Brief Summary of Minutes of Annual Meeting

See attached minutes file for 2011 annual report.

NC-1170 Accomplishments 2011 Overall summary. The project has jointly (with NRSP-8) annually organized a very well-attended scientific session at the Plant and Animal Genomic meeting, which included invited international speakers and attracts many national and international scientists from outside of NC-1170, and including industry scientists. Project outputs have included the improvement of comprehensive genetic and physical maps for chicken and turkey; development and testing of novel techniques for gene transfer in chickens; greater insight into how genomic sequence variation affects phenotypic variation in poultry; and identification of QTL, specific genes and pathways associated with important biological traits in poultry. Research resources, such as poultry genetic lines and cells, and high-density SNP chips, have been developed and widely used across the stations. Examples of specific accomplishments in 2011 are listed below by project objective. Objective 1. Create and share data and technology to enhance the development and application of genomics and systems biology in poultry. MHC class I target recognition, immunophenotypes and proteomic profiles for natural killer cells and monocytes within the spleens of day-14 of chick embryos were characterized. The miRNA targeting of the 225 bp insert in BG1*R4 allele of BG1 were identified. Additional genes were mapped to chicken chromosome 16. We have expanded the core turkey MHC sequence; measured MHC haplotype diversity within the species; and quantified expression of genes in the expanded MHC by examining immune system tissues. To enhance sequence coverage of the unassigned portion of the turkey genome we have sequenced pools of BACs from the CHORI and Texas A&M turkey BAC libraries using next-gen sequencing. Specifically, this effort identified BACs with end sequences that physically mapped to either the ChrUN portion of the 2.01 genome build or to the Z chromosome. To investigate the response to aflatoxin exposure, we are using RNA-Seq approaches to characterize the transcriptome level changes in the liver of birds exposed to AFB1 with and without probiotics (Lactobacilli). An introgression line is being generated by simultaneously introgressing four QTL alleles from the Virginia low-line into a Virginia high-line background. The introgression is being performed by marker assisted back-crossing to the high-line. A panel of 40 SNP, closely linked to the 4 QTL, was developed. Four generations of introgression have been completed. To employ GWMAS to address social and ethical concerns while at the same time demonstrating the power and limitations of GWMAS in a multi-generational selection experiment, we utilized 2 broiler and 3 layer lines with different selection objectives, but primary among the traits for selection were leg angle in broilers and livability in layers. We have completed re-sequencing ADOL Lines 6 and 7 based on a pool of 6 individuals each, to 44X per line, on an ABI SOLiD machine at Purdue. The KGB and DXL lines have been completed. We have developed software to call SNPs, find LD, Fst, Fis, and Tajima D from sequencing data. This software is being used as a pipeline to find signatures of selection for a number of traits, including aggression and MD resistance. Microarray analysis in early- and late passage (passage 4 and 18, respectively) of primary CEF cells was performed with 4X44K chicken oligo microarray. A total of 2149 differentially expressed genes were identified. To elucidate cellular and molecular mechanisms of resistance to C. jejuni colonization in chickens, two genetically distinct broiler lines (Line A (resistant) colonized less by C. jejuni compared to Line B (susceptible)) werei noculated with wild type (WT) or mutant (MT) C. jejuni or PBS at day of hatch. Total RNA was isolated from the bursa of Fabricius harvested at 1, 4, 24, and 36 hrs post-inoculation and a chicken whole genome 44K array was used to profile the host innate response to C. jejuni. We presented scenarios and basic principles of how HTC can be used in genomic selection,implemented using various techniques from simple batch processing to pipelining in distributed computer clusters. The analysis in several in vitro cell systems indicated that ALT is operating within immortalized cells as per the key markers: absence of telomerase activity, heterogeneous telomeric DNA profiles, and presence of APB foci. Results suggest that the telomerase and telomere-recombination (ALT) pathways may coexist in chicken cells; however, depending on the cell type, one or the other mechanism may dominate. Analysis of MDV integration through a temporal profile of immune cells and tumors was completed. Developmental mutants were studied through targeted capture array/next generation sequencing. We also employed whole embryo in situ hybridization to assess the expression patterns of genes within the causative regions during the relevant developmental stages and locations to assist in prioritizing candidate genes and to learn more about gene expression during chicken development. A key cytogenomic finding of the chicken:turkey species comparison was the stability of the two genomes relative to each other, with a number of inversions and placement of the centromere more terminally in turkey chromosomes than the chicken orthologs. Study of the Japanese quail genome affirmed multiple NOR loci (three versus one in both turkey and chicken) and showed cytogenetic linkage of one of the NORs with the MHC. Electrophoretic mobility shift assays demonstrated that the isoforms differ in their ability to bind Pit-1 binding sites on the cGH promoter. From our results, we conclude that Pit-1 isoforms differ in their ability to activate the chicken GH promoter, and this may be due to differences in their DNA-binding abilities. Our results indicate that like in mice, Shh must be down-regulated in order to allow for OE evagination and RP formation and that a specific dosage of FGF8 is required for proper development of RP. By testing temporal transcriptome changes using three representative chicken lines with different resistance to MD, we identified 163 candidate genes for MD-resistance and 175 candidate genes for MD-susceptibility over the three time points. We utilized microarrays to screen miRNAs that were sensitive to Mareks disease virus (MDV) infection. QRT-PCR analysis confirmed the microarray data and revealed expression patterns of some miRNAs in tumor samples. Chicken miRNA gga-miR-15b, which is reduced in infected susceptible chickens and splenic tumors, controls the expression of ATF2 (activating transcription factor 2). ATF2 is significantly increased in the same group. Using immunoblotting technique we noted that chicken macrophages express a variety of DUBs, while our bioinformatic search identified only eight of these enzymes, demonstrating usefulness of this targeted proteomic technique in comparison to complementary bioinformatic tools that can be used alongside proteomics. We have developed and deployed Genome2Seq, a tool that rapidly looks up genome co-ordinates generated from RNA-Seq data and returns genes and Gene Ontology (GO) annotation when the co-ordinates map to annotated genes and a fasta sequence files when co-ordinates do not map to previously annotated genes. Two existing AgBase ID mapping tools (ArrayIDer and AffyIDer) are being reconfigured to handle a larger number of accession types and combined for ease of use. In addition to developing these tools we are also providing online support for functional modeling and access to biocomputing (including high performance computing) via request through AgBAse. We have developed guidelines for providing standardized gene nomenclature for chicken genes. We are liaising with NCBI to ensure that chicken nomenclature is displayed in NCBIs Entrez Gene database and are working to expand this collaboration to include eChickAtlas and Ensembl. Chickspress is being developed as a tissue specific compendium of gene expression for chicken gene products. Via BirdBase we are creating a comparative genome browser for bird genomes, initially including the three public bird genomes (chicken, turkey, zebra finch). To facilitate turkey genetics research and better understand avian genome evolution, a BAC-based integrated physical, genetic, and comparative map was developed for this important agricultural species. The turkey genome physical map was constructed based on 74,013 BAC fingerprints (11.9x coverage) from two independent libraries. We have obtained deep next generation sequence data for the DF1 and DT40 chicken cell lines. 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. Many unique experimental lines of poultry were developed, maintained and characterized; and shared among researchers. A non-cytolytic Clostridium alpha toxin vaccine, engineered to lack the cytolytic 28 amino acid domain in the middle of the polypeptide, was developed and tested to protect against turkey cellulitis. We combined trisomy mapping with array Comparative Genomic Hybridization (aCGH) to identify additional GGA 16 sequences among unassigned genomic sequence contigs. Working on adult chicken anatomy we have developed almost 700 terms (and definitions) to describe adult chicken musculoskeletal system and integument. Objective 3. Elucidate genetic mechanisms that underlie economic traits and develop new methods to apply that knowledge to poultry breeding practices. The expression of nutrient transporters and digestive enzymes in the yolk sac membrane and embryonic intestine were defined. Proteomic analysis of chicken breast muscle demonstrated differential protein expression with varied growth rate and water-holding capacity. Integration of nutritional phenotypes, duodenal transcriptomics and plasma metabolites in divergently selected broiler chickens (high vs. low feed efficiency lines) at 35 and 42 days of age demonstrated likely mechanisms linking feed efficiency