RNA-sequence reveals differentially expressed genes affecting the crested trait of Wumeng crested chicken

Abstract

Wumeng crested chicken has a cluster of slender feathers on its head, and the underlying skull region exhibits an obvious tumor-like protrusion. This is the typical skull structure of crested chickens. The associated regulatory genes are located on autosomes and are incompletely dominant. This trait is related to brain herniation, but the genetic mechanisms of its formation and development are unclear. In this study, RNA sequencing (RNA-Seq) analysis was conducted on 6 skull tissue samples from 3 Wumeng crested chickens with prominent skull protrusions and 3 without a prominent skull protrusion phenotype. A total of 46,376,934 to 43,729,046 clean reads were obtained, the percentage of uniquely mapped reads compared with the reference genome was between 89.73%-91.00%, and 39,795,458-41,836,502 unique reads were obtained. Among different genomic regions, the highest frequency of sequencing reads occurred in exon regions (85.44-88.28%). Additionally, a total of 423 new transcripts and 26,999 alternative splicings (AS) events were discovered in this sequencing analysis. This study identified 1,089 differentially expressed genes (DEGs), among which 485 were upregulated and 604 were downregulated. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses indicated that the DEGs were enriched in terms related to signal transduction, cell development, cell differentiation, the lysosome, serine, and threonine metabolism, and the interaction of cytokines with cytokine receptors. Based on the comprehensive analysis of DEGs combined with reported quantitative trait loci (QTLs), the expression of BMP2, EPHA3, EPHB1, HOXC6, SCN2B, BMP7, and HOXC10 was verified by real-time quantitative polymerase chain reaction (qRT-PCR). The qRT-PCR results were consistent with the RNA-Seq results, indicating that these 7 genes may be candidates genes regulating the crested trait.

Keywords: RNA-Seq; Wumeng crested chicken; crested traits; differentially expressed gene; epigenetic mechanism.

Figure 1

(A) Heat map of the correlations between samples. The horizontal and vertical coordinates are the squared values of the correlation coefficients of each sample. (B) Differentially expressed gene clustering heat map. The abscissa is the sample name, and the ordinate is the normalized value of the differential gene FPKM. The redder the color is, the higher the expression level, and the greener the color is, the lower the expression level.

Figure 2

Volcano map of differential gene expression. The abscissa is the log2FoldChange value, the ordinate is the -log10p value and the blue dotted line represents the threshold of the differential gene screening criteria.

Figure 3

GO enrichment analysis histogram. The abscissa is the GO term, and the ordinate is the number of enriched genes for the GO term. Abbreviation: GO, Gene Ontology.

Figure 4

Correlations of mRNA expression levels of 7 DEGs between C and NC using RNA-Seq and qRT-PCR. The x- and y-axis correspond to the log2 (ratio of C/NC) measured from RNA-Seq and qRT-PCR, respