Abstract
Marek’s disease (MD) is a commercially important neoplastic disease of chickens caused by Marek’s disease virus (MDV), a naturally occurring oncogenic alphaherpesvirus. Selecting for increased genetic resistance to MD is a control strategy that can augment vaccinal control measures. To identify high-confidence candidate MD resistance genes, we conducted a genome-wide screen for allele-specific expression (ASE) amongst F1 progeny of two inbred chicken lines that differ substantially in MD resistance. High throughput sequencing was initially used to profile transcriptomes from pools of uninfected and infected individuals at 4 days post-infection to identify any genes showing ASE in response to MDV infection. RNA sequencing identified 22,655 single nucleotide polymorphisms (SNPs) of which 5,360 in 3,773 genes exhibited significant allelic imbalance. Illumina GoldenGate assays were subsequently used to quantify regulatory variation controlled at the gene (cis) and elsewhere in the genome (trans) by examining differences in expression between F1 individuals and artificial F1 RNA pools over six time periods in 1,536 of the most significant SNPs identified by RNA sequencing. Allelic imbalance as a result of cis-regulatory changes was confirmed in 861 of the 1,233 GoldenGate assays successfully examined. Furthermore we have identified seven genes that display trans-regulation only in infected animals and ∼500 SNP that show a complex interaction between cis- and trans-regulatory changes. Our results indicate ASE analyses are a powerful approach to identify regulatory variation responsible for differences in transcript abundance in genes underlying complex traits. And the genes with SNPs exhibiting ASE provide a strong foundation to further investigate the causative polymorphisms and genetic mechanisms for MD resistance. Finally, the methods used here for identifying specific genes and SNPs have practical implications for applying marker-assisted selection to complex traits that are difficult to measure in agricultural species, when expression differences are expected to control a portion of the phenotypic variance.
Highlights
Marek’s disease virus (MDV, Gallid herpesvirus 2) is a naturally occurring oncogenic alphaherpesvirus of chickens that targets lymphoid tissues like the bursa of Fabricius, thymus, and the spleen, which is an important lymphatic organ and repository of T and B cells
Genes with at least one single nucleotide polymorphisms (SNPs) displaying signs of allelic imbalance were further queried for an allele-specific response to infection as determined by a 2 × 2 factorial test, which examined the effect of disease status on allele-specific expression (ASE) rates
We found a significant impact of each SNP on the degree of allelic imbalance, which would be expected if a large number of these genes and the extent to which they are regulated explain some of the genetic resistance to MD
Summary
Marek’s disease virus (MDV, Gallid herpesvirus 2) is a naturally occurring oncogenic alphaherpesvirus of chickens that targets lymphoid tissues like the bursa of Fabricius, thymus, and the spleen, which is an important lymphatic organ and repository of T and B cells. MDV infects both T and B cells, which may result in lymphoid tumors, nerve lesions, and immunosuppression. Virtually every bird is exposed to MDV at a very young age. MD vaccines do not eliminate MDV infection, replication, or spread, the virus is believed to have evolved for increased virulence in response to vaccination (Witter, 1997). The lack of new vaccines and the expectation that the virus will evolve to overcome existing vaccines means the identification of genes involved in natural immunity is of major interest to the poultry industry
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