Genome-Wide Identification and Quantification of cis- and trans-Regulated Genes Responding to Marek’s Disease Virus Infection via Analysis of Allele-Specific Expression

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.

Transcriptional Profiling of MEq-Dependent Genes in Marek’s Disease Resistant and Susceptible Inbred Chicken Lines

“…Our group has previously demonstrated that Jak-STAT is an important cellular pathway involved in genetic basis of MD resistance [20,30]. Similarly, we also noted that genes in Jak-STAT pathway are transcriptionally up-regulated in susceptible line 7 and not in the resistant line 6.…”

“…However, it is worth noting transcript profiling using spleens and other organs suffers from the potential confounding influence of differences in cellular composition and reagents to properly quantify each cell subtype are limiting in chicken. Thus, the high degree of overlap between our current results, which utilized cultured cells from defined chicken lines, and those from our other studies that used actual birds [11,12,20,30] for the identification of critical pathways suggests that CEF are reasonable models. This agreement also suggests an important contribution of innate immunity towards MD genetic resistance.…”

“…These results suggest that the genes related to cell death and proliferation is one of the major determinants of genetic variability in resistance/susceptibility to MD. In one of our previous studies using RNA sequencing analysis, we have shown that apoptotic signaling is significantly overrepresented in spleens of birds infected with MDV [30]. …”

Genomic Variation between Genetic Lines of White Leghorns Differed in Resistance to Marek’s Disease

“…Host genetic resistance is considered as an attractive approach to augment the current control measure against MD [4,[13][14][15]. Host genetic resistance to MD was first noticed in the 1940s [16][17][18].…”

Fine mapping of QTL and genomic prediction using allele-specific expression SNPs demonstrates that the complex trait of genetic resistance to Marek’s disease is predominantly determined by transcriptional regulation

“…The second goal was to demonstrate the power and utility of using ASE to identify specific genes impacting disease and genetic resistance. Previously, we identified 4528 SNPs in 3718 genes that exhibit ASE in response to MDV infection using F 1 progeny from experimental inbred White Leghorn layer lines that differ greatly in MD genetic resistance [ 13 , 14 ]; lines 6 and 7 are MD resistant and susceptible, respectively. Here we show that these genes with cis-regulatory elements account for the majority of the genetic variation between these two experimental White Leghorn (egg laying) lines and verify our genomic predictions by creating lines differentiated for the identified cis-regulatory elements that demonstrate associated changes in MD resistance.…”