Fig 1.
Graphical representation of the experimental strategy.
The oviposition cycle of all birds was measured daily, and birds were slaughtered 18 h after ovulation. This stage generally represents the fast growth stage during the eggshell calcification. Individuals from the same sire family that laid eggs with one of two extremely different eggshell qualities were selected. We selected eight birds with normal eggshell strength (NES) and eight birds with very low eggshell strength (LES) for further study. Two biological replicate samples from each group were selected for transcriptome sequencing, and one pooled DNA library for each group was made for whole genome re-sequencing. Finally, we integrated transcriptome and whole genome re-sequencing data to discover the genetic information related to eggshell quality.
Fig 2.
The eggshell strength (A) and eggshell thickness (B) in the normal eggshell strength (NES) group and the low eggshell strength (LES) group.
There are 154 and 159 individuals for the NES group and LES group, respectively.
Table 1.
Eggshell quality in Rhode Island White hens.
Fig 3.
Differentially expressed genes (DEGs) between the low eggshell strength (LES) group and the normal eggshell strength (NES) group.
The red spots represent DEGs belonging to a calcium signaling pathway (KO: 04020) (P < 0.01).
Fig 4.
Enriched biological process GO term among differentially expressed genes.
Numbers in parentheses indicate the number of differentially expressed genes.
Fig 5.
Validation of gene expression in the calcium signaling pathway.
The qPCR was performed to quantify gene expression level. The 2−ΔΔCT method was used analyze changes in gene expression relative to chicken β-actin. Fold changes between the low eggshell strength (LES) group and the normal eggshell strength (NES) group were calculated for each gene. * represent the significant differentially expressed genes by the qPCR (P < 0.05, one way ANOVA analysis). Fold-changes based on RNA-Seq and qPCR were highly correlated (r = 0.71).
Table 2.
KEGG pathway enriched in differentially expressed genes.
Table 3.
Eggshell qualities of different genotypes for CACNA1H gene.
Table 4.
Non-synonymous mutations of the differentially expressed genes in calcium signaling pathways and matrix proteins.