 females was available for total egg production (TEP), fertility (FERT), hatch of fertile eggs (HOF), egg weight (EW), and body weight at 18 weeks of age (BW18). A total of 1,911 hens were genotyped using a 65K single nucleotide polymorphism (SNP) array, from which 49,289 SNP were available after quality control. Variance components, heritabilities, and genetic correlations were estimated using a multi-trait single- step genomic BLUP model (ssGBLUP) via Restricted Maximum Likelihood (REML). In addition, a genome- wide association study (GWAS) was implemented for TEP. A relatively high heritability was obtained for EW (h2 = 0.52), a moderate value for TEP (h2 = 0.35), and much lower values for FERT and HOF (h2 = 0.10 and h2 = 0.13, respectively). EW was positively correlated with BW18 (0.20 ± 0.03), whereas it was negatively correlated with FERT (-0.04 ± 0.05), HOF (-0.51 ± 0.04) and TEP (-0.12 ± 0.04). FERT was found positively correlated with HOF (0.40 ± 0.08) and TEP (0.28 ± 0.06). HOF was negatively correlated with BW18 (-0.20 ± 0.05) and positively correlated with TEP (0.23 ± 0.06). TEP was negatively correlated with BW18 (-0.17 ± 0.05). Such high negative (and genetically unfavorable) correlation between EW and HOF, as well as the negative correlation between TEP and BW18 should be carefully considered or the development of a multiple trait breeding strategy. The GWAS detected some interesting genomic regions, including an SNP located in GREB1L on chromosome 3 (q-value < 0.05, p-value = 1.10543e-06), which might play a role on TEP. The region located 0.5 Mb upstream and 0.5 Mb downstream of that SNP (1 Mb window including significant SNP) harbors candidate genes related to egg formation (i.e. maturation of follicle/oocyte, ovulation or reproductive track development). The current study aimed to shed some light on the genetic background of reproductive traits in Turkeys. It combined pedigree and genomic information for the estimation of genetic parameters, and presented the first GWAS analysis for total egg production on a male turkey line. Including Phenotypic Causal Networks in Genome-Wide Association Studies Using Mixed Effects Structural Equation Models Network based statistical models accounting for putative causal relationships among multiple phenotypes can be used to infer single-nucleotide polymorphism (SNP) effect which transmitting through a given causal path in genome-wide association studies (GWAS). In GWAS with multiple phenotypes, reconstructing underlying causal structures among traits and SNPs using a single statistical framework is essential for understanding the entirety of genotype-phenotype maps. A structural equation model (SEM) can be used for such purposes. We applied SEM to GWAS (SEM-GWAS) in chickens, taking into account putative causal relationships among breast meat (BM), body weight (BW), hen-house production (HHP), and SNPs. We assessed the performance of SEM-GWAS by comparing the model results with those obtained from traditional multi-trait association analyses (MTM-GWAS). Three different putative causal path diagrams were inferred from highest posterior density (HPD) intervals of 0.75, 0.85, and 0.95 using the inductive causation algorithm. A positive path coefficient was estimated for BM→ BW, and negative values were obtained for BM→ HHP and BW→ HHP in all implemented scenarios. Further, the application of SEM-GWAS enabled the decomposition of SNP effects into direct, indirect, and total effects, identifying whether a SNP effect is acting directly or indirectly on a given trait. In contrast, MTM-GWAS only captured overall genetic effects on traits, which is equivalent to combining the direct and indirect SNP effects from SEM-GWAS. Although MTM-GWAS and SEM-GWAS use the similar probabilistic models, we provide evidence that SEM-GWAS captures complex relationships in terms of causal meaning and mediation and delivers a more comprehensive understanding of SNP effects compared to MTM-GWAS. Our results showed that SEM-GWAS provides important insight regarding the mechanism by which identified SNPs control traits by partitioning them into direct, indirect, and total SNP effects. Predictive ability of genome-assisted statistical models under various forms of gene action Recent work has suggested that the performance of prediction models for complex traits may depend on the architecture of the target traits. Here we compared several prediction models with respect to their ability of predicting phenotypes under various statistical architectures of gene action: (1) purely additive, (2) additive and dominance, (3) additive, dominance, and two-locus epistasis, and (4) purely epistatic settings. Simulation and a real chicken dataset were used. Fourteen prediction models were compared: BayesA, BayesB, BayesC, Bayesian LASSO, Bayesian ridge regression, elastic net, genomic best linear unbiased prediction, a Gaussian process, LASSO, random forests, reproducing kernel Hilbert spaces regression, ridge regression (best linear unbiased prediction), relevance vector machines, and support vector machines. When the trait was under additive gene action, the parametric prediction models outperformed non-parametric ones. Conversely, when the trait was under epistatic gene action, the non-parametric prediction models provided more accurate predictions. Thus, prediction models must be selected according to the most probably underlying architecture of traits. In the chicken dataset examined, most models had similar prediction performance. Our results corroborate the view that there is no universally best prediction models, and that the development of robust prediction models is an important research objective.   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 ADOL ADOL chicken populations: A major strength of ADOL is the large number of chicken lines that are characterized for a number of traits, especially those associated with viral diseases, and maintained under specific pathogen free (SPF) conditions. Besides providing unique genetic resources to ADOL, ~1,500 embryos or chicks are supplied yearly to academic institutions or companies in the United States. The lines and maintenance are briefly summarized below. ADOL maintains 35 chicken lines with special genetic characteristics for tumor or viral susceptibility that also differ remarkably for immunological and physiological traits. All but 3 (C, N and P) were developed at the ADOL over the last 67 years. These include 4 of the world’s most highly inbred lines (63, 71, 72,and 15I5,), all of which are well defined for avian leukosis virus (ALV) receptor genes, endogenous virus loci (EV), and resistance to MD. Two of the lines are outbred, 2 of which are highly utilized worldwide for ALV analyses (0 and 15B1). Four congenic lines exist for analysis of EV genes; 3 (0.44-TVBS1- EV21, 0.44-TVBS3-EV21, and RFS) were developed from line 0 and 1 (100B) from line 72. Eight congenic lines exist for analysis of the influence of the MHC (B haplotype) on resistance to tumor diseases, immune responses or vaccinal immunity; 7 (15.6-2, 15.7-2, 15.15I-5, 15.C-12, 15.P-13, 15.P-19, and 15.N-21) were developed from line 15I5, and 1 (15.N-21) from line 0. Lines 63 and 72 differ markedly for MD resistance and immune function traits, as well as ALV and EV genes, but have the same B haplotype. Nineteen recombinant congenic strains (RCS) are under development to identify non-MHC genes that influence traits differing between lines 63 and 72. ADOL also developed one transgenic chicken line (0.ALV6) that is very beneficial for analysis of ALV. ADOL lines are routinely tested by blood-typing using 40 antisera either to ensure purity or to maintain heterozygosity (EV21, 100B, and O.P-13) during annual line reproduction. The breeders are unique in that they are maintained in a quarantined state and, on the basis of frequent serologic tests for 11 pathogens, are considered free of infection from common poultry pathogens. Estimates for the completion of the Athens, GA facilities is fall 2022. Unfortunately, despite promises that ADOL and all of its resources would remain intact, it is apparent that the buildings to house the ADOL lines are not sufficient. Thus, the number of lines kept will have to be reduced. Still worrisome is that no plans have been established to move the lines, which will require at least 2 years of having lines at both ADOL and Athens. COH Evaluating MHC-Y haplotypes segregating in experimental inbred and highly-selected lines (Zhang, Goto, Lamont, Siegel, Honaker, and Miller). PCR-based genotyping (see below) has allowed us to readily define MHC-Y haplotypes in different populations. We are investigating the relationship between MHC-Y haplotype and immune responses in a variety of experimental populations. In the Fayoumi and white leghorn lines maintained at Iowa State University, we found only one MHC-Y haplotype present in each line, consistent with the highly inbred nature of these two lines. In a retrospective analysis of RNASeq data from an earlier challenge trial of these lines with Newcastle disease virus (NDV), we found major differences of MHC-Y class I gene expression in spleen with greater expression in the Fayoumi line. Another recently reported study shows enhanced expression of MHC-Y after NDV challenge in the respiratory tract of the Fayoumi birds (Deist et al., 2018). In the closed Virginia Tech HAS and LAS lines, we have defined five haplotypes. The HAS and LAS lines, maintained by matings among individuals at each generation, exhibiting high antibody responses in HAS and low antibody responses in LAS (mate-selection is restricted by no sib matings and no sire or dam families over represented). Selection is still ongoing (Lillie et al., 2017). We have MHC-Y genotyped the 44th and 45th generations (nearly 200 birds in each generation). We first defined patterns and then used these guided by pedigree information for the two generations to define haplotypes. Among the five haplotypes, one is found exclusively within the HAS line. Three others are very common in the LAS line. MHC- Y could be guiding immune responses such that MHC-Y haplotypes have become asymmetrically distributed during the multiple rounds of selection for antibody response. From our experience with typing, we have learned that multiple MHC-Y haplotypes are typically segregating in non-selected lines. We are working on ways to assign MHC-Y haplotypes in these lines in the absence of pedigree data. Typing is ongoing of birds in Campylobacter colonization trials (Roslin and Alberta) and a study of infection-related lameness (Arkansas). IA Iowa State University chicken resource populations maintained, but reduced in number. For most of the year, Iowa State University maintained 13 unique chicken research lines [including highly inbred, MHC-congenic, closed populations; and advanced intercross lines (AIL)] to serve as resources for identifying genes, genetic elements and genomic regions of economic importance; as well as defining unique aspects of chicken genomic architecture. All adult breeders were housed in individual cages and matings done by artificial insemination to ensure pedigree accuracy. All MHC-defined lines were blood-typed to verify MHC serologic haplotype. Two AILs (now at generation F28) were maintained to facilitate fine-mapping of QTL with the goal of identifying genomic regions and candidate genes controlling important phenotypes. Because of reduction in space allotted to the genetics program in the new poultry farm (construction to begin in 2019), the number of genetic lines was reduced from 13 to eight lines. The eight remaining lines are: one AIL (broiler X Fayoumi), two inbred Fayoumi lines, two inbred Leghorn lines, one inbred Spanish line, one antique inbred line (inbreeding started in 1925) and one closed population of broilers from 1980s industry genetics. Utilization and sharing of research populations. The ISU genetic lines formed a discovery platform for research on the genomics of heat resistance in a USDA-AFRI-NIFA project (PD: C Schmidt, U Del) and a USAID project on genomics of resistance to Newcastle disease virus and heat (PD: H Zhou, UC-Davis) because of defined, distinct responses among lines. Genetic material (chicks, fertile eggs, blood, tissues, DNA or RNA) was shared with many cooperating investigators to expand studies on the chicken genome. Active collaborations utilizing ISU chicken genetic lines or biological materials include H Zhou, UC-Davis (NDV and heat-stress response); C. Schmidt, U Delaware (heat stress); R Coulombe, Utah State (aflatoxin sensitivity); B Abasht, U Delaware (allele-specific expression); E Wong, Virginia Tech (Eimeria response) and V Kapur, Penn State (NDV-embryo assays). TX Athrey lab is maintaining a Red Junglefowl population of the Richardson strain. The current population size stands at 42, of which 14 are males. We completed an undergraduate project that used microsatellite markers to assess population genetic status, and pedigree information in this captive population, which resulted in a publication. The well documented research population has become key to a new project we started with Prof. Leif Andersson, to look at the developmental biology of morphological traits in chicken. We will be generating F2 crosses between RJF and heritage breeds.  Also, as the captive RJF are generating more eggs than we can hatch out and place in the colony (due to space limitations), we are able to collect and ship fertile RJF eggs.   