Investigation of genomic imprinting through allelic expression analysis of mRNA in chicken embryonic brain and liver

Abstract Text: Genomic imprinting refers to the epigenetic phenomenon that some autosomal genes are exclusively expressed from either the maternal or paternal allele whereas, based on Mendelian inheritance, expression of alleles are expected to be in equal amount and independent of their parental origin. DNA methylation in cis-acting manner is the major mechanism for genomic imprinting. Imprinted genes have been identified in several animal species and are frequently associated with embryonic growth and survival functions. Yet whether genomic imprinting exists in chickens is still debatable, as previous studies reported conflicting evidence regarding the topic. Albeit no genomic imprinting has been found in the chicken embryo as a whole, we investigated whether certain embryonic tissues exhibit genomic imprinting. In this study we interrogated the existence or absence of genomic imprinting in chicken embryonic brain and liver by examining the mRNA expression of parental alleles in an F1 generation. Eggs from two highly inbred chicken lines (Fayoumi and Leghorn) and their reciprocal crosses were collected and incubated for 12 days; then brain and liver were harvested from embryos for cDNA library preparation. To establish the genotypes of the inbred lines and the F1 hybrids and to minimize reference bias of RNA-Seq sequence alignment, genomic DNA from inbred Fayoumi and Leghorn chickens was pooled separately and each pool was sequenced at 20X coverage. The SNP loci identified from DNA-Seq data were masked to create a customized reference genome (based on Ensembl Galgal4) for RNA-Seq reads mapping. Of 65 million RNA-Seq reads per sample generated using the Illumina HiSeq 2000 sequencer, 88% were mapped to the customized reference genome. The genome-wide ratio of mapped reads containing reference allele was reduced by 1.5% when comparing with the results from the original reference genome. Our analyses indicated that genomic imprinting is absent in chicken 12-day embryonic brain and liver. In genome-wide and chromosome-wide scales, we observed a balanced expression of maternal and paternal alleles. About 9.2% of the heterozygous loci showed allele specific expression independent of their parental origin (binominal test, p value ≤ 0.05). Certain alleles showed consistent expression pattern across all 8 F1 individuals indicating possible presence of cis-acting regulatory mutations or epigenetic modifications influencing expression of these alleles.

Keywords: genomic imprinting, chicken, RNA-Seq