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ARS Home » Southeast Area » Athens, Georgia » U.S. National Poultry Research Center » Endemic Poultry Viral Diseases Research » Research » Research Project #434384

Research Project: Genomic Screens to Identify Regulatory Elements with Causative Polymorphisms Accounting for Marek’s Disease Genetic Resistance in Chicken

Location: Endemic Poultry Viral Diseases Research

Project Number: 6040-31320-010-05-I
Project Type: Interagency Reimbursable Agreement

Start Date: Jul 1, 2018
End Date: Jun 30, 2021

Objective:
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 approximately $2 billion per year. Despite control measures including biosecurity and MD vaccines, new and more virulent Marek's disease virus (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. In this submission, utilizing and integrating genomic techniques that identify chromatin marks for promoters and enhancers, and ribonucleic acid (RNA) sequencing 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 using MDV that lacks Meq (MDV EcoQ fragment), the viral oncogene and a 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. If successful, the resulting information will be combined with our existing information and ongoing experiments, which should further increase the accuracy of genomic selection for enhanced MD genetic resistance when applied to commercial flocks, plus provide a significant increase in fundamental biological knowledge on gene regulation in chicken for MD genetic resistance and pathology.

Approach:
Objective 1 is our major effort and is designed to identify candidate cis-regulatory elements that account for the allele-specific expression (ASE) in response to Marek's disease virus (MDV) infection. To achieve this goal, we will collect splenic-derived lymphocytes from uninfected and MDV-infected line 6 (resistant) x line 7 (susceptible) F1 birds; these are the same experimental genetic lines used to identify 83% of the genetic variation for MD resistance determined previously. Then the resulting samples will be subjected to several genomic screens and RNA sequencing, and the resulting large datasets integrated to identify promoter tethered regions (PTRs), which should identify promoters and enhancers for each expressed gene. In other words, the goal is to identify and connect regulatory elements (promoters and enhancers) to each expressed gene, and ideally to address whether there are PTRs specific to MDV infection or an allele of a gene. Promoter and enhancer regions for all the genes will be screened for specific sequence motifs and candidate transcription factors (TFs). If successful, for the genes showing ASE in response to MDV infection, we will identify sequence motifs and candidate TFs that may reveal the causative mutations. Objective 2, addressing a concern of prior submissions regarding the role of MDV in the context of the entire virus, we repeat the same studies in Objective 1 using MDV delta Meq, a recombinant virus that lacks both copies of Meq. While not our major goal, this information should help to interpret promoter-enhancer interactions, especially those thought to involve Meq in Objective 1 as there will be no Meq present or expressed from this virus. And as MDV delta Meq is a candidate MD vaccine, these results may inform on genes and pathways involved in the immune response that promote protection. Objective 3 also addresses a prior concern, which is to translate our results to commercial chickens. To begin to do so, using the results from Objective 1 and 200 progeny tested commercial layer (egg-type birds) males for MD genetic resistance, we will sequence regulatory regions for 10 genes showing ASE and the highest association with MD resistance. If the proposed TF-binding sites are causal, then we should identify sequence polymorphisms in these regions as it is likely that the same biological pathways are being used when responding to viral infection. Furthermore, we will test if the called genotype for the TF-binding site is or is not in perfect linkage disequilibrium (LD) with the original ASE SNP, which will help inform on whether this additional information (i.e., the putative causative mutation) might provide increased accuracy in genomic predictions compared to linked ASE single nucleotide polymorphisms (SNPs).