Objective 3. Elucidate genetic mechanisms that underlie economic traits and develop new methods to apply that knowledge to poultry breeding practices. ADOL Characterizing the genomic landscape of Marek’s disease virus-induced lymphomas Meq, a bZIP transcription factor and the viral oncogene for pathogenic strains of Marek’s disease virus (MDV), is required to induce CD4 T cell lymphomas that characterize Marek’s disease (MD) in chickens. However, Meq is not sufficient for neoplastic transformation as not all birds infected with pathogenic strains of MDV developed MD. We hypothesize that additional drivers or somatic mutations in the chicken genome are required for MDV-induced transformation. Using and integrating DNA and RNA genomic screens of MD tumors from genetically-defined experimental layers, our analyses reveal 0.3 somatic mutations per megabase consisting primarily of somatic single nucleotide variants (SNVs) and small insertions and deletions (Indels). Somatic deletions, insertions, and point mutations were enriched in IKZF1 (Ikaros), the first driver gene of MD lymphomas. Ikaros, a Zn-finger transcription factor and the master regulator of lymphocyte development, is a known tumor suppressor in human and murine acute leukemias and lymphomas. In our surveyed MD tumors, 41% of the samples had somatic mutations in key N-terminal Zn-finger binding domains, strongly suggesting perturbed Ikaros function in its ability to bind DNA and regulate transcription. Interesting, somatic mutations in IKZF1 were preferentially found in tumors of gonadal tissues as well as their metastatic clones. IKZF1 mutant MD tumors revealed gene expression profiles indicative of Ikaros perturbation. In addition to IKZF1, other putative somatic mutations reside in FLT3, SOX1, VWF, ROBO1, and ROBO2 and warrant future evaluation. Our results suggest MDV-induced tumors are driven by both Meq expression and Ikaros somatic mutations, which in combination lead to unregulated proliferation, increased cell adhesion, increased migration, and dedifferentiation Contribution of the T cell receptor (TCR) repertoire to Marek’s disease resistance Marek’s disease (MD) is a commonly diagnosed herpesviral-induced T cell lymphoproliferative disease of chickens, which has increased in virulence over time and prompted the search for continued improvements in control, both through improved vaccines and increased flock genetic resistance. Model pairs of genetically MD-resistant and susceptible chickens that are either B2 MHC-matched or B21 and B19 MHC-congenic has allowed the study of both non-MHC-linked and MHC-linked genetic resistance; here, we have applied these models to characterizing the T cell receptor (TCR) repertoires in MDV infection. Chickens resistant to MD showed higher usage of Vbeta-1 TCRs than susceptible chickens, in both the CD8 and CD4 subsets in the MHC-matched model, and in CD8 subset only in the MHC-congenic model; and Vbeta-1+ CD8 cells expanded during MDV infection. The TCR locus was found to be divergent between MD-resistant and susceptible chickens in the MHC-matched model, with MD-resistant chickens expressing a greater number of Vbeta-1 TCRs and an increased representation of Vbeta-1 CDR1 loops with an aromatic residue at position 45. TCR Vbeta-1 CDR1 usage in resistant x susceptible F1 birds indicated that the most commonly used CDR1 variant was present only in the susceptible line, suggesting that selection for resistance in the MHC-matched model has optimized the TCR repertoire away from dominant recognition of one of the B2 MHC molecules. Finally, TCR downregulation during MDV infection in the MHC-matched model was observed most strongly in the MD-susceptible line, and TCR downregulation due to viral reactivation in a tumor cell line could be demonstrated to be virus-specific and not due to apoptosis induction. Neurovirulence of a Meq-deleted Marek’s disease virus in early in ovo challenge Marek’s disease virus is an important pathogen of chickens which causes neuropathic as well as lymphoproliferative disease. Virulent MDV strains include an oncogene, Meq, while avirulent MDV and closely related viruses are nononcogenic and apparently non-neuropathic, thus allowing there use as vaccine strains. Here, we describe a fatal neuropathy of chicks induced by early in ovo injection (prior to 15 days of embryogenesis) of non-oncogenic Meq-deleted MDV, which induces severe bursa and thymic atrophy as well as mild lymphocytic peripheral nerve lesions. We suggest that the previously identified correlation between neuropathogenicity and a single MDV gene (pp14) presents a strong case for an immune-mediated component to MDV neuropathy, and that other vaccine strains may be capable of inducing neuropathic disease in immune- dysregulated birds. Identifying the molecular basis for MD vaccine synergy Marek’s disease (MD) vaccines utilize protective synergism, a phenomenon where the protective efficacy of two vaccines in combination is greater than either vaccine when administered alone. A key example is the bivalent MD vaccine of serotype 2 (SB-1) and serotype 3 HVT (FC-126). Despite this widespread usage, the biological mechanism of the synergistic effect has never been elucidated. We previously found that SB-1 replicated to the highest levels in spleen, bursa, and thymus, respectively, while HVT showed replication only in bursa at 1 dpi and could not be detected at any other time point or tissue type. Looking at later time points, we find that only SB-1 + HVT (bivalent MD vaccine) can control MDV replication compared to the other two MD vaccines administered alone. Currently, we are screening cytokines to determine if unique or enhanced expression of one or more might account for protective synergism. Hypothesizing that CD8 T cells may play a role in the protective ability of MD vaccines, we injected chicks with anti-CD8 antibody, which reduced CD8 T cell populations by 50-80%. Regardless of the MD vaccine administered, disease incidence was significantly higher in CD8-depleted birds, indicating that CD8 is responsible for at least part of the protection afforded by MD vaccines. The question is whether CD8 T cells are essential for anti-viral or anti-tumor response. Comparative transcriptome analysis of spontaneous avian leukosis virus (ALV)-like bursal lymphoma and normal bursal tissues to explore genomic basis associated with the tumor- incidence in chicken. Sporadic ALV-like bursal lymphoma, also known as spontaneous lymphoid leukosis (LL)-like tumors, were identified in primary breeders’ elite, grandparent and parent hens of a commercial broiler breeder in the absence of exogenous ALV infection and a few experimental lines of White Leghorn. The role of ALV-E field isolates in development of spontaneous LL-like tumors and the potential joint impact in conjunction of a Marek’s disease (MD) vaccine (SB-1) on enhancement of the spontaneous LL-like tumor formation were characterized in chickens of an experimental line of White Leghorn. The spontaneous LL-like tumor incidences of 8%, 17%, 14%, and 42% were observed in the negative control, SB-1 inoculated, ALV-E inoculated, and SB-1 plus ALV-E inoculated groups, respectively, under control conditions. The spontaneous tumor and normal bursa tissues were sampled for total RNAs followed by deep RNA sequencing. After bioinformatical analyses, a total of 923 genes was identified with significantly differentiated expression, which are reportedly involved in over 100 gene ontology terms and pathways, including KEGG pathways, NOD-like receptor signaling pathways, and Toll-like receptor signaling pathways. AR Identification of genes affecting ascites susceptibility AR used whole genome resequencing to identify 31 chromosomal regions as candidate QTLs for affecting ascites.  Three of these regions have been further validated, representing the first validated QTLs for ascites. We are now working on genotyping for all 31 regions in multiple lines including commercial products.  We are preparing breeders for marker assisted selection using two regions to assess utility for selection for resistance to ascites.  AR evaluated the developmental, gender and ontological aspects of mitochondrial biogenesis in broilers, primarily focused on skeletal muscle and the cardio-pulmonary system.  Significant differences were found for gender, tissue, age, and ascites susceptibility.  Muscle mitochondrial content was positively correlated with ascites phenotype.  This may be an easily evaluated trait for selection against ascites.  Bacterial chondronecrosis with osteomyelitis and lameness in broilers AR performed one trial for reducing bacterial chondronecrosis with osteomyelitis (BCO) lameness with a commercial organic supplement.  We determined the specific treatment significantly reduced BCO lameness.  The effects of the supplement on specific immune functions were determined.  AR has been evaluating bacterial isolates from BCO lesions to understand the virulence determinants specific to colonization and pathogenesis in broilers. Virulence has been assessed in direct challenge to live broilers as well as phagocytosis assays using chicken macrophage in culture.  Macrophage response may be a simple test to select for resistance to bacterial colonization. We have also been evaluating embryo lethality assays to assess pathogenicity.  We have completed two rounds of directed genome evolution to identify particular pathogenicity determinants in BCO isolates. Broiler breast muscle and myopathies  AR performed analyses for differential abundance of microRNA (miRNA) in bigger breast muscle of modern pedigree male broilers and unselected counterparts. A total of 994 miRNA were identified in breast muscle tissues from small RNA sequencing method. Eight higher abundant miRNAs (miR-2131-5p, miR-221-5p, miR-126-3p, miR-146b-5p, miR-10a-5p, let-7b, miR-125b-5p, and miR-146c-5p) and a lower abundant miRNA (miR-206) were identified in modern broiler breast muscles compared to unselected counterparts. Results were integrated with differentially expressed (DE) mRNA in the same tissues and 118 down-regulated mRNAs may be targeted by the up-regulated miRNAs, while 35 up-regulated mRNAs appear to be due to a down-regulated miRNA (i.e., miR-206). Functional network analyses of target genes of DE miRNAs showed their involvement in calcium signaling, axonal guidance signaling, and NRF2-mediated oxidative stress response pathways suggesting their involvement in breast muscle growth in chickens. Genes and proteins involved in the stress response of broilers AR- in collaboration with the University of Missouri has completed studies investigating the function of a newly discovered structure in the broiler brain that is involved in stress. The structure, the nucleus of the hippocampal commissure (NHpC), contains a significant population of corticotropin-releasing hormone (CRH) neurons. Using food deprivation (FD) as a gradual stressor that was imposed on male broiler chicks beginning at two weeks of age, chicks were sampled every hour for the first  4 h and at 8 h.  Brain, anterior pituitary gland (PIT) and a blood sample was taken for each time period. Within the brain two structures were dissected, the NHpC as well as the paraventricular nucleus (PVN), the latter a main hypothalamic nucleus involved in the stress response.  Gene expression for CRH neurohormone and its major receptors of CRH, CRHR1 and CRHr2, were determined for each sampling period and compared between the NHpC and PVN. Additionally, gene expression of corticotropes in the PIT that expressed the pre-prohormone proopiomelanocortin (POMC) was determined.  Plasma corticosterone (CORT) was determined by radioimmunoassay.  The NHpC showed the first peak of CRH mRNA within the first 2h of FD.  In contrast, CRH neurons in the PVN showed peak gene expression for CRH at 8h following FD.  CRHR1, the major receptor of CRH neurons within the NhpC showed a negative relationship with CRH mRNA.  In contrast, CRHR1, in the PVN, showed a positive relationship. In other words, as CRH mRNA increased, CRHR1 also increased in the PVN throughout the 8 h of sampling.  Both CRH neurons in the NHpC and PVN were responsible for the significant increase in pituitary POMC mRNA as well as the first detectable plasma increase in the stress hormone CORT.  To date, data support the hypothesis that the NHpC appears to be part of the neuroendocrine system working with the hypothalamic PVN that activates the anterior pituitary and ultimately the adrenal gland to produce the stress hormone CORT during the stress response.  CA Improving food security in Africa by enhancing resistance to Newcastle disease virus and heat stress in chickens Within a USAID funded Feed the Future Innovation Lab for Genomics to Improve Poultry project (H. Zhou, PI) through a partnership of the University of California at Davis (H. Zhou, D. Bunn, R. Gallardo, T. Kelly), Iowa State University (S.J. Lamont, J. Dekkers), Sokoine University of Agriculture (SUA) -Tanzania, the University of Ghana (UOG), and the University of Delaware (C. Schmidt). The five-year research program applied advanced genetics and genomics approaches to sustainably enhance innate resistance to Newcastle disease virus (NDV) and heat stress in chickens to improve poultry production in Africa. We are investigating two stressors (biotic: NDV and abiotic: heat stress). Birds of two genetically distinct and highly inbred lines (Fayoumi and Leghorn), and Hy-Line Brown were either exposed to NDV only (Iowa State) or NDV and heat stress (UCD). Measures of body temperature, blood gas parameters, NDV titers from tears, and antibody response in serum were taken on the live birds, and tissues were collected for transcriptome analysis. Three ecotypes each in Ghana and Tanzania were exposed to La sota NDV and natural exposure to velogenic NDV. DNA isolated from Hy-Line Brown and African ecotypes were genotyped using chicken 600K SNP for GW AS. At UCD, three manuscripts are prepared on transcriptome analysis in three tissues: Harderian gland, lung and trachea. Harderian gland transcriptome analysis revealed that a limited early response in both Fayoumi and Leghorn lines occurred at 2 dpi. Leghorns eventually had a substantially stronger response at 6 dpi, while Fayoumi had a robust response at the later stages of infection under heat stress. In addition, very few differentially expressed genes overlapped between the lines at each time point suggesting a distinct, line-specific host response to NDV. Especially, GP6 signaling pathway plays significant role in the disease resistance in chickens. Harderian gland manuscript is published. The other two manuscripts are expected to be submitted in the next few months. Completed genome-wide association analysis for challenge experiments on Hy-Line Brown: All three replicate trials with over 1,100 challenged birds were completed at UCD and ISU. NDV titers were measured in tears collected at 2 and 6 days post infection. In addition, NDV serum antibody response was measured at 10 days post-infection. The major findings were that the 600K SNP panel and GWAS identified few significant regions associated with the measured phenotypes and these were generally of low genetic effect, emphasizing the highly polygenic nature of the NDV response traits. Additional regions of marginal significance were supported by independent studies that demonstrated differential expression in resistant- susceptible line contrasts of genes that were in the QTL regions. One manuscript reporting these results has been submitted for journal review and another one (ISU) has been published. For African ecotype NDV challenges, replicate trials involving a total of 2,653 chicks (UOG) and 1789 chicks (SUA) were completed in the challenge facilities. For each replicate, blood was collected for DNA isolation. At four weeks of age, the chicks were challenged with NDV. Tear samples were collected at 2 and 6 dpi for NDV titers. Serum samples were also collected pre-challenge and at 10 dpi for NDV antibody titers. Body weight data was also collected at hatch, weekly until challenge, and at 6 and 10 dpi. To utilize replicates of previously challenged birds by La Sota NDV strain with available 600K SNP genotype data, the NDV resistance of indigenous African chickens in Tanzania and Ghana undergo further evaluation under field conditions. A RT-qPCR assay that specifically detects mesogenic and velogenic NDV strains was established and validated at SUA. The assay is being used to confirm natural exposure to velogenic NDV. Following natural NDV exposure, data on survival times, body weight, antibody response, and pathological lesion scores were collected. Data analyses are underway. Serum isolated from the blood samples of 800 birds (~800 birds) have been analyzed to determine antibody levels at pre-exposure and at two time points during the natural exposure trial. ND viral RNA isolation from tear samples and determination of virus titers by RT-qPCR is ongoing. We have currently completed 1436 birds at 2 days post-infection (dpi) and 1301 birds at 6 dpi at UoG and a total of 2,954 birds at SUA. Using SNP genotypes, birds from each ecotype were reassigned to the emergent population structure, which was then used for estimation of genetic parameters and GWAS. Estimates of heritabilities were moderate to high (0.20 to 0.55) for all traits measured, including pre- and post-infection growth rate, antibody response, and viral levels. Viral levels were, however, not yet available for all birds. Estimates of genetic correlations between antibody level and viral levels were moderately positive but with large standard errors because of the yet small data size for viral levels. Genome-wide Association Studies were completed on the available data using both single-SNP analyses and multi-SNP Bayesian variable selection analyses. Multiple genomic regions with suggestive significance on traits were identified but no QTL with major effects were identified. Overall, response to infection was found to be highly polygenic, with many QTL with small effects but that add up to a substantial genetic component that can be capitalized on using phenotypic or genomic selection with SNPs across the genome. These analyses will be finalized and the identified regions will be explored for underlying genes once the full data is available. COH PCR-based method for MHC-Y genotyping (Miller, Goto, Zhang, Fulton, Psifidi, Stevens). We are working to further improve MHC-Y typing methods so that they can be even more easily used in typing large populations of birds. Initially, MHC-Y haplotypes were defined through the patterns revealed in Southern hybridizations. Southern hybridizations are especially time-consuming and not well-suited for typing large numbers of birds. Currently, we are using a PCR-fragment typing method based on microsatellite sequences that are present upstream of the MHC-Y class I genes. These microsatellite sequences vary in repeat number between loci and among haplotypes. Fragments produced from PCR amplification across this microsatellite sequences make it possible to distinguish MHC-Y genotypes on the basis of patterns revealed in ethidium bromide-stained agarose gels. Over the past year, we have improved this typing method by adopting a hot-start polymerase suitable for GC-rich DNA, improving resolution through optimizing gel conditions, recording images with a higher-resolution camera, and routinely typing with two similar but not identical primer pairs. We are working on additional methods that may further reduce the challenge of MHC-Y typing in large populations of chickens. CU The liver is particularly important in birds because yolk is produced in the liver. Our preliminary data indicate that full fed broiler breeders develop a fatty liver with an associated decreased egg production. Generally, when an animal is in good health, the somatotropic axis is coupled and there is an abundant population of growth hormone receptor (GHR) in the liver as well as high circulating growth hormone (GH) and insulin-like growth factor (IGF1). We hypothesize that excessive feed intake uncouples the GH/IGF1 axis and disrupts the usual relation between feed intake and ovarian function. Follicle development in the laying hen is a highly efficient and regulated process. Maintenance of a well ordered follicular hierarchy is essential for optimum follicle selection and subsequent egg production in hens. We have previously found that IGF1 is significantly elevated in full fed (FF) broiler breeder hens as compared to restricted fed (RF) broiler breeder hens. Associated with this is excessive and disorganized development of follicles. The relationship between the metabolic and reproductive axis in broiler breeder hens was examined by investigating the in vitro response of granulosa cells to treatment with IGF1. IGF1 does not affect granulosa mRNA expression of FSHR and AMH, factors associated with follicle selection. FSH decreases AMH mRNA expression in cultured granulosa cells from 3-5 mm follicles. IGF1R mRNA is present in the oocyte, a proposed site of IGF1 action. IGF1R mRNA is higher in ooplasm of 3 mm follicles as compared to granulosa cells of 3 mm follicles and to ooplasm and granulosa cells of 5 mm follicles. IGF1 treatment of whole follicles does not alter E2 secretion, although these follicles maintain responsiveness to LH. Preliminary results show no significant effect of IGF1 on mRNA expression of BMP15 or FSHR. In conclusion, localization of IGF1R in the oocyte suggests that IGF1 may have important effects in the oocyte to promote follicle development. One Ph.D student has initiated her program while working on this project. An undergraduate student was supported by this project during the academic year. DE Transcriptomic Analysis of Pectoral Muscles in Broiler Chickens during the Early Phase of Wooden Breast Disorder. Wooden Breast (WB) is a muscle/meat quality disorder in modern broiler chickens that is clinically distinguished by abnormally firm consistency of the pectoral muscles. To characterize the transcriptome associated with the early pathogenesis of WB in commercial broiler chickens, a time-series study was conducted on the Pectoralis (P.) major muscles between affected and unaffected chickens from a purebred broiler line. To accomplish this objective, chickens were raised for up to 7 weeks of age with muscle biopsy samples from the cranial or the caudal aspect of the P. major muscle belly harvested at week 2, 3 and 4 time points. Cranial P. major muscle specimens from the 3rd week of age are the focus of the present study as this time point precedes the onset of clinically detectable gross lesions at approximately 4 weeks of age. Biopsy samples, including 4 unaffected (U) and 11 affected (A) samples, were processed for RNA-sequencing producing 618 differentially expressed (DE) genes at fold-change (A/U or U/A) >1.3 and False Discovery Ratio (FDR) Blood Analysis and Proportional Muscle and Organ Weights in Broilers with Wooden Breast. Wooden Breast is a myopathy of fast growing, commercial broilers causing myofiber necrosis, vasculitis (phlebitis), myoregeneration, and fibrosis with extensive fibrillar collagen deposition in the superficial part of the pectoralis major, presenting clinically as palpably firm breast muscle. Rapid growth, high feed efficiency, and large breast muscle yield are predisposing factors, although the etiology of the disease is still poorly understood. A group of 103 7-week-old Cobb 500 broilers were used to determine the effect of Wooden Breast on 13 blood parameters and the relative weights of the pectoralis major muscle, pectoralis minor muscle, external oblique muscle, wing, heart, lungs, liver, and spleen. Blood analysis performed with the i- STAT handheld blood analyzer on samples taken from the brachial vein of live birds revealed significant differences in blood gases between affected and unaffected chickens, with affected chickens exhibiting higher partial pressure of CO2, total CO2, bicarbonate, and base excess, and lower partial pressure of O2, oxygen saturation, and pH. Affected chickens also possessed a significantly larger pectoralis major muscle and wing relative to body weight. Hypercapnia and hypoxemia in affected chickens suggest greater metabolic demand and insufficient gas exchange, potentially caused by disturbances in circulation, cardiac output, or respiration. Disproportionately slower growth in the external oblique muscle may indicate inadequate development of respiratory muscles relative to the larger breast muscle size. These results support tissue hypoxia and the buildup of metabolic wastes as major contributors to Wooden Breast development and give a more systemic view of Wooden Breast pathology. The metabolic basis of susceptibility to Wooden Breast Disease in chickens with high feed efficiency. This study was conducted to characterize metabolic differences between high feed efficiency (HFE) and low feed efficiency (LFE) chickens to investigate why feed efficient chickens are more susceptible to muscle abnormalities such as Wooden Breast Disease. Gene expression profiles were generated by RNA-sequencing of pectoralis major muscle samples from 10 HFE and 13 LFE broiler chickens selected from a modern broiler population. Metabolism-associated differentially expressed genes were identified and interpreted by Ingenuity Pathway Analysis and literature mining. Our RNA-seq data indicates decreased glycolytic capacity, increased fatty acid uptake, mitochondrial oxidation of fatty acids and several other metabolic alterations in the pectoralis major muscle of HFE chickens. We also quantified glycogen content of the pectoralis major muscle and found that the HFE chickens had a significantly (P ≤ 0.05) lower glycogen content. Collectively, this study indicates extensive metabolic differences in the pectoralis major muscle between HFE and LFE chickens and helps identify metabolic features of susceptibility to muscle disorders in modern broiler chickens.  GA Molecular and cellular mechanisms that underlie genes and antioxidant enzyme activities during heat stress Heat stress causes critical molecular dysfunction and cellular changes that affect productivity and potentially compromises bird's welfare. Global temperatures have increased in the past few decades, and climate change will lead to frequent heat waves and longer hot seasons. Molecular mechanisms that underlie nutrient partitioning and metabolism of poultry under heat stress would allow for strategies to mitigate the effects of heat stress. We investigated the immediate and long term transcriptomics changes in chickens under heat stress. Forty- eight Cobb500 male birds were divided into two groups and raised under either constant 25oC or 35oC from 14-26 days of age in individual cages and fed ad libitum on a diet containing 21% CP and 3100kcal ME/kg. Five birds per treatment at 1 and 12 days after heat treatment were euthanized and the liver was sampled for gene expression analysis. We evaluated effect of heat stress (HS) on the expression of select genes in the oxidation/antioxidation pathway in the liver of chickens and further assess changes in antioxidant enzyme activity and biomarkers for oxidative stress in the liver and the Pectoralis (P.) major muscle. mRNA expression of Nrf2, oxidants NADPH(NOX): [NOX1, NOX2, NOX3, NOX4, NOX5 and DUOX2], and antioxidants [SOD1, CAT, GR, GPx1, NQO1] in the liver were analyzed at 1 and 12 days post-HS. We show that, HS changes the mRNA expression of oxidants thereby increasing cellular reactive oxygen species (ROS). Additionally, persistent HS up- regulates SOD which converts superoxides to hydrogen peroxide. We further demonstrated the dynamic relationship between catalase, GSH peroxidase (GPx) and NADPH under both acute and chronic heat stress. The pentose phosphate pathway could be important under HS since it generates NADPH which serves as a cofactor for GPx. Also, methionine, a precursor of cysteine has been shown to have reducing properties and thereby makes for an alternative fuel for redox processes. Genes in the ROS and antioxidant generation pathways may provide insight into nutritional intervention strategies, especially the use of methionine and/or cysteine when birds are suffering from heat stress. Heat stress was also associated with increased lipid and protein oxidations in the P. major. Molecular and cellular changes in the oxidation/antioxidation pathway may provide insight into interventional strategies. Effect of Heat stress on Eimeria replication in broiler chickens Eimeria infection is one of the most important diseases affecting poultry production, and is characterized by bloody or watery diarrhea, weight loss, poor feed conversion and moderate to high mortality. Heat stress (HS) is among the major environmental stressors in poultry, predisposing broiler chickens to immunosuppression and rendering them susceptible to diseases. There are some suggestions that HS reduces Eimeria oocyst output in chickens, however, the relationship between HS and coccidiosis is not elucidated. Our objective was to investigate the effect of HS on the development of E. tenella. Fifty-four 21 day old Cobb500 broiler chickens were infected via gavage with 15x104 E. tenella sporulated oocysts suspended in water and raised in either a thermoneutral (control: 25oC) or a heat-stressed (treatment: 35oC) environment. At 6-days post-infection (dpi), 9 birds in each group were euthanized humanely, and the caecal lesion scores, merozoite and oocyst counts were evaluated. The rectal temperatures were also taken. The HS group had significantly higher cloacal temperature (43.03±0.45oC versus 40.72±0.40; P<0.001) as compared to the control group. At 6 dpi, merozoites, caeca lesion scores and oocyst counts were evaluated in both groups. The HS chickens had lower caeca lesion scores (0.33±0.16 versus 1.89±0.45; P=0.014), merozoite (26.67±24.26 versus 823.21±262.31; P=0.0002) and oocyst counts (80.40±24.36 versus 1802±266.34; P=0.008) as compared to control chickens. Overall our results indicate an interruption of the cycle of E. tenella in chickens housed under heat stress conditions. High density marker panels, SNPs prioritizing and accuracy of genomic selection The availability of high-density (HD) marker panels, genome wide variants and sequence data creates an unprecedented opportunity to dissect the genetic basis of complex traits, enhance genomic selection (GS) and identify causal variants of traits of economic importance. The disproportional increase in the number of parameters in the genetic association model compared to the number of phenotypes has led to further deterioration in statistical power and an increase in co-linearity and false positive rates. High density panels do not significantly improve GS accuracy and, in some instances reduce accuracy. This is true for both regression and variance component approaches. To remedy this situation, some form of SNP filtering or external information is needed. Current methods for prioritizing SNP markers (i.e. BayesB, BayesC, etc) are sensitive to the increased co-linearity in HD panels which could limit their performance. Knowledge of genetic diversity based on evolutionary forces is beneficial for tracking loci influenced by selection. The fixation index (FST), as a measure of allele frequency variation among sub- populations, provides a tool to reveal genomic regions under selection pressure. The utility of FST, a measure of allele frequency variation among populations, as an external source of information in GS was evaluated. A simulation was carried out for a trait with heritability of 0.4. Data was divided into three subpopulations based on phenotype distribution (bottom 5%, middle 90%, top 5%). Marker data were simulated to mimic a 770 K and 1.5 million SNP marker panel. A ten- chromosome genome with 200 K and 400 K SNPs was simulated. Several scenarios with varying distributions for the quantitative trait loci (QTL) effects were simulated. Using all 200 K markers and no filtering, the accuracy of genomic prediction was 0.77. When marker effects were simulated from a gamma distribution, SNPs pre-selected based on the 99.5, 99.0 and 97.5% quantile of the FST score distribution resulted in an accuracy of 0.725, 0.797, and 0.853, respectively. Similar results were observed under other simulation scenarios. The accuracy obtained using all SNPs can be easily achieved using only 0.5 to 1% of all markers. These results indicate that SNP filtering using already available external information could increase the accuracy of GS. This is especially important as next-generation sequencing technology becomes more affordable and accessible to poultry applications. IA Liver and breast muscle transcriptome of laying hens altered by exposure to high ambient temperature. To maintain productivity, chickens undergo changes in gene expression to maintain metabolic homeostasis during heat exposure. Although previously described for broiler chickens, little is known of the effects of heat on the transcriptome in mature laying hens. We profiled the transcriptome of breast muscle and liver, two major metabolic tissues in poultry, during a 4-week cyclic heating study performed on layers in egg production. Both treatment versus control contrasts and time-based contrasts were analyzed to determine di

Publications

Impact Statements

WI This project generates turkeys with selenium status that ranges from Se-deficient to high-Se by feeding turkey poults a very low Se basal diet supplemented with graded levels of Se, and then analyzes tissues for selenoenzyme activity and transcript expression. These studies show that the dietary selenium requirement of the young turkey poult should be raised to 0.4 µg Se/g as inorganic selenium, and that the turkey is resistant to high dietary Se. These studies further indicate that the FDA limit of dietary selenium supplementation could be safely raised to 0.5 µg Se/g as inorganic selenium, at least for young turkey poults. There are, however, no good biomarkers for excess Se and toxic Se status. We are continuing to analyze these turkey samples to identify molecular biomarkers for high Se status that could better characterize safe upper limits for dietary Se for turkeys, for other production animals, and for humans

Date of Annual Report: 03/08/2020

Report Information

Annual Meeting Dates: 01/11/2020 - 01/12/2020
Period the Report Covers: 01/13/2019 - 01/12/2020

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WorkshopAttendeeList.pdf

Brief Summary of Minutes

See attachment below for full annual report.

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Accomplishments

Objective 1: <h1>CA</h1> <h2>Identification of Regulatory Elements in Livestock Species</h2> The recent international FAANG (Functional Annotation of ANimal Genomes) initiative has stimulated efforts to functionally annotated important livestock species, which will ultimately be leveraged to improve production efficiency, animal welfare, and food safety. As one of the FAANG pilot projects coordinated by UC Davis focus on chicken, cattle, and pig, Samples were collected from adipose, cerebellum, cortex, hypothalamus, liver, lung, muscle, and spleen in two male biological replicates from each species, allowing the identification of both universal and tissue-specific functional elements. We have completed data generation for all ChIP-seq marks (H3K4me3, H3K27ac, H3K27me3, H3K4me1, and CTCF) for eight tissues and two male biological replicates of chicken, pig, and cattle. Across all species, an average of 24,986,402 aligned and filtered reads were obtained per H3K4me3 library, 27,428,564 per H3K4me1 library, 26,874,867 per H3K27ac library, 30,394,847 per CTCF library, and 48,702,912 per H3K27me3 library. Many quality metrics were used to ensure high data quality, including the ENCODE data standards. We found the Jensen-Shannon distance (JSD) metric, which is not part of the ENCODE standards, to be the most informative in determining high data quality. All libraries were required to exceed a JSD of 0.1 for narrow marks (all except H3K27me3) and a JSD of 0.05 for H3K27me3. These cut-offs were determined by manual inspection of a wide variety of data. An average JSD of 0.37, 0.17, 0.25, 0.15, and 0.09 were obtained for the H3K4me3, H3K4me1, H3K27ac, CTCF, and H3K27me3 marks respectively. The fraction of reads in peaks (FRiP) was another key data quality metric and is widely used in ChIP-seq studies. Average FRiPs of 0.38, 0.22, 0.26, 0.14, and 0.23 were achieved for the H3K4me3, H3K4me1, H3K27ac, CTCF, and H3K27me3 marks respectively. ChromHMM was used to build a 14-state model integrating all the ChIP-seq data to predict genome-wide chromatin states per-tissue in each species. The chromatin states were used to annotate an average of 16,653 active enhancers (2,718 tissue-specific) in each chicken tissue, 39,811 (6,729 tissue-specific) in each pig tissue, and 31,339 (7,883 tissue-specific) in each cattle tissue. An average of 13,413, 13,962, and 15,345 active promoters were identified in chicken, pig, and cattle respectively, with 409 being tissue-specific on average across all tissues and species. Insulators, marked by CTCF, were identified with an average of 29,192 per tissue (9,395 tissue-specific) across all species. Open chromatin data (DNase-seq in chicken, ATAC-seq in pig and cattle) and RRBS-seq data were also generated for all samples used to generate the ChIP-seq data. Comparative analysis across species has begun, including additional data from horse (Equine FAANG) as well as human and mouse (ENCODE) in addition to the chicken, pig, and cattle data generated for this project in order to investigate the evolutionary conservation of these regulatory elements. <h1>COH</h1>   <ul> <li>Evaluating MHC-Y haplotypes segregating in chicken lines selected for high and low antibody responses to an experimental antigen (Zhang, Goto, Siegel, Honaker, Taylor, Parmentier, and Miller). </li> <li>With advances made in the last year improving the MHC-Y genotyping method (see below), we have greatly expanded studies to define the MHC-Y haplotypes segregating in experimental populations that have been selected for high and low antibody responses to the experimental antigen – sheep red blood cells (sRBC).</li> <li>Confirmed now in detailed typing is the skewed distribution of MHC-Y haplotypes in the Virginia Tech (VT) HAS and LAS lines that have been selected for over 45 generations for high and low antibody responses. In typing HAS and LAS lines at Generations 44 and 45 and individuals from additional specific matings designed to verify MHC-Y haplotypes, five haplotypes (a, b, c, d, e) have been defined in fully pedigreed families. Two haplotypes are exclusive to the HAS line (a & e, with a nearly four times more common than the next most frequent haplotype b). In contrast, one haplotype (d) is exclusively found in the LAS line. Two haplotypes (b & c) are found in both HAS and LAS.</li> <li>To begin to evaluate whether the highly skewed distribution of MHC-Y genotypes observed in the HAS and LAS lines is related to function or whether it could be the result of chance selection, we tested additional selected lines, including lines at VT in which high (HAR) and low (LAR) antibody selection was stopped 23 generations ago at Generation 22. Haplotype b, which is common in HAS and LAS, is also frequent in the HAR and LAR lines. Haplotype d, which is found exclusively in the LAS line, is the second most frequent haplotype in the LAR line after b. The most noticeable difference is in the frequency of a. While a is the most common haplotype in the HAS line, in the HAR line a is less common than two other haplotypes (b and an additional haplotype designated g). In addition, a is also found in LAR. An additional haplotype, f, is also present in HAR and LAR. These findings, particularly the rarity haplotype a in HAR, suggest that MHC-Y haplotypes could be under selection because of their contributions to immune responsiveness the phenotype under continuous selection in the HAS and LAS lines.</li> <li>We also examined the MHC-Y genotypes present in an additional set of lines at Wageningen University and Research (WUR) also selected for antibody responses to SRBC for twenty generations. In this population the MHC-Y genotypes are also skewed. Like VT HAS and LAS line, the WUR HA and LA lines have distinctly different in MHC-Y genotype distributions. One genotype is highly frequent in the high antibody line (WUR HA) and another is highly frequent in low antibody line (WUR LA). As with HAS and LAS, the WUR lines share one genotype in common. In a related, non- selected WUR control line, no genotype dominates. Overall the findings with the WUR lines are similar to the findings for VT lines. In both, a single MHC-Y genotype is highly frequent in lines selected for high antibody titer. This finding supports the likelihood that MHC-Y genetics influence immune responses.</li> </ul> Evaluating MHC-Y haplotypes in experimental Campylobacter colonization trials (Zhang, Goto, Psifidi, Stevens, and Miller). <ul> <li>Work defining the MHC-Y genotypes of birds in a series of Campylobacter colonization trials has also accelerated as the result of the moving to high-resolution chromatograms generated by microcapillary electrophoresis for scoring MHC-Y It is now possible to type with greater accuracy the large sample sets for in the backcross and intercross populations in the Campylobacter challenge trials described in Psifidi et al. 2016. These trials were conducted with crosses of Line 61 and Line N, two White Leghorn-derived lines that show heritable differences in resistance to Campylobacter colonization. The backcross experiments, [(Line 61 x Line N) x Line N] were conducted in three replicate experiments/hatches. Only one had a range of low to high colonization (others had either little colonization or all were heavily colonized) and was suitable for testing for association between MHC-Y haplotype and Campylobacter load. The MHC-Y haplotype Roslin-Y1 was significantly more frequent in low colonization birds (p-value 0.005), while two other haplotypes (Roslin-Y4 and Roslin-Y5) were significantly more common in the high colonization group (p-values 0.015 and 0.003, respectively). Three additional haplotypes, present at lower frequencies, showed no significant association with the levels of colonization. This finding suggests that MHC-Y may have a major gene effect in Campylobacter colonization. Additional typing is underway with samples from the intercross population to evaluate the apparent linkage between MHC-Y type and Campylobacter colonization.</li> </ul> <h1>IA</h1>   Transcriptome data made public. Several datasets from RNAseq experiments on chickens were deposited in public databases upon submission for journal publication of the manuscripts describing those studies. Genetics and genomics of response to Newcastle Disease virus (NDV). Newcastle Disease is a global threat to domestic poultry, especially in the developing countries, where entire small holder flocks are often lost to the disease. Local chicken ecotypes are important to rural family households through provision of high-quality protein in the form of eggs and meat and serve as a source of income. Studies were conducted in two countries, Ghana and Tanzania. In each country, three popular chicken ecotypes were challenged with a lentogenic (vaccine) strain of NDV. Various host response phenotypes, including viral load at 2 and 6 dpi, anti-NDV antibody levels (pre-infection and 10 days post-infection, dpi), and growth to 38 days of age, were measured. All birds were genotyped using a 600K Single Nucleotide Polymorphism (SNP) panel. We estimated genetic parameters and performed genome-wide association study (GWAS) analyses, using data on about 1400 birds per country. Heritability estimates for the various traits ranged from moderate to high (0.18 – 0.55). Six and twelve quantitative trait loci (QTL) were identified by single-SNP analyses for growth and/or response to NDV for Tanzania and Ghana, respectively. Several locations of these QTL corresponded in location with genomic regions explaining >1% of the genetic variance identified by the Bayes B GWAS analysis method. Immune related genes were located in the QTL regions for some response traits. Significant SNPs from GWAS and other important SNPs from separate studies, along with SNPs spread across the genome were used in the development of a 5K SNP panel for use in imputation. The moderate estimates of heritability and identified QTL suggest that host response to NDV can be improved through selective breeding of Africa local chicken ecotypes to enhance increased NDV resilience and vaccine efficacy. (Work conducted with H. Zhou of UC-Davis, and collaborators.) <h1>MN</h1> Additional RNAseq datasets compiled for the turkey have been accessioned in NCBI’s Gene Expression Omnibus (GEO) repository.       <h1>TX</h1> Structural variants are an important source of phenotypic variation in domestic species, including chicken, standardized databases for comparison across phenotypes are not available at present. Athrey Lab at continued development and testing of new structural variant cataloging tools, Girar.  One main approach to validate structural variants has been a partnership with Bionano. Using the Bionano Saphyr system, we generated data from Red Junglefowl and Commercial broilers to validate SV that we initially identified using sequence data. These data have generated high-resolution, phased SV data with new knowledge on the type, and location of shared and unique variants. These data were generated with the help of students Travis Williams and James Alfieri.   <h1>VA</h1> Long chain omega-3 polyunsaturated fatty acids (LC n-3 PUFA) regulate an array of pathways that are relevant to poultry production, including fat accretion, energy utilization, bone density, immunity, and the inflammatory response. This class of fatty acids, which includes the fatty acids (eicosapentaenoic acid (EPA; 20:5 n-3) and docosahexaenoic acid (DHA; 22:6 n-3) that are characteristic of fish oil, are considered essential in the diets of vertebrates because of a limited capacity to synthesize them from their precursor alpha-linolenic acid (ALA; 18:3 n-3). Unlike most species, chickens have the ability to synthesize significant amounts of EPA and DHA from ALA, which can be provided in diets by sources such as flax seed. This creates the potential to enrich tissues in specific fatty acids that can have beneficial effects on physiology by feeding flax and other sources of ALA, which are much more economical and sustainable than fish and other marines oils. However, despite the potential value of exploiting the LC n-3 PUFA synthesis pathway in broiler production, the expression and regulation of the chicken elongase and desaturase enzymes in this pathway are poorly characterized. To address this gap in knowledge, we profiled expression of six fatty acid elongase genes (ELOVLS 2-7), three fatty acid desaturase genes (FADS1, 2 and 6), and two stearoyl-CoA desaturase genes (SCD and SCD5) during adipose development in the broiler chick, spanning from E13 to D14. We focused on adipose tissue because we have previously shown that LC n-3 PUFA reduce fat accretion and suppress adipogenesis in the growing broiler chick. In addition, fatty acid utilization and availability are dynamically regulated during this window as developing adipose first stores and then mobilizes fatty acids in preparation for hatch. Subcutaneous adipose was harvested from broiler embryos at E13, E15, and E17, and subcutaneous, abdominal and crop adipose were collected at D7 and D14, as well as liver (n=5-6/age). We developed a targeted RNAseq panel that represents 171 chicken genes to efficiently profile expression of this pathway, in the context of other genes selected for their roles in metabolism and adipose biology. We found significant developmental changes in expression of almost all of the 11 genes in the LC n-3 PUFA synthesis pathway. Expression patterns in subcutaneous adipose were also associated with tissue fatty acid abundance, to infer enzymatic roles of specific enzymes. Collectively, these data provide new insight into regulation of this pathway and its genes in broiler chicks and during development of adipose and liver. In addition, we created a tool that will be utilized in ongoing studies in our lab and is available for use by collaborators. An important feature of this tool is that it can be easily expanded to encompass other pathways and genes of interest.     <h1>WI-Madison</h1> <h2> 1. Causal phenotypic networks for egg traits in an F2 chicken population</h2> Traditional single-trait genetic analyses, such as quantitative trait locus (QTL) and genome-wide association studies (GWAS), have been used to understand genotype–phenotype relationships for egg traits in chickens. Even though these techniques can detect potential genes of major effect, they cannot reveal cryptic causal relationships among QTLs and phenotypes. Thus, to better understand the relationships involving multiple genes and phenotypes of interest, other data analysis techniques must be used. Here, we utilized a QTL-directed dependency graph (QDG) mapping approach for a joint analysis of chicken egg traits, so that functional relationships and potential causal effects between them could be investigated. The QDG mapping identified a total of 17 QTLs affecting 24 egg traits that formed three independent networks of phenotypic trait groups (eggshell color, egg production, and size and weight of egg components), clearly distinguishing direct and indirect effects of QTLs towards correlated traits. For example, the network of size and weight of egg components contained 13 QTLs and 18 traits that are densely connected to each other. This indicates complex relationships between genotype and phenotype involving both direct and indirect effects of QTLs on the studied traits. Most of the QTLs were commonly identified by both the traditional (single-trait) mapping and the QDG approach. The network analysis, however, offers additional insight regarding the source and characterization of pleiotropy affecting egg traits. As such, the QDG analysis provides a substantial step forward, revealing cryptic relationships among QTLs and phenotypes, especially regarding direct and indirect QTL effects as well as potential causal relationships between traits, which can be used, for example, to optimize management practices and breeding strategies for the improvement of the traits. <h2>2. Effect of quality control, density and allele frequency of markers on the accuracy of genomic prediction for complex traits</h2> The project goal was to assess the impact of quality control, density and allele frequency of single nucleotide polymorphisms (SNP) markers on the accuracy of genomic predictions. A dataset on beef cattle genotyped with the Illumina BovineHDAssay was used as a case study, with three traits with different heritabilities and considering two methods of prediction. A total of 1756; 3150 and 3119 records of age at first calving (AFC); weaning weight (WW) and yearling weight (YW), respectively, were available. Three scenarios with different exclusion thresholds for minor allele frequency (MAF), deviation from Hardy–Weinberg equilibrium (HWE) and correlation between SNP pairs (r2) were constructed for all traits: (1) high rigor (S1): call rate <0.98, MAF <0.05, HWE with P <10-5, and r2 >0.999; (2) Moderate rigor (S2): call rate <0.85 and MAF <0.01; (3) Low rigor (S3): only non-autosomal SNP and those mapped on the same position were excluded. Additionally, to assess the prediction accuracy from different markers density, six panels (10K, 50K, 100K, 300K, 500K and 700K) were customized using the high-density genotyping assay as reference. Finally, from the markers available in high-density genotyping assay, six groups (G) with different minor allele frequency bins were defined to estimate the accuracy of genomic prediction. The range of MAF bins was approximately equal for the traits studied: G1 (0.000–0.009), G2 (0.010–0.064), G3 (0.065–0.174), G4 (0.175–0.325), G5 (0.326–0.500) and G6 (0.000–0.500). The Genomic Best Linear Unbiased Predictor and BayesCp methods were used to estimate the SNP marker effects. Five-fold cross-validation was used to measure the accuracy of genomic prediction for all scenarios. There were no effects of genotypes quality control criteria on the accuracies of genomic predictions. For all traits, the higher density panel did not provide greater prediction accuracies than the low density one (10K panel). The groups of SNP with low MAF (MAF <0.007 for AFC, MAF <0.009 for WW and MAF <0.008 for YW) provided lower prediction accuracies than the groups with higher allele frequencies. A corrected and updated revised FASTA transcript sequences for the complete turkey selenogene transcriptome (n=25) was assembled to include the selenocysteine encoding in-frame UGA and the 3’UTR containing the SECIS elements. This set was used by K. Reed (U Minnesota) to remap and obtain the RNA Seq datasets for the turkey selenotranscriptome. Analysis and publication is underway.     Objective 2: <h1>ADOL</h1>   <h2>ADOL chicken populations.</h2> A major strength of ADOL is the large number of chicken lines that are characterized for a number of traits, especially those associated with viral diseases, and maintained under specific pathogen free (SPF) conditions. Besides providing unique genetic resources to ADOL, ~1,500 embryos or chicks are supplied yearly to academic institutions or companies in the United States. The lines and maintenance are briefly summarized below. ADOL maintains 35 chicken lines with special genetic characteristics for tumor or viral susceptibility that also differ remarkably for immunological and physiological traits. All but 3 (C, N and P) were developed at the ADOL over the last 67 years. These include 4 of the world’s most highly inbred lines (63, 71, 72,and 15I5,), all of which are well defined for avian leukosis virus (ALV) receptor genes, endogenous virus loci (EV), and resistance to MD. Two of the lines are outbred, 2 of which are highly utilized worldwide for ALV analyses (0 and 15B1). Four congenic lines exist for analysis of EV genes; 3 (0.44-TVBS1- EV21, 0.44-TVBS3-EV21, and RFS) were developed from line 0 and 1 (100B) from line 72. Eight congenic lines exist for analysis of the influence of the MHC (B haplotype) on resistance to tumor diseases, immune responses or vaccinal immunity; 7 (15.6-2, 15.7-2, 15.15I-5, 15.C-12, 15.P-13, 15.P-19, and 15.N-21) were developed from line 15I5, and 1 (15.N-21) from line 0. Lines 63 and 72 differ markedly for MD resistance and immune function traits, as well as ALV and EV genes, but have the same B haplotype. Nineteen recombinant congenic strains (RCS) are under development to identify non-MHC genes that influence traits differing between lines 63 and 72. ADOL also developed one transgenic chicken line (0.ALV6) that is very beneficial for analysis of ALV. ADOL lines are routinely tested by blood-typing using 40 antisera either to ensure purity or to maintain heterozygosity (EV21, 100B, and O.P-13) during annual line reproduction. The breeders are unique in that they are maintained in a quarantined state and, on the basis of frequent serologic tests for 11 pathogens, are considered free of infection from common poultry pathogens. Estimates for the completion of the Athens, GA facilities is fall 2022. Unfortunately, despite promises that ADOL and all of its resources would remain intact, it is apparent that the buildings to house the ADOL lines are not sufficient. Thus, the number of lines kept will have to be reduced. Still worrisome is that no plans have been established to move the lines, which will require at least 2 years of having lines at both ADOL and Athens, and the new buildings that were built did not follow ADOL specifications, thus, require significant and costly renovations. <h1>IA</h1> Iowa State University chicken resource populations maintained, but reduced in number. Iowa State University maintained eight unique chicken research lines [including highly inbred, MHC-congenic, and a closed population; and an advanced intercross line (AIL)] to serve as resources for identifying genes, genetic elements and genomic regions of economic importance; as well as defining unique aspects of chicken genomic architecture. The eight lines are: one AIL (broiler X Fayoumi), two inbred Fayoumi lines, two inbred Leghorn lines, one inbred Spanish line, one antique inbred line (inbreeding started in 1925) and one closed population of broilers from 1980s industry genetics. All adult breeders were housed in individual cages and matings done by artificial insemination to ensure pedigree accuracy. All MHC-defined lines were blood-typed to verify MHC serologic haplotype. The AILs (now at generation F30) was maintained to facilitate fine-mapping of QTL with the goal of identifying genomic regions and candidate genes controlling important phenotypes. Sharing and utilization of research populations. The ISU genetic lines formed a discovery platform for research because of defined, distinct responses among lines. Genetic material (chicks, fertile eggs, blood, tissues, DNA or RNA) was shared with several cooperating investigators to expand studies on the chicken genome. Active collaborations utilizing ISU chicken genetic lines or biological materials include H Zhou, UC-Davis (NDV and heat-stress response); C. Schmidt, U Delaware (heat stress); R Coulombe, Utah State (aflatoxin sensitivity); B Abasht, U Delaware (allele-specific expression); Jim Kaufman, Univ Edinburgh (MHC structure).   <h1>TX -Athrey</h1> Athrey lab is maintaining a Red Junglefowl population of the Richardson strain. The current population size stands at 85. The well documented research population has become key to a new project we started with Prof. Leif Andersson, to look at the developmental biology of morphological traits in chicken. In 2019 we generated RJF x Silver Sebright crosses, that will be used for a mapping project to determine the genetic basis of feathering and pigmentation traits. Also, as the captive RJF are generating more eggs than we can hatch out and place in the colony (due to space limitations), we are able to collect and ship fertile RJF eggs   <h1>TX- Walzem</h1>   Began flock of Greater Prairie Chickens for fertile egg production with the aim of isolating primordial germ cells for use in gene editing studies.     Objective 3:     <h1>ADOL</h1>   <h2>3.2.a. Characterizing the genomic landscape of Marek’s disease virus-induced lymphomas</h2> Uncovering the Marek’s disease virus (MDV) pathogenesis process has been a major goal of the Marek’s disease (MD) community as this knowledge should translate to improved MD control strategies as well as inspiring the biomedical scientific community. Herein, we present a snapshot of the cancer genome of MD lymphomas to encourage these pursuits. It has long been acknowledged that the MDV oncogene Meq drives lymphomas; and we’ve shown that MD lymphomas are additionally driven by somatic mutations in the DNA-binding domains of IKZF1. Although Meq and mutant IKZF1 are the most recurrent oncogenic drivers discovered, they do not explain all phenotypic variance. To fill this gap, we surveyed diverse somatic mutation types across 22 gonadal tumor genomes and transcriptomes. Common somatic variant types—SNVs, indels, insertions, deletions, copy number variants, rearrangements, and gene fusions—scatter infrequently across the MD cancer genome and IKZF1 represents the most significantly (nonsynonymously) mutated gene in our cohort. However, examination of the ‘dark matter’ of the MD cancer genome reveals intronic hotspots—clusters of somatic mutations within intronic regions. Together, the ensemble of somatic mutation types (intronic hotspots, truncal drivers in IKZF1, and the remaining infrequent mutation types) augments differentially expressed pathways contributing to common themes in MD cancer genomes, which provides new insights. 3.2.b. Marek’s disease lymphomas with neuro-like transcription profiles independent of IKZF1 mutations   Uncovering the Marek’s disease virus (MDV) pathogenesis process has been a major A phenomenon exists in certain cancer types between their genomes and transcriptomes, i.e., tumors driven by different somatic mutations result in nearly identical gene expression profiles. CD4+ T-cell MD lymphomas (seeded in gonad) also demonstrate this phenomenon, suggesting that there are multiple somatic mutation strategies to get to their reprogrammed transcriptome of MD tumors. However, the most common strategy (at least in our cohort) involved dominant negative mutations in the IKZF1 gene, which encodes IKAROS, the master regulator of B and T cell development. We present a snapshot of the nearly identical transcriptomes of IKZF1-mutant and non-IKZF1-mutant tumors. These tumors demonstrate low somatic mutation frequencies and founding somatic truncal mutations. Truncal mutations in IKZF1 make up the majority of nonsynonymous variants and demonstrating mutual exclusivity to other nonsynonymous founding events across hosts. Therefore, regardless of mutation status, MD tumors (seeded in the gonads) demonstrate similar transcriptome characteristics, especially regarding gene expression and alternative splicing. For example, MD tumors commonly demonstrate down-regulation of T-cell differentiation pathways and (as expected) enrichment in gene sets associated with stem-like characteristics. It was, therefore, expected that hematopoetic or leukemic stem-cells would most similarly characterize MD transcriptome profiles; however, all tumors demonstrate enrichment for neural-stem-like transcriptomes—characterized by up- regulation of genes in the axon-guidance pathway. Preliminary interrogations of neural growth pathways (beyond differential gene expression) also demonstrate enrichment for alternatively spliced isoforms. We suggest that MD tumors stimulate neural progenitor growth pathways as a strategy for progression.   3.2.c.  Genomic screens to identify causative polymorphisms accounting for Marek’s disease genetic resistance in chicken  Marek’s disease (MD), a lymphoproliferative disease of chickens caused by the highly pathogenic Marek’s disease virus (MDV), is the most serious chronic disease problem that costs the worldwide poultry industry ~$2 billion per year. Despite control measures including biosecurity and MD vaccines, new and more virulent MDV strains have repeatedly arisen and is predicted to continue in the future. Consequently, alternative control methods, especially improving MD genetic resistance, are needed and highly desired.  Utilizing and integrating Hi-C, ChIP seq for MDV Meq and chromatin marks that identify promoters and/or enhancers, and RNA seq to identify transcripts, we will identify candidate regulatory elements that contain the causative polymorphisms. In Experiment 1, we use splenic-derived lymphocytes from uninfected and MDV-infected experimental chickens to reveal promoters and/or enhancers with specific transcription factors (TF) motifs that regulate gene expression in response to viral infection. In Experiment 2, the same design will be conducted except MDV will lack Meq, the viral oncogene and a bZIP transcription factor. Results from this experiment should help identify genes that are regulated by Meq. In Objective 3, we validate our experimental predictions by screening key regions in progeny-tested commercial layer sires. Since the inception of this project (July 2018), all samples have been collected. Hi- C and RNA seq datasets have been generated and ChIP seq experiments are underway. Upon collection of all of the dataset, they will be further analyzed and integrated as planned. <h2>3.2.c. Contribution of the T cell receptor (TCR) repertoire to Marek’s disease resistance</h2> Marek’s disease (MD) is a commonly diagnosed herpesviral-induced T cell lymphoproliferative disease of chickens, which has increased in virulence over time and prompted the search for continued improvements in control, both through improved vaccines and increased flock genetic resistance. Model pairs of genetically MD-resistant and susceptible chickens that are either B2 MHC-matched or B21 and B19 MHC-congenic has allowed the study of both non-MHC-linked and MHC-linked genetic resistance; here, we have applied these models to characterizing the T cell receptor (TCR) repertoires in MDV infection. Chickens resistant to MD showed higher usage of Vbeta-1 TCRs than susceptible chickens, in both the CD8 and CD4 subsets in the MHC-matched model, and in CD8 subset only in the MHC-congenic model; and Vbeta-1+ CD8 cells expanded during MDV infection. The TCR locus was found to be divergent between MD-resistant and susceptible chickens in the MHC-matched model, with MD-resistant chickens expressing a greater number of Vbeta-1 TCRs and an increased representation of Vbeta-1 CDR1 loops with an aromatic residue at position 45. TCR Vbeta-1 CDR1 usage in resistant x susceptible F1 birds indicated that the most commonly used CDR1 variant was present only in the susceptible line, suggesting that selection for resistance in the MHC-matched model has optimized the TCR repertoire away from dominant recognition of one of the B2 MHC molecules. Finally, TCR downregulation during MDV infection in the MHC-matched model was observed most strongly in the MD-susceptible line, and TCR downregulation due to viral reactivation in a tumor cell line could be demonstrated to be virus-specific and not due to apoptosis induction. <h2>3.3.d. Marek’s disease vaccines-induced differential expression of microRNAs in primary lymphoid organ bursae.</h2> Marek’s disease (MD) is a contagious disease of domestic chickens caused by MD viruses (MDV). MD has been controlled primarily by wide use of vaccines, yet sporadic outbreaks of MD take place worldwide. Commonly used MD vaccines include HVT, SB- 1 and CVI988/Rispens. MD vaccine efficacy is dependent of multiple factors including host genetics. Our previous studies showed that the protective efficacy of a MD vaccine can differ drastically from one genetic line of chickens to the next. Two highly inbred lines of White Leghorn were inoculated with MD vaccine HVT and CVI988/Rispens. Bursa samples were taken 26 days post vaccination and subjected to small RNA sequencing analysis to profile microRNAs. A total of 589 and 519 microRNAs were identified in one line, known as line 63 birds, 490 and 630 microRNAs were identified in the other, known as line 72, in response to HVT and CVI988/Rispens inoculation, respectively. HVT and CVI988/Rispens induced mutually exclusive 4 and 13 differentially expressed (DE) microRNAs in line 63 birds in contrast to a non-vaccinated group of the same line. HVT failed to induce any DE microRNA and CVI988/Rispens induced a single DE microRNA in line 72 birds. Thousands of target genes for the DE microRNAs were predicted, which were enriched in a variety of gene ontology terms and pathways.   <h1>AR</h1> <h2>3.1 Identification of genes affecting ascites susceptibility</h2> Utilizing a NIFA funded project on genetics of ascites, AR has generated a population using marker assisted selection (MAS) from our resistant line (REL) for ascites research. We used two loci previously determined to be associated with ascites resistance to generate the MAS line as homozygous for the non-reference, resistant associated, alleles. We are now evaluating the MAS line using a hypobaric chamber located at the Poultry Farm and at the Poultry Processing Plant to document changes in production traits. We have also completed a WGR analysis of a commercial broiler for mapping QTLs for ascites in a hypobaric chamber challenge. <h2>3.2 Bacterial chondronecrosis with osteomyelitis and lameness in broilers</h2> AR performed one trial for QTL mapping for resistance to bacterial chondronecrosis with osteomyelitis (BCO) lameness in 2 commercial crosses. We used whole genome resequencing (WGR) on lame and healthy birds after a challenge with isolate Staphylococcus agnetis 908 in the drinking water. The results are under a Non-Disclosure Agreement. The results will be published soon. We continue to pursue the genetics for macrophage survival using isolate 908. We have used Directed Genome Evolution from isolate 908 to a hypovirulent cattle isolate of the same species. The transformants are selected by passage through an immortalized chicken macrophage line. The work has suggested that a single amino acid residue in a nucleotide recycling protein is responsible. Confirmation work is underway. AR performed a series of in vivo and functional in vitro studies and identified a key role of Dicer 1 dysregulation and dsRNA accumulation in BCO pathogenesis. <h2>3.3 Broiler breast muscle and myopathies</h2> A current issue in the poultry industry is the occurrence of woody breast (WB) in broilers, particularly in fast growing lines of birds. Data from studies suggest localized hypoxia and metabolic stress may be involved in this myopathy. A study was completed examining plasma levels of the stress hormone, corticosterone (CORT) of birds scored with high levels of WB compared to controls. Thereafter liver and breast muscle were sampled to determine gene expression levels utilizing quantitative RT-PCR. Results showed that WB birds had significantly higher plasma CORT compared to controls. Additionally, glucocorticoid receptor (GR) expression and 11β- hydroxysteroid dehydrogenase 1 (11β-HSD1) were significantly reduced in breast muscle samples from WB birds. In constrast, GR gene expression was significantly elevated in liver samples taken from WB birds compared to controls while no significant differences were observed for liver 11β-HSD1 between the two groups. Results suggest that CORT, GR and 11β-HSD1 may be involved in the etiology of the WB syndrome in broilers. Studies conducted in AR have shown that systemic and breast muscle local hypoxia induce woody breast myopathy and that supplementation of quantum blue reduces the severity of WB by 5% via modulation of oxygen homeostasis-related networks. <h2>3.4 Genes and proteins involved in the stress response of broilers</h2> AR currently maintains two genetically distinct lines of Japanese quail named as High Stress (HS) and Low Stress (LS), which were selected for divergent plasma corticosterone response to restraint stress in the 1980s through 2009 by Dr. Dan Satterlee. Since then these two lines have been transferred to UA and utilized as genetic models for stress studies. In the LS line, the mean corticosterone level is approximately one-third lower compared to HS line. To understand genetic components underlying stress related traits, we performed whole genome re-sequencing of pooled DNA samples of 20 birds each from HS and LS lines using the Illumina HiSeq 2×150 bp paired end method. Sequencing data were aligned to the quail genome and CNVnator was used to detect CNVs in the aligned data sets. The depth of coverage for the data attained 41.4x and 42.6x for the HS and LS birds, respectively. We identified 262 and 168 CNV regions affecting 1.6 and 1.9% of the reference genome that completely overlapped 454 and 493 unique genes in HS and LS birds, respectively. Ingenuity pathway analysis showed that the CNV genes were significantly enriched to phospholipase C signaling, neuregulin signaling, reelin signaling in neurons, endocrine and nervous development, humoral immune response, and carbohydrate and amino acid metabolisms in HS birds, whereas CNV genes in LS birds were enriched in cell-mediated immune response, and protein and lipid metabolisms. AR continued studies of the nucleus of the hippocampal commissure (NHpC), a structure containing a significant population of corticotropin-releasing hormone (CRH) neurons. Using food deprivation (FD) as a stressor provided additional evidence that the NHpC appears to be an early responder to stress in birds contributing to a significant increase in the stress hormone corticosterone (CORT). Increased gene expression of CRH neurons in the paraventricular nucleus (PVN) of the hypothalamus appeared to function secondarily to continue an increase in CORT plasma levels. A positive feedback between CRH neurons and its major receptor, CRHR1, occurs in the PVN. A subsequent study, examining arginine vasotocin (AVT) and its major receptors, the V1aR and V1bR, showed a delay in hours that followed the significant increase in gene expression of CRH in both the NHpC and PVN. The delay in the change in AVT gene expression followed by its robust, significant increase supports a key role of AVT to sustain the significant, increased level of CORT. Of interest is a positive feedback shown between AVT and both of its key receptors, the V1a and V1b receptors, both located within the PVN. The overall data support our previous suggestion that the NHpC appears to function within the traditional HPA axis to initiate the neuroendocrine stress response followed by CRH neurons in the PVN. A positive feedback occurs in the PVN between CRH and its major receptors. The delayed, but significant increase in AVT and its two receptors in the PVN likewise show a positive feedback response revealing an overall mechanism accounting for the continued release of AVT that directly stimulates activation of the pituitary stress hormone, pro-opiomelanocortin (POMC), the pre-prohormone that produces the major pituitary stress hormone, ACTH, responsible for stimulating CORT release by the adrenal gland. To date, data support the hypothesis that the NHpC appears to be part of the neuroendocrine system working with the hypothalamic PVN to activate the anterior pituitary and ultimately the adrenal gland to produce the stress hormone CORT during the stress response in poultry. Studies on heat stress provided evidence for the use of a non-invasive method to monitor stress in poultry by determining gene expression of Feather HSP70. A second molecular marker for heat stress was shown to be 75 kDa glucose regulated protein (GRP75). <h1>COH</h1> <h2>PCR-based method for MHC-Y genotyping (Miller, Goto, Zhang).</h2> During the past year we adapted STR typing to capillary electrophoresis on an ABI Prism 3730 DNA analyzer. We thank NC1170 members, especially Chris Ashwell, for suggesting we do this. We tested a series of primers allowing variability across MHC-Y haplotypes to be revealed. The primers are anchored at the start site of the many MHC class I-like genes in MHC-Y. The number of MHC-Y class I-like genes in a single haplotype may be large. For example, there are at least 49 loci are present in the MHC-Y haplotype in the RJF reference genome. The STR patterns are highly reproducible. For acceptance as suitable for typing, the primer pairs must reflect inheritance patterns predicted in fully pedigreed families and be reproducible within defined inbred lines (Miller et al. 2014; Zhang et al, submitted).   <h1>CA</h1> 3.3.1. Improving food security in Africa by enhancing resistance to Newcastle disease virus and heat stress in chickens This USAID funded Feed the Future Innovation Lab for Genomics to Improve Poultry project (H. Zhou, PI) through a partnership of the University of California at Davis (H. Zhou, R. Gallardo, T. Kelly), Iowa State University (S.J. Lamont, J. Dekkers), Sokoine University of Agriculture (SUA) -Tanzania, the University of Ghana (UOG), and International Livestock Research Institute (I. Baltenweck, E. Ouma) was renewed for the second phase (2018-2023). The five-year research program applied advanced genetics and genomics approaches to sustainably enhance innate resistance to Newcastle disease virus (NDV) and heat stress in chickens to improve poultry production in Africa. There are five specific objectives on the second phase: Assess correlations of crucial production traits with disease resistance traits: egg production, growth rate; Select and breed genetically enhanced local ecotypes;Characterize circulating strains of NDV; Determine and monitor the effectiveness of genomic selection; Conduct value chain assessment and business plan development; Develop a training toolkit on application of genetic selection platform. The 600K SNP panel and two GWAS methods (single-SNP and multi-SNP Bayesian selection analyses) were used to identify regions of the chicken genome associated with NDV response traits, including pre- and post-infection growth rates, anti-NDV antibody levels, and viral load at 2 and 6 dpi. Estimates of heritabilities were moderate to high, 0.18 to 0.35 and 0.23 to 0.55, for Tanzania and Ghana, respectively. Estimates of genetic correlation between anti-NDV antibody levels and NDV titers (viral load) were positive and low-moderate. These results suggest that genetic improvement on these disease resistance parameters is feasible and promising. Multiple genomic regions with genetic variance >1% and significant SNPs associated with growth and NDV response traits were identified (20% suggestive adjusted Bonferroni correction). Genome-wide association (GWAS) analyses performed on Tanzania chicken ecotypes revealed five genomic regions and 7 QTLs (9 significant SNPs) associated growth and NDV response traits. For Ghana GWAS analyses, 20 genomic regions and 13 QTLs (11 significant SNPs) were identified. Some of the identified genomic regions (genes) were supported by collaborative studies conducted at UC Davis. Development of low density SNP Panel: To develop a 5K low-density SNP panel, SNPs were acquired from various sources, including 4500 SNPs across the chicken genome, significant/suggestive SNPs from GWAS analyses, MHC SNPs, SNPs from important genomic regions from other collaborative studies on GWAS on Hy-Line Brown and RNA-seq on inbred lines at Iowa State University and UC Davis, and SNPs that directly affect a gene product. All SNPs from the various sources were acquired using a variety of genomic software/programs. Our goal is to develop a low-density SNP panel using the new genotyping platform, genotyping by sequencing (GBS). In order to evaluate the GBS platform, the initial genotyping phase involved both Affymetrix and GBS platforms to identify any inconsistences. A second set of birds was genotyped by GBS and sequence data is currently under evaluation before all birds are genotyped. Upon completion of the low-density panel genotyping, we plan to impute it to a high whole genome 600K SNP panel that will be used for selective breeding. <h2>3.3.2 Knockout of transcription factor IRF7 reveals its novel role on modulating host response against avian influenza virus</h2> Interferon regulatory factor 7 (IRF7) is known as the master transcription factor of type I interferon response in mammalian species along with IRF3. Birds yet only have IRF7 while missing IRF3 with a smaller repertoire of immune-related genes which leads to a distinctive immune response of chickens compared to mammals. In order to understand the functional role of IRF7 in the regulation of antiviral response against avian influenza virus in chickens, we generated IRF7-/- chicken embryonic fibroblast (DF-1) cell lines and respective control (IRF7wt) by utilizing the CRISPR/Cas9 system. IRF7 knockout resulted in increased viral titers of low pathogenic avian influenza viruses. Further RNA-sequencing performed on H6N2 infected IRF7-/- and IRF7wt cell lines revealed that deletion of IRF7 resulted in significant down-regulation of antiviral effectors and differential expression of genes in MAPK and mTOR signaling pathways. Dynamic gene expression profiling of host response between the wildtype and IRF7 knockout revealed the potential signaling pathways involving AP1, NF-κB and inflammatory cytokines that may complement IRF7. Our findings in this study have provided novel insights that have not been reported previously and have laid solid foundation on enhancing our understanding of host antiviral response against avian influenza virus in chickens. <h1>DE</h1> Role of Lipoprotein Lipase (LPL) And Slow Muscle Fiber Gene Expression In Wooden Breast. Previous transcriptomic studies have hypothesized the occurrence of slow myofiber-phenotype, and dysregulation of lipid metabolism as being associated with the development of Wooden Breast (WB), a meat quality defect in commercial broiler chickens. To gain a deep understanding of the manifestation and implication of these two biological processes in health and disease states in chickens, cellular and global expression of specific genes related to the respective processes were examined in pectoralis major muscles of modern fast-growing and unselected slow-growing chickens. Using RNAin situ hybridization, lipoprotein lipase (LPL) was found to be expressed in endothelial cells of capillaries and small-caliber veins in chickens. RNA-seq analysis revealed upregulation of lipid-related genes in WB-affected chickens at week 3 and downregulation at week 7 of age. On the other hand, cellular localization of slow myofiber-type genes revealed their increased expression in mature myofibers of WB-affected chickens. Similarly, global expression of slow myofiber-type genes showed upregulation in affected chickens at both timepoints. Parallels between Myopathy of Broilers and Metabolic Syndrome in Humans. Most studies have focused on advanced stages of wooden breast apparent at market age, resulting in limited insights into the etiology and early pathogenesis of the myopathy. Therefore, the objective of this study was to identify early molecular signals in the wooden breast transcriptional cascade by performing gene expression analysis on the pectoralis major muscle of two-week-old birds that may later exhibit the wooden breast phenotype by market age at 7 weeks. Biopsy samples of the left pectoralis major muscle were collected from 101 birds at 14 days of age. Birds were subsequently raised to 7 weeks of age to allow sample selection based on the wooden breast phenotype at market age. RNA-sequencing was performed on 5 unaffected and 8 affected female chicken samples, selected based on wooden breast scores (0 to 4) assigned at necropsy where affected birds had scores of 2 or 3 (mildly or moderately affected) while unaffected birds had scores of 0 (no apparent gross lesions). Differential expression analysis identified 60 genes found to be significant at an FDR-adjusted p-value of 0.05. Of these, 26 were previously demonstrated to exhibit altered expression or genetic polymorphisms related to glucose tolerance or diabetes mellitus in mammals. Additionally, 9 genes have functions directly related to lipid metabolism and 11 genes are associated with adiposity traits such as intramuscular fat and body mass index. <h1>GA</h1> <h2>Dietary methionine levels alter digestibility and gene expression of amino acid transporters</h2> Imbalance in nutrients can affect digestibility of amino acids by altering gene expression of amino acid transporters. Methionine is an essential amino acid required for protein synthesis and is also the precursor of S- adenosylmethionine (SAM). Methionine is involved in 5 metabolic pathways: transmethylation, transsulfuration, re-methylation, aminopropylation, and salvage. In transmethylation, methionine acts as the sole methyl donor to a variety of acceptors including nucleic acids, proteins, CpG islands in DNA and biological amines. We investigated digestibility and molecular transporters of essential amino acids in chickens fed a methionine-deficient diet. A total of 40 chicks (23 D old) were randomly assigned to either a control (0.49% methionine) or a deficient (0.28%) diet until 41 D when they were sampled for Pectoralis (P.) major, kidney, ileum, and hypothalamus for mRNA expression analysis. The ileal content was collected for apparent ileal digestibility (AID) analysis. The duodenum, skeletal muscle from the superficial pectoral muscle, spleen, liver, and bursa of each bird (N = 10 per group) were collected and fixed in 10% buffered formalin. After fixation, samples were trimmed, routinely processed (Tissue-Tek VIP Sakura, Torrance, CA), embedded in paraffin (Leica EG1150), sectioned at 4 microns (Leica RM2255), and stained with hematoxylin and eosin (Leica Autostainer XL). Slides were examined by light microscopy (Leica DMR). Duodenum samples were used for villus height and crypt depth measurements using photomicrographs (Leica DC 500 camera) and Image J (NIH download) program for measurements. A total of 3 intact villi and the 3 corresponding crypts were randomly sampled thrice and measured in microns and averaged for each duodenum. Villus height/crypt depth ratios were calculated. All tissues were examined for microscopic changes and scores were assigned to major alterations using the scoring method of Henry et al. (1980). Birds fed the deficient diet had reduced growth and worse feed efficiency compared to control. The AID of methionine was similar between both groups. The AID of other essential amino acids was higher in the deficient group than control. mRNA expression of b0,+AT and LAT4 were upregulated in the ileum and kidney but LAT1 was downregulated only in kidney of the deficient group compared to control. In the P. major, SNAT1, SNAT2, and CAT1 were upregulated in the deficient group compared to control. A diet deficiency in methionine affects digestibility of essential amino acids and cysteine, but not the digestibility of methionine. The change in digestibility is reflected in the mRNA expression of amino acid transporters across different tissues. Dietay methionine did not affect villi height and crypt depth <h2> </h2> <h2>Host Genotype and Emeria acervulina infection: metabolomics</h2> Eimeria infection is one of the most important diseases affecting poultry production, and is characterized by bloody or watery diarrhea, weight loss, poor feed conversion and moderate to high mortality. A study was conducted to identify metabolic biochemical differences between two chicken genotypes infected with Eimeria acervulina and to ascertain the underlying mechanisms for these metabolic alterations and to further delineate genotype-specific effects during merozoite formation and oocyst shedding. Fourteen day old chicks of an unimproved (ACRB) and improved (COBB) genotype were orally infected with 2.5 x 105 sporulated E. acervulina oocysts. At 4 and 6 day-post infection, 5 birds from each treatment group and their controls were bled for serum. Global metabolomic profiles were assessed using ultra performance liquid chromatography/tandem mass spectrometry (metabolon, Inc.,). Statistical analyses were based on analysis of variance to identify which biochemicals differed significantly between experimental groups. Pathway enrichment analysis was conducted to identify significant pathways associated with response to E. acervulina infection. A total of 752 metabolites were identified across genotype, treatment and time post infection. Altered fatty acid (FA) metabolism and β-oxidation were identified as dominant metabolic signatures associated with E. acervulina infection. Key metabolite changes in FA metabolism included stearoylcarnitine, palmitoylcarnitine and linoleoylcarnitine. The infection induced changes in nucleotide metabolism and elicited inflammatory reaction as evidenced by changes in thromboxane B2, 12-HHTrE and itaconate. Serum metabolome of two chicken genotypes infected with E. acervulina demonstrated significant changes that were treatment-, time post-infection- and genotype-dependent. Distinct metabolic signatures were identified in fatty acid, nucleotide, inflammation and oxidative stress biochemicals. Significant microbial associated product alterations are likely to be associated with malabsorption of nutrients during infection. <h2> </h2> <h2>A Weighted Genomic Relationship Matrix Based on FST Prioritized SNPs for Genomic Selection</h2> Recent advances in high-throughput genotyping and sequencing techniques led to the generation of dense marker panels and facilitated the genotyping of large numbers of individuals. Because of the availability of these cost-effective genotyping technologies and the increase in sequencing speed, large-scale genotyping for single- nucleotide polymorphisms (SNP) has become more affordable and accessible. Genomic data provides an unprecedented opportunity to dissect the genetic basis of complex traits and to identify relevant functional associations. From animal breeding perspective, the use of genomic information allows for a substantial reduction in generation interval and in the increase of the accuracy of predicted breeding values, leading undoubtedly to an improvement in the genetic response. Genomic selection (GS) is often carried out using multiple regression or mixed linear models. For both methods, the density of the SNP marker panel and the LD structure between markers and QTL have a great impact on accuracy. Regression based methods directly model the association between the phenotypes and all or a subset of the genotyped variants. Thus, their problems stem mainly from the high dimensionality of the parameter space. As the effect of a QTL (often small for complex traits) is distributed in a non-trivial manner between all markers that are in LD with the causal mutation, there is little statistical power to accurately estimate its effect. Genomic best linear unbiased prediction (GBLUP) assumes equal weight for all SNPs. Weighted single-step GBLUP (WssGBLUP) was developed to allow for estimating weights within single-step GBLUP (ssGBLUP) process. However, the challenge is how to derive the optimum set of weights to compute the genomic relationship matrix. Due to these limitations, variant prioritization has become a necessity to improve accuracy. FST as a measure of population differentiation has been used to identify genome segments and variants under selection pressure. Using prioritized variants has increased the accuracy of GS. Additionally, FST can be used to weight the relative contribution of prioritized SNPs in computing G. In this study, relative weights based on FST scores were developed and incorporated into the calculation of G and their impact on the estimation of  of variance components and accuracy was assessed. The results showed that prioritizing SNPs based on their FST scores resulted in an increase in the genetic similarity between training and validations animals and improved the accuracy of GS by more than 5%.   <h1>IA</h1> Transcriptome of gut tissue in fat and lean selected broiler lines. Broiler production has improved greatly over the past several decades, but excessive abdominal fat deposition remains a problem. Fat deposition is a result of excess energy intake, in which the intestine plays a role by digesting feed and absorbing nutrients. The association of the intestine with broiler abdominal fat deposition has not been investigated at the transcriptome level. Therefore, to explore intestinal gene expression associated with broiler lines selected for abdominal fat deposition, we collected the duodenum, jejunum, ileum, and cecum of 10 high- and 10 low-abdominal fat line (HL and LL) male broilers from Generation 21 of the Northeast Agricultural University High- and Low-Fat (NEAUHLF) broiler lines that had been divergently selected for abdominal fat. We identified differentially expressed genes (DEGs) in the four intestine tissues in comparisons of the HL vs LL, and in comparisons across tissues within the HL and LL using RNA-seq. Ingenuity Pathway Analysis (IPA) predicted that duodenal cell turnover functions would be inhibited of the HL vs LL. IPA predicted that ileal transport of lipids would be inhibited in the HL vs LL. Catabolism of lipids and transport of lipids were significantly predicted to be inhibited in ileum vs duodenum and ileum vs jejunum within the HL, but no differences were predicted within the LL. Our data suggest that more lipids might be absorbed in the duodenum and jejunum within the HL. The current study’s results provide a foundation for understanding of transcriptional regulation of broiler abdominal fat deposition and intestinal lipid digestion and absorption.   Results: Estimates of heritability were intermediate (0.23-0.46) for all disease phenotypes with the exception of antibody responses to Fowl Typhoid where the estimate was low (0.08). GWAS identified genomic markers significantly associated with response to infectious diseases at genome-wide and chromosome-wide level. Some of the putative Quantitative Trait Loci (QTL) regions for antibody responses were common for different diseases. The average accuracy of GEBVs was relatively poor (0.30-0.45), but this could be attributed to the limited sample size. Conclusions: Results underpin the potential of genetic selection for enhanced antibody response and disease resistance across Ethiopian indigenous chicken ecotypes since all the studied traits found to be heritable and common QTLs segregating in the two populations. However, future studies are needed to establish the required sample size to derive GEBVs with good accuracy in these settings. <h1>MI</h1> <h2>Influence of thermal challenge on turkey muscle development and meat quality.</h2> This project in collaboration with the University of Minnesota and Ohio State University is designed to evaluate the impact of temperature extremes resulting from climate change on poultry breast muscle growth and development and consequent effects on meat quality. Based on this information, our goal is to develop strategies that will result in improved avian thermotolerance that will mitigate the undesirable changes on turkey meat quality.  This project ended on March 14, 2019 and we are working to submit manuscripts for publication. We have also completed pilot experiments on metabolomic analysis of breast muscle from birds whose breast muscles were classified as normal or PSE. Funding for this seed grant ended on February 28, 2019, but data analysis is ongoing. <h2>Related studies</h2> In collaboration with researchers at the National Center for Genetic Engineering and Biotechnology in Thailand, we have investigated changes in gene expression in chicken breast muscle as a function of white striping and wooden breast myopathies. We have recently published a manuscript showing differences in absolute expression of hypoxia-inducible factor-1 alpha subunit (HIF1A) and in genes involved in stress responses and muscle repair using a droplet digital polymerase chain reaction. <h1>MN</h1> <h2>Influence of thermal challenge on turkey muscle development and meat quality.</h2> This project in collaboration with Michigan State University and Ohio State University seeks to quantify climate change impacts on poultry breast muscle growth and development, morphological structure, intramuscular fat deposition, and protein functionality to develop appropriate strategies to mitigate the undesirable changes in meat quality. To this effect we completed analysis of data from RNAseq experiments and have published these results. Two manuscripts examining aspects of transcriptome changes in proliferating and differentiating muscle satellite cells and PSE have been submitted. We have initiated a study of circRNAs using the data generated from this project and presented preliminary results at ISAG 2019. <h2>Genomics to increase aflatoxin resistance in turkeys.</h2> To investigate the response to aflatoxin exposure we are using RNA-Seq approaches to characterize the transcriptome level changes in the liver, intestine and spleen of birds exposed to AFB1. We have completed analysis of a liver, intestine, and spleen RNAseq databsets from an AFB1 challenge of 16wk wild and domestic turkeys conducted at our collaborating institution (Utah State University, RA Coulombe). Manuscripts detailing these studies have been published. <h2>Antibiotic-free alternatives to improve health and performance in commercial turkeys</h2> The goal of this project is to advance our understanding of the interactions between the turkey gastrointestinal microbiome and host during maturation and microbiome modulation. We seek to change the paradigm by which alternatives to antibiotics are developed, using systematic and science-grounded approaches. A manuscript reporting on a combined mega analysis was published this year (Ward et al. 2019). <h2>Related studies</h2> Determining Turkey Selenium Nutrition and Requirements Using Molecular Biology, University of Wisconsin, Multi-state Hatch Project. Roger Sunde (PD). This project utilizes RNA-Seq to characterize the effect of selenium on the turkey liver transcriptome. We continue to work with Dr. Sunde and his graduate student Rachel Taylor to provide bioinformatics support for data analysis and developed a custom transcript database for read mapping.   <h1>NY</h1> The problem with the declining productivity of hens in a laying cycle (over 72 weeks) is that there is variability in the flock. That is, some hens continue to lay at an acceptable rate and others decline dramatically. The decision whether to force molt the hens and synchronize them or to replace the flock is based on economics. Knowledge about ovar

Publications

Impact Statements

Impact Statements: ADOL • Determining the purity of tumor samples has aided our efforts to identify Ikaros and other candidates as the first driver genes for MD. This supports our hypothesis that somatic mutations are required in addition to MDV infection to get tumors in susceptible birds.   • The TCR genes and usage play a role in response to MDV infection. As the TCR interactions with the MHC, this makes sense as the MHC has a major influence on MD genetic resistance.   • Advancement in understanding the underlying genetic and epigenetic factors that modulate vaccine efficacy would greatly improve the development of strategy in design of new vaccines, and therefore better control of the disease. The findings that MD vaccines-induced differentially expressed microRNAs in primary lymphoid organ, bursae, suggest the epigenetic factors are highly likely involved in modulating vaccine protective efficacy in chicken.   AR • Additional data have been published to support our proposed suggestion that the nucleus of the hippocampal commissure (NHpC) be added to the classical hypothalamo-pituitary-adrenal (HPA) axis in avian species due to its early activation of corticotropin releasing hormone gene expression within that structure following an imposed stressor.   • A new molecular marker for heat stress, 75 kDa glucose regulated protein (GRP75) and a non- invasive molecular marker to monitor stress in poultry, feather HSP70, have been identified.   • Providing new molecules and additional key mechanisms into the cellular pathways for muscle growth and muscle mass development in breast muscle of broilers will improve production efficiency and hopefully prevent metabolic myopathy such as ‘woody breast’. • Identification of the genetics of ascites will allow breeders to select against ascites and reduce production losses   • Development of management strategies to reduce lameness caused by BCO is critical for reducing a significant animal welfare issue in broilers.   CA • Identification of genes that are associated with resistance to heat stress and Newcastle disease virus and can be used to genetic enhancement of disease resistance of chicken in adaption to hot climate.   • Knowledge of genes associated with enhanced immune response may inform further information on vaccine efficacy in poultry production.   • ChIP-seq and ATAC-seq assays developed and other omic data generated for regulatory elements annotation will be important for animal genome community.   COH • The improved typing method makes it feasible to expand efforts to understand the impact of MHC-Y genetic variability on immunity and disease resistance in chickens.   • Evidence continues to accumulate supporting the likelihood that MHC-Y contributes to the genetics of immune responses in chickens.   DE To our knowledge, our study is the first to show the expression of LPL from the vascular endothelium in chickens. Our study also confirms the existence of slow myofiber-phenotype and provides mechanistic insights into increased lipid uptake and metabolism in Wooden Breast disease process. We also found intriguing parallels between myopathy of broilers and metabolic syndrome in humans, suggesting chickens as a potential model for studying complications caused by diabetes in humans. IA • Genetic variation was characterized in commercial, research and indigenous lines of chickens.   • Genes, pathways and genomic regions associated with important biological traits in chickens were  identified.   • The feasibility of applying molecular genetics and genomics to analysis of variation in structure,  function and gene expression within the chicken genome was demonstrated.   MI • Our efforts are focused on projects that directly impact poultry production and the quality of muscle as a food, i.e., meat. Temperature extremes are predicted to increase in frequency and extent according to most climate models. These thermal challenges threaten the quality of poultry muscle as a healthy, high quality food product by increasing fat deposition and altering muscle protein organization, especially in the breast muscle which is the most valuable source of poultry meat. Identification of molecular mechanisms altered by these temperature extremes will inform development of mitigation strategies based on breeding, nutritional intervention, and other strategies to improve poultry muscle food quality and quantity. • Our studies on white striping and wooden breast suggest that hypoxia within the abnormal breasts is potentially associated with oversized muscle fibers. Between white-striping and wooden breast myopathies, divergent glucose metabolism, cellular detoxification and myo-regeneration at the transcriptional level could be anticipated. These results form the basis for future studies designed to mitigate the problems associated with these myopathies through improved breeding and management strategies. MN • Our efforts are focused on projects that directly impact poultry health and production. Extreme temperature variations threaten the quality of poultry muscle as a healthy, high quality food product. Identification of molecular mechanisms associated with altered muscle development will result in development of mitigation strategies based on improved genetic selection, nutritional intervention, and other strategies to improve poultry muscle food quality and quantity. Likewise, AFB1 causes annual industry losses estimated in excess of $500 M. Increasing innate resistance to AFB1 could result in numerous health benefits. Transformational improvements in AFB1 resistance require a multidisciplinary approach to identify protective alleles with potential to reduce disease. Genetic markers to improve AFB1-resistance have a potentially high commercial value and positive economic impact to industry, owing to improvements in health and well-being, productivity, and a safer product for consumers. The gastrointestinal health of an animal is key to its successful growth and development. Elimination of subtherapeutic antibiotics for growth promotion and health in poultry will leave a critical void. This project will improve our mechanistic understanding of host-microbiome interactions in the avian host, and identify feasible approaches towards modulating the turkey intestinal microbiome resulting in enhanced health and performance. RVC • Campylobacteriosis the leading cause of human foodborne diarrhoea. The main source of infection is consumption or handling of contaminated poultry meat. While there are a range of biosecurity strategies at farm level, there are no effective vaccines or inhibitors. Breeding for enhanced resistance to Campylobacter colonisation is an attractive option to control the disease in humans and based on our results, this is feasible. • Poultry play an important role in the agriculture of many African countries. The majority of chickens in sub-Saharan Africa are indigenous raised under scavenging conditions. Although these birds are well adapted to local conditions, vaccinations and biosecurity measures are rarely applied and infectious diseases remain a major cause of mortality and reduced productivity. Breeding for increased resistance to infectious diseases offers a potentially sustainable solution. However, successful genomic selection programmes require sufficient numbers of individual birds with genomic and phenotypic information, which may be a challenge to achieve in the numerically small populations of indigenous chicken ecotypes. Therefore, the use of information across multiple populations emerges an attractive approach to increase the relevant numbers and the accuracy of genomic predictions. TN • We have characterized expression patterns of genes encoding each of the elongase and desaturase genes annotated in the Gallus gallus genome. These data provide an important foundation for understanding the LC n-3 PUFA synthesis pathway in chicken. • We have developed an approach that complements QPCR and RNAseq and allows efficient expression profiling of multiple genes in large sets of samples.   • Next year we will utilize the targeted RNAseq platform to identify pathways that are regulated by EPA and DHA  during broiler chick, adipose and muscle development.   TX (Athrey) • Identification of the structural variants that are associated with wooden breast myopathy in broilers • Knowledge of microbiota acquisition and structure in chicken in early life as a function of the photoperiods they are raised under • Creation and curation of a database on structural variants unique to commercial chicken varieties. TX (Walzem) Genetic methods used to modify mammals do not work for a major branch on the tree of life: birds. The tremendous insight into mammalian biology, including human health, arising from genetic modification of animal models such as mice persuades that great benefit would develop if methods for genetic modification of birds were readily available. Birds are crucial contributors to ecosystems, the source of basic biological insights; have many utilities including food, vaccine production, and recreation. However, aviculture and bird conservation face increasing challenges, especially from disease. Advances in methods for work with bird “germ lines” have reached a tipping point and drive our goal to establish robust technical protocols for gene editing in all types of birds. Global, methods to manipulate primordial germline cells (PGCs) in birds and transfer them to host embryonic gonads prior to incubation coupled with education and training opportunities for students and scientists will serve to lower the entrance bar for use of gene modification technologies in avians for biologic discovery. VA • The identification of Muc2 mRNA expressing cells in the crypt may represent pre-goblet cells that have started to differentiate but have not yet migrated out of the crypts. Understanding the development of goblet cells is important for the production of the mucus layer that protects the intestinal epithelial cells from pathogens.   • Delayed access to feed affects the number and distribution of goblet cells, which may result in a decrease in the mucus layer and an increase in the risk of infection from pathogens.   • Since graduate students (and impactful undergrads) continue to represent a significant expense item in research, our approach of research and science education may be of interest to others since we are now in our 17th year, 85% matriculation rate of our PREP, and since November 2019, 68 PhD completions. Research in our lab focused on genotypic-phenotype relationships also continued and would define a heritage strain useful for introgression as well as genes that may be responsible for differences in inflammation among turkeys. WI • This project generates turkeys with selenium status that ranges from Se-deficient to high-Se by feeding turkey poults a very low Se basal diet supplemented with graded levels of Se, and then analyzes tissues for selenoenzyme activity and transcript expression. These studies show that the dietary selenium requirement of the young turkey poult should be raised to 0.4 µg Se/g as inorganic selenium, and that the turkey is resistant to high dietary Se. These studies further indicate that the FDA limit of dietary selenium supplementation could be safely raised to 0.5 µg Se/g as inorganic selenium, at least for young turkey poults. • There are, however, no good biomarkers for excess Se and toxic Se status. Because turkeys appear to be more resistant to Se toxicity, we focused on assessing transcripts in turkeys to identify molecular biomarkers for high Se status. We found, however, few transcript changes indicating that turkeys do not alter gene expression as a homeostatic mechanism to adapt to high Se. • Because turkeys fed high Se accumulate Se in the liver without raising selenoprotein levels, we now are focusing on the nature of the selenometabolites. Using HPLC-MS, we found that turkeys accumulate Se in liver as a unique selenosugar which is bound both to low molecular weight thiols but is also bound to general cysteine-containing proteins. These species have potential as biomarkers to help to determine safe upper limits for dietary Se for turkeys, for other production animals, and for humans.

Date of Annual Report: 04/23/2021

Report Information

Annual Meeting Dates: 02/23/2021 - 02/24/2021
Period the Report Covers: 01/01/2020 - 12/31/2020

Participants

Institutional Stations (Institutional Abbreviation: Members)
Beckman Research Institute at the City of Hope (COH1: M. Miller1,2,3), Cornell University (CU: P. Johnson), Iowa State University (IA1: S. Lamont1,2,3, J. Dekkers1), Michigan State University (MI1: G. Strasburg1,2,3), Mississippi State University (MS: C. McDaniel, B. Nanduri1,2,3), North Carolina State University (NC: C. Ashwell1,2,3, J. Petitte), Pennsylvania State University (PA: A. Johnson, R. Ramachandran), Royal Veterinary College (RVC1: A. Psifidi1,2), Texas AgriLife Research (TX1: G. Athrey1,3, R. Walzem1,3), University of Arizona (AZ1: F. McCarthy1,2,3,S. Burgess), University of Arkansas (AR1: A. Alrubaye1, W. Kuenzel1,3, B. Kong1,2,3, D. Rhoads1), University of California, Davis (CA1: M. Delany2,3, H. Zhou1,2,3), University of Delaware (DE1: B. Abasht1,3), University of Florida (FL1: M. Edelmann), University of Georgia (GA1: S. Aggrey1,3) University of Maryland (MD: T. Porter, J. Song), University of Minnesota (MN: K. Reed2,3) University of Tennessee (TN1: B. Voy1,3), University of Wisconsin (WI1: G. Rosa1,2,3, R. Sunde1), USDA-ARS-Avian Disease and Oncology Lab (ADOL1: H. Cheng1,2,3, H. Zhang) , Virginia Tech (VA1: E. Wong1,2,3, E. Smith), Western University (WU1, Yvonne Dreshler1,2).
1 Submitted written report
2 Presented at the workshop
3 Attended business meeting

Brief Summary of Minutes

Abridged version of minutes from annual business meeting

Welcome, introductions, review of the agenda (Sue Lamont)–

Briefed the group about the importance of a concise annual report that clearly illustrates how NC1170 facilitates productive, cooperative research activities.

Prompted members to begin thinking about the next proposal and noted that responsibility to write the proposal should be rotated among members. Noted that objectives will need to be tweaked to be more contemporary, while retaining the focus on understanding genetics.

NRSP8 Update (Huaijun Zhou):

Informed group that seed funds are available to support very small projects; e.g., have invested in creation of stable cell lines that will be available to members

Encouraged members to seek support for preliminary data generation.

Bioinformatics (Fiona McCarthy):

Noted that they are still providing a curated QTLs database.

Funds support other resources, including OMIA (Online Mendelian Inheritance in Animals), and noted that DBSNP stopped supporting animal SNPs.

USDA Update (Frank Siewerdt, NIFA Representative):

Sees a couple of trends in funding: 1) Insuring support for underrepresented scientist groups, especially for new investigators; 2) Movement toward integrated projects; integration needs to be meaningful for both teaching and extension.

There is a push against good science that never leaves the shelf.

Group Moved to hold next annual meeting in conjunction with PAG in San Diego:

Accomplishments

Overview: Across stations, significant research progress was in the areas of 1) the genetic and functional basis disease resistance mechanisms, 2) heat stress and responses to climate change, 3) genetics and biology of muscle disorders in chicken and turkeys, 4) novel genomic resources for annotation and gene editing, 5) gut microbiota of poultry, and 6) development of new computational tools and approaches.   Unique project-related findings:             Demonstrated that marker assisted selection against ascites is effective and does not affect production traits.             Improved assembly, nomenclature, and functional annotation of the chicken genome             Developed genetic panel to assist in breeding lines of chickens that are more resilient chickens to hot climate for production in developing countries             Developed a novel chicken cell line for in vitro studies             Identified genes and pathways associated with susceptibility to viral infection   Summaries: <ul> <li>IA, ADOL and TAES stations maintained research populations to serve as resources for identifying genes, genetic elements and genomic regions of economic importance; as well as defining unique aspects of chicken genomic architecture. Active collaborations utilizing ISU chicken genetic lines or biological materials include H Zhou, UC- Davis (NDV and heat-stress response). TAES maintained flock of Greater Prairie Chickens for fertile egg production with the aim of isolating primordial germ cells for use in gene editing studies.</li> <li>Multiple groups are involved in investigating the muscle biology of chicken, focusing on wooden breast disorder, including DE, TAES, AR, and AZ stations, while MN and MI stations focused on turkey breast muscle development and satellite cells.</li> <li>Various groups carried out projects focusing on the genetics of infectious diseases in poultry. VA and GA investigated physiological changes during Eimeria maxima RVC investigated host genomes and microbiomes to develop complementary strategies to control Campylobacter infection. COH is developing genetic maps of the MHC region. ADOL investigated the underlying genetic and epigenetic factors to control MDV. AR studied BCO and lameness in broilers. CA and IA stations are working together on enhancing resistance to NDV virus.</li> <li>Heat stress is an important issue facing the poultry industry and there is intensive research on this topic among NC1170 members. MI and MN stations are focusing on muscle development and thermal challenge in turkeys, whereas CA and IA are focusing on heat-stress in chicken. GA studied effect of heat stress on sexual development of Eimeria maxima</li> </ul>  Outputs and ACTIVITIES: Publications: NC1170 members published 85 reports, and peer-reviewed articles and 8 book chapters in the year 2020. Additionally, six graduate and one undergraduate theses/dissertations were compiled in 2020. See Appendix for complete list, including those representing collaborations across members and entities of NC1170. Funds: The NC1170 committee leveraged external funding of $29,210,126 from funders such as NIFA, NSF and private industry, with much of that amount resulting from collaborations among stations. This is likely an underestimate as numbers were not reported by all members. Workshop: Members from four collaborating institutions organized and managed the group’s annual poultry workshop, which was delivered virtually for the first time due to the COVID pandemic.  Milestones: not applicable

Publications

Impact Statements

The impact of this group’s collective efforts toward genetic improvements in poultry are reflected in ongoing improvements in genome annotation and in tools that facilitate the use of genetic information. The annotation of regulatory elements in livestock species continues to be a core effort and generates new knowledge for chicken as well as for swine and cattle. These efforts impact stakeholders across several industries. The development of new statistical methods for genomic prediction is directly improving livestock breeding (WI) and the development of new Neural Networks for body weight prediction promise to usher in a new era of machine learning in animal breeding. In this year, group members also moved the field forward on the topic of muscle myopathies in broilers and turkeys. These conditions have both economical and food security significance and the new knowledge will benefit poultry producers, primary breeders, and consumers.

Date of Annual Report: 02/16/2022

Report Information

Annual Meeting Dates: 01/08/2022 - 01/09/2022
Period the Report Covers: 01/01/2021 - 12/31/2021

Participants

Abdulkarim Shwani, University of Arkansas/ Fayetteville
Adnan Alrubaye*, University of Arkansas
Behnam Abasht*, University of Delaware
Bindu Nanduri*, Mississippi State University
Brandi Sparling, Western University of Health Sciences
Brynn Voy*, Univ of Tennessee
Byungwhi Kong*, University of Arkansas
Carl Kroger, Purdue University
Claire Prowse-Wilkins, Agriculture Victoria
Dailu Guan, UC Davis
Douglas Rhoads*, University of Arkansas
Duc Lu American Angus Association
Ellie Duan, Cornell
Eric Wong*, Virginia Tech
Fiona McCarthy, University of Arizona
Francoise Thibaud-Nissen, NCBI
Gale Strasburg*, Michigan State Univ
Giri Athrey*, Texas A&M University
Hans Cheng*, USDA, ARS
Hao Cheng, UC Davis
Huaijun Zhou*, UC Davis
Jack Dekkers*, Iowa State University
Janet Fulton, Hy-Line International
Jiuzhou Song*, University of Maryland
Kent Reed*, University of Minnesota
Liqi An, UC Davis
Max Rothschild, Iowa State University
Michael Kaiser, ISU
Minjeong Kim, University of Tennessee
Oladipupo Ridwan Bello, University of Maryland
Paul Doran, Twist Bioscience
Pengxin Yang, ISU
Roger Sunde*, University of Wisconsin
Ronald Okimoto, Cobb-Vantress Inc.
Sammy Aggrey*, University of Georgia
Sébastien Guizard, The Roslin Institute
Susan Lamont*, Iowa State University
Tae Hyun Kim, Penn State University
Theros Ng, Western University of Health Sciences
Tom Porter*, University of Maryland
Usuk Jung, University of Tennessee
Vamsi Kodali, NCBI
Wayne Kuenzel*, University of Arkansas
Ying Wang, UC Davis
Yvonne Drechsler*, Western University of Health Sciences
Zhangyuan Pan, UC Davis
Ziqing Wang, University of Delaware
* designates NC1170 Members

Brief Summary of Minutes

Abridged version of minutes from annual business meeting

Welcome, introductions, review of the agenda (Sue Lamont, Administrative Advisor)–

F. Siewerdt, NIFA rep, was unable to attend and distributed report to the committee in advance

Reviewed the process by which new members can join the group; discussed this in light of the need to recruit new members

Reminded the group of the three types of reports to be submitted over the course of the year:
- SAES442 – required; based on the questionnaire that documents collaborative activities among group members and stations
- REEport – describes individual station activities; required by local experiment stations
- Station report – a self-imposed requirement of this group
  - Group discussed the usefulness of this report; noted that some stations never submit; collective feeling is that it is helpful and should be continued
- Prompted members to roles, responsibilities, and plan for writing the next proposal
  - Discussed objectives – need to be modified to be more contemporary
  - Writing team established: Yvonne Drecshler, Fiona McCarthy, Bindu Nanduri, Hans Cheng, Huaijun Zhou, Giri Athrey, and Brynn Voy; 2-3 group members per objective, with an overall coordinator
  - Issues, justification, new objectives sections need to be uploaded by October 15 but should be done sooner. Once this step is complete, the invitation to participate will be submitted to the experiment stations.
  - Sue has a list of writing instructions and will send to the writing team.

NRSP8 Update (Huaijun Zhou):

Informed group that seed funds are available to NRSP-8 members to support very small projects

Encouraged NRSP-8 members to seek support for preliminary data generation.

Group Moved to hold next annual meeting in conjunction with PAG in San Diego. Members also discussed the advantages of having the option to attend the meeting virtually.

Accomplishments

Overview: Across stations, significant research progress was in the areas of 1) the genetic and functional basis disease resistance mechanisms, 2) heat stress and responses to climate change, 3) genetics and biology of muscle disorders in chicken and turkeys, 4) novel genomic resources for annotation and gene editing, 5) gut microbiota of poultry, and 6) development of new computational tools and approaches   Short-term Outcomes: None to report   Outputs:  <ul style="list-style-type: circle;"> <li>Unique project-related findings <ul> <li>Identified circulating proteins and fatty acids that are diagnostic markers of ascites</li> <li>Identified circulating metabolites that are markers of incidence of breast muscle myopathies</li> <li>Functionally annotated gene families encoding novel deubiquitinase enzymes in the chicken genome</li> <li>Identified traits that can be modified throuhg selective breeding to improve disease resistance</li> <li>Established high heritability of breast muscle myopathies and identified QTL and candidate genes associated with susceptibility</li> <li>Defined the transcriptional effects of dietary selenium in turkey liver</li> <li>Characterized the role of host defense peptides in the yolk sac for their role in susceptibility to pathogens</li> <li>Established routine ability to isolate and culture primordial germs cells from chickens</li> <li>Improved workflows for predicting lncRNA and miRNA functions in chicken</li> </ul> </li> <li>Publications <ul> <li>Members published 57 peer-reviewed journal articles, including those co-authored collaboratively by members of multiple stations within this group,as well as 3 book chapters (see attached publication list)</li> </ul> </li> <li>Workshop <ul> <li>The annual meeting was organized and held virtually, allowing participants to share their work with both members and non-members in attendance.</li> </ul> </li> <li> Education and workforce training <ul> <li>Collectively, members of NC1170 trained numerous undergraduate and graduate students, postdoctoral fellows, and junior and visiting scientists during this reporting period.</li> </ul> </li> <li>Funding <ul> <li>Not all members reported funding. However, based on those who did report, membership continues to leverage the activities and collaborations of this group to garner significant external funding.</li> </ul> </li> </ul> Activities: <ul style="list-style-type: circle;"> <li>IA, ADOL and TAES stations maintained research populations to serve as resources for identifying genes, genetic elements and genomic regions of economic importance; as well as defining unique aspects of chicken genomic architecture.  TAES maintained flock of Greater Prairie Chickens for fertile egg production with the aim of isolating primordial germ cells for use in gene editing studies.</li> <li>DE, RVC, AK, CA conducted genome-wide association studies to identify loci associated with susceptibility to disease, stress tolerance, and composition of the gut microbiome.</li> <li>TN, WI used RNAseq to enhance functional annotation of the chicken and turkey genomes.</li> <li>AZ and CA developed and applied tools to further identify regulatory regions of poultry genomes.</li> </ul>

Publications

Impact Statements

NC1170 members improved the functional annotation of poultry genomes, which is critical for efforts to use genetics to maintain cost-effective poultry production and thus the supply of a key source of affordable dietary protein worldwide.

Date of Annual Report: 03/20/2023

Report Information

Annual Meeting Dates: 01/14/2023 - 01/15/2023
Period the Report Covers: 01/03/2022 - 01/15/2024

Participants

Sue Lamont
Adnan Alrubaye
Doug Rhoads
Eric Wong
Huaijun Zhou
Wayne Kuenzel
Brynn Voy
Fiona McCarthy
Giri Athrey
Yvonne Dreschler
Nisha Pillai
Darrell Kapzynski
Wes Warren
Behnam Abasht
Samuel Aggrey
Jiuzhou Song
Tom Porter
Gale Strasburg
Kent Reed
Bindu Nanduri
Hans Cheng

Brief Summary of Minutes

NIFA rep Angilca Van Goor National Program Leader in the division of Animal System, 2^nd level rep. Frank was unable to come due to a flight cancellation
- No notes from Frank to relate to this group.
- The new AFRI RFA was supposed to be released last month. It was held up in the chain of command. The RFA will be released in 2 months.
- The next RFA will be for 2 years and they expect it to be for 2 years moving forward. The RFA will be released in the next 2 months, and it will have information for 2023 and 2024.
- AFRI got an increase of $10 million dollars for a total of $455 million. We expect flat funding.
- The dual purpose dual benefit is only applicable to large animals.

Sue Lamont report:
- Quick assessment: Great project productivity; thank you to Brynn. Timeline, experimental station directors are reviewing the proposal expect minor revisions. revisions are due June 01^st; expect approval by mid-July. Look for that information in mid-July.
- We have lost 11 members compared to last cycle. Some members were inactive, changed from 37 to 26.
- The experimental station director will help. We need to encourage scientists to apply and become members. We welcome relevant members who are not members in experimental stations. Send the experimental station leader an email and let them know that there is a new NC1170 project and that you want to join.
- Local units may have funding, and some may have no funding If you are a member, some experimental stations may direct some multistate money to members.
- Election of next secretary for NC1170: Chair Adnan, Secretary: Yvonne selected.
- Kent Reed NRSP8: the project meeting is this afternoon. Plan is to have another NRSP8 meeting in PAG. One of the major changes is that it won’t be the same project. The organizational structure will focus on the three aims. More in the meeting this afternoon. NRSP8 funding available for junior scientists and students.
- Voting on NC1170 meeting location. Meeting next year will be at PaG over the weekend.

Accomplishments

<ul> <li>Dr. Lamont collaboration with Drs. Zhou, UC-Davis; Abasht and C. Schmidt from the U Delaware resulted in the identification of genomic regions associated with response of indigenous African chickens to Newcastle disease virus.</li> <li>Zhou collaboration with ADOL and ISU, H.H. Cheng, H. Cheng, J. Dekkers, M. Delany, C. Ernst, J. Fulton, S.J. Lamont, Cooperating scientists: R. Gallardo, T. Kelly, A. Muhairwa, P. Msoffe, B. Kayang, A. Naazie. S. Aggrey, R. Hawken, R. Okimoto, L. Fang, N. Yang, X. Hu resulted in identifying genetic variants associated with economically important traits in chickens and can be used to genetically improve productivity and poultry health.</li> <li>Fiona McCarthy, University of Arizona; and Dr. Wesley Warren, University of Missouri research collaboration resulted in the characterization and annotation of immune cells and immunoglobulin-like receptors in the chicken genome that contribute to disease resistance.</li> <li>Collaborative work between Dr. Malheiros of the North Carolina State University and his multistate partners resulted in four projects.</li> <li>Dr. Strasburg of the Michigan Station collaborated with Dr. Kent Reed of the Minnesota station and Dr. Sandra Velleman from Ohio State University.</li> </ul>

Publications

Walugembe, M., Naazie, A., Mushi, J.S., Akwoviah, G.A., Mollel, E., Mang'enya, J.A., Wang, Y., Chouicha, N., Kelly, T., Msoffe, P.L.M. Otsyina, H.R., Gallardo, R.A., Lamont, S., Muhairwa, A.P., Kayang, B.B., Zhou, H., Dekkers, J.C.M. 2022. Genetic analyses of response of local Ghanaian and Tanzanian chicken ecotypes to a natural challenge with velogenic Newcastle disease virus. Animals 12:2755. doi.org/10.3390/ani12202755 Guan D, Halstead MM, Islas-Trejo AD, Goszczynski DE, Cheng HH, Ross PJ, Zhou H. Prediction of transcript isoforms in 19 chicken tissues by Oxford Nanopore long-read sequencing. Front Genet. 2022 doi: 10.3389/fgene.2022.997460.  Botchway ,P.K., Amuzu-Aweh, E.N., Naazie, A., Aning, G. K., Otsyina, H.R., Saelao, P., Wang, Y., Zhou, H., Walugembe, M., Dekkers, J., Lamont, S.J., Gallardo, R.A., Kelly, T.R., Bunn, D. and Kayang, B.B. 2022. Host response to successive challenges with lentogenic and velogenic Newcastle disease virus in local chickens of Ghana. Poultry Science 101:102138. doi.org/10.1016/j.psj.2022.102138. Walugembe, M., Naazie, A., Mushi, J.S., Akwoviah, G.A., Mollel, E., Mang'enya, J.A., Wang, Y., Chouicha, N., Kelly, T., Msoffe, P.L.M. Otsyina, H.R., Gallardo, R.A., Lamont, S., Muhairwa, A.P., Kayang, B.B., Zhou, H., Dekkers, J.C.M. 2022. Genetic analyses of response of local Ghanaian and Tanzanian chicken ecotypes to a natural challenge with velogenic Newcastle disease virus. Animals 12:2755. doi.org/10.3390/ani12202755 Botchway ,P.K., Amuzu-Aweh, E.N., Naazie, A., Aning, G. K., Otsyina, H.R., Saelao, P., Wang, Y., Zhou, H.,  Walugembe, M., Dekkers, J., Lamont, S.J., Gallardo, R.A., Kelly, T.R., Bunn, D. and Kayang, B.B. 2022. Host response to successive challenges with lentogenic and velogenic Newcastle disease virus in local chickens of Ghana.  Poultry Science 101:102138. doi.org/10.1016/j.psj.2022.102138  Tudeka, C.K., Aning, G.K., Naazie, A., Botchway, P.K., Amuzu-Aweh, E.N., Agbenyegah, G.K., Enyetornye, B., Fiadzomor, D., Saelao, P., Wang, Y., Kelly, T.R., Gallardo, R., Dekkers, J.C.M., Lamont, S.J., Zhou, H., and Kayang, B.B. 2022. Response of three local chicken ecotypes of Ghana to lentogenic and velogenic Newcastle disease virus challenge. Tropical Animal Health and Production 54:134. doi.org/10.1007/s11250-022-03124-8  Lake, J.A., Yan, Y., Dekkers, J.C., Qiu, J., Brannick, E.M. and Abasht, B., 2022. Identification of circulating metabolites associated with wooden breast and white striping. PloS one, 17(9), p.e0274208. <a href="https://doi.org/10.1371/journal.pone.0274208">https://doi.org/10.1371/journal.pone.0274208</a> Smith, J. M. Alfieri, N. Anthony, P. Arensburger, G. N. Athrey, J. Balacco, et al.: “Fourth Report on chicken genes and chromosomes.” Cytogenet Genome Res. 2023. DOI: 10.1159/000529376. Christina L. Swaggerty, Ramon D. Malheiros, Ludovic Lahaye, Hector Hernandes Salgado, J. Allen Byrd II, Kenneth J. Genovese, Haiqi He, Elizabeth Santin, Michael H. Kogut. Addition of a protected complex of biofactors and antioxidants to breeder hen diets confers transgenerational protection against Salmonella enterica serovar Enteritidis in progeny chicks. Poultry Science (accepted, in press). Carvalho, L.C., Malheiros, D.,Lima, M.B., Mani, T.S.A., Pavanini, J.A., Malheiros, R.D., Silva, E.P. Determination of the Optimal Dietary Amino Acid Ratio Based on Egg Quality for Japanese Quail Breeder, Agriculture, 13, 173. Malheiros, R.D., V. M. B. Moraes, K. E. Anderson, F. L. S. Castro, and J. E. Ferrel. Influence of dietary dacitic tuff breccia on laying hen performance and egg quality parameters and bone structure at 85 weeks of age after a non-anorexic molt program at 73 to 77 weeks. 2022, Poultry Science 101:101718. Reed KM, Mendoza KM, Xu J, Strasburg GM, Velleman SG. Transcriptome Response of Differentiating Muscle Satellite Cells to Thermal Challenge in Commercial Turkey. Genes (Basel). 2022 Oct 14;13(10):1857. doi: 10.3390/genes13101857. PMID: 36292741 Reed KM, Mendoza KM, Strasburg GM, Velleman SG. Transcriptome response of proliferating muscle satellite cells to thermal challenge in commercial turkey. Frontiers in Physiology. 2022. Aug 25;13:970243. doi: 10.3389/fphys.2022.970243. PMID: 36091406 Xu J, Strasburg GM, Reed KM, Velleman SG. Thermal stress and selection for growth affect myogenic satellite cell lipid accumulation and adipogenic gene expression through mechanistic target of rapamycin pathway. Journal of Animal Science. 2022 Aug 1;100(8):skac001. doi: 10.1093/jas/skac001. PMID: 35908789. Xu J, Strasburg GM, Reed KM, Velleman SG. Temperature and Growth Selection Effects on Proliferation, Differentiation, and Adipogenic Potential of Turkey Myogenic Satellite Cells Through Frizzled-7-Mediated Wnt Planar Cell Polarity Pathway. Frontiers in Physiology. 2022. May 23;13:892887. doi: 10.3389/fphys.2022.892887. PMID: 35677087 Xu J, Strasburg GM, Reed KM, Velleman SG. Thermal stress affects proliferation and differentiation of turkey satellite cells through the mTOR/S6K pathway in a growth-dependent manner. PLoS One. 2022 Jan 13;17(1):e0262576. doi: 10.1371/journal.pone.0262576. PMID: 35025965.

Impact Statements

Understanding role of early heat or cold stress on gene expression in the turkey hatchling from slow-growing and modern commercial turkey lines. We investigated the effects of exposure of satellite cells, which are progenitors of muscle cells in the turkey hatchling, to hot or cold temperatures.