Brief Summary of Minutes of Annual Meeting

See attachment below for full annual report.

Objective 1:

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 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.

COH

• 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).
• 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).
• 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.
• 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.
• 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.

Evaluating MHC-Y haplotypes in experimental Campylobacter colonization trials (Zhang, Goto, Psifidi, Stevens, and Miller).

• 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.

IA

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.)

MN

Additional RNAseq datasets compiled for the turkey have been accessioned in NCBI’s Gene Expression Omnibus (GEO) repository. 


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.  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.

VA

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.

WI-Madison

 1. Causal phenotypic networks for egg traits in an F2 chicken population

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.

2. Effect of quality control, density and allele frequency of markers on the accuracy of genomic prediction for complex traits

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:

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, and the new buildings that were built did not follow ADOL specifications, thus, require significant and costly renovations.

IA

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).

TX -Athrey

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

TX- Walzem

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:

ADOL

3.2.a. Characterizing the genomic landscape of Marek’s disease virus-induced lymphomas

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.

3.2.c. 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.

3.3.d. Marek’s disease vaccines-induced differential expression of microRNAs in primary lymphoid organ bursae.

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.

AR

3.1 Identification of genes affecting ascites susceptibility

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.

3.2 Bacterial chondronecrosis with osteomyelitis and lameness in broilers

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.

3.3 Broiler breast muscle and myopathies

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.

3.4 Genes and proteins involved in the stress response of broilers

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).

COH

PCR-based method for MHC-Y genotyping (Miller, Goto, Zhang).

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).

CA

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.

3.3.2 Knockout of transcription factor IRF7 reveals its novel role on modulating host response against avian influenza virus

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.

DE

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.

GA

Dietary methionine levels alter digestibility and gene expression of amino acid transporters

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

Host Genotype and Emeria acervulina infection: metabolomics

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.

A Weighted Genomic Relationship Matrix Based on FST Prioritized SNPs for Genomic Selection

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%.

IA

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.

MI

Influence of thermal challenge on turkey muscle development and meat quality.

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.

Related studies

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.

MN

Influence of thermal challenge on turkey muscle development and meat quality.

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.

Genomics to increase aflatoxin resistance in turkeys.

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.

Antibiotic-free alternatives to improve health and performance in commercial turkeys

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).

Related studies

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.

NY

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