phenotypes with their genotypes. We used qPCR to determine that reduced protein levels in sperm are not reflected at the level of the testis transcriptome. We performed RNAseq on testis RNA and over 3000 genes are significantly altered for expression, and we have identified SNPs in 300 of those genes that are differentially represented with respect to mobility phenotype. We have identified informative SNPs in HTR2B, UTS2D, and AGTR1. SNPs in the former three genes have been applied to different experimental lines and found to show strong association with ascites phenotype. Employing immobilization stress, and examining a second vasotocin receptor subtype, the VT4R, we have found that it participates in the avian stress response. Between HGFL and LGLL lines, in overlapping windows of 100 SNP along autosomes, we have identified several potential genomic targets of divergent selection and putative candidate genes subjected to divergent selection within these regions. High-throughput proteomic analysis in chicken lung with avian influenza virus infection was conducted by MASS spectrometry. Using simulation, it was found that RBF had better ability (higher predictive correlations and lower predictive mean square errors) of predicting merit of individuals in future generations in the presence of non-additive effects than a linear additive model, the Bayesian Lasso. We evaluated the effects of directional selection based on estimated genomic breeding values (GEBVs) for a quantitative trait. Comparison between a structural equation model (SEM) with the selected structure and a Multiple Trait Animal Model using deviance information criterion indicated that the SEM is more plausible. Coupling prior knowledge with the output provided by the inductive causation algorithm allowed further learning regarding phenotypic causal structures when compared to standard mixed effects SEM applications. Screening for allele-specific expression (ASE) in response to MDV infection, a simple and novel method was used for identifying polymorphic cis-acting regulatory (genetic) elements, which may contain strong candidate genes with specific alleles that confer MD genetic resistance. The DNA-binding sites and the genes that are directly regulated by Meq, the likely MDV oncogene, were defined. Sequence datasets of the genomes of lines 6 and 7 were further defined. A vaccine by chicken line interaction affecting vaccine protective efficacy was identified. One SNP accounting for more than 6% of the disease/non-disease variation in MD infection was determined. We have further characterized the functional role of the Wnt target gene Annexin A6 during neural crest cell development. A global microarray was used to measure gene expression in spleen and peripheral blood leukocytes (PBL) from the same 40 birds, either infected or not with E. coli. Birds showing severe pathology had a greater induction of gene response than repression. DAVID analysis identified many terms related to immune response and metabolic processes. In addition to the differentially expressed genes and networks identified in individual tissues, the combined bi-analysis of two tissues from the same birds gave insights into potential systemic signaling between tissues. TLR15 RNA expression was found to be increased in response to APEC challenge in the bursa of broilers. Heat-stressed chicks were found to have significantly lower blood hemoglobin, hematocrit, carbon dioxide partial pressure, and higher pH and saturated oxygen, than control chicks. There were several interactions of genetic line with heat treatment effects. A total of 65 single nucleotide polymorphisms (SNPs) were identified in chicken FTO, and 18 tested SNPs were significantly associated with traitsof body weight, body composition and fatness. A high-density SNP study detected a number of genomic regions showing association with egg defects, some of which explain a sizeable proportion of genetic variance. A QTL region on chromosome 4 was found to explain a large proportion of the genetic variance for the mean (30%) and uniformity (up to 16%) for egg weight. A high-density SNP study of markers associated with body weight (BW) and hen house production (HHP) showed that both traits are highly polymorphic with no major QTL. A steady decline of accuracy was observed as the number of generations with low-density (LD) genotyped dams increased, which suggests that some dams may need to be strategically high-density (HD) genotyped to maintain accuracy close to that achieved using HD platforms. Comparison of the accuracy of genotype imputation using different methods suggested that a combined approach using the best features of the methods may be needed to optimally utilize the information and maximize accuracy within defined computing resources. We continue to provide training, outreach and support for poultry researchers via AgBase to ensure that they are able to better leverage their functional genomics data to understand key economic traits for poultry.

See attached file.