Abstract
Eggshell damages lead to economic losses in the egg production industry and are a threat to human health. We examined 49-wk-old Rhode Island White hens (Gallus gallus) that laid eggs having shells with significantly different strengths and thicknesses. We used HiSeq 2000 (Illumina) sequencing to characterize the chicken transcriptome and whole genome to identify the key genes and genetic mutations associated with eggshell calcification. We identified a total of 14,234 genes expressed in the chicken uterus, representing 89% of all annotated chicken genes. A total of 889 differentially expressed genes were identified by comparing low eggshell strength (LES) and normal eggshell strength (NES) genomes. The DEGs are enriched in calcification-related processes, including calcium ion transport and calcium signaling pathways as reveled by gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis. Some important matrix proteins, such as OC-116, LTF and SPP1, were also expressed differentially between two groups. A total of 3,671,919 single-nucleotide polymorphisms (SNPs) and 508,035 Indels were detected in protein coding genes by whole-genome re-sequencing, including 1775 non-synonymous variations and 19 frame-shift Indels in DEGs. SNPs and Indels found in this study could be further investigated for eggshell traits. This is the first report to integrate the transcriptome and genome re-sequencing to target the genetic variations which decreased the eggshell qualities. These findings further advance our understanding of eggshell calcification in the chicken uterus.
Highlights
Calcified eggshells provide protection to egg contents, prevent contamination by microorganisms, and protect the embryo [1]
Identification of differentially expressed genes and novel isoforms The total number of reads varied from 37 to 71 million, and 78.09–80.72% of filtered reads were mapped on the reference chicken genome (Table C in S1 File)
This discrepancy did not significantly affect gene expression analysis because the RPKM values were corrected for the total number of read counts for each sample
Summary
Calcified eggshells provide protection to egg contents, prevent contamination by microorganisms, and protect the embryo [1]. Eggshells regulate the exchange of metabolic gases and water, and provide calcium to the embryo. Eggshells are composed of two shell membranes, a calcium carbonate layer, and a foamy layer of cuticle [3]. Mineralization of the avian eggshell is a complex process involving the precipitation of calcium carbonate that results from interaction between this mineral and the organic matrix during the nucleation and growth phases [4,5,6,7]. Calcification occurs on the shell membrane in the cellular uterine fluid, which contains the inorganic minerals and organic matrix precursors of the eggshell [8]. Calcification is the lengthiest phase of egg formation and occurs in three stages over a 6–22h period following ovulation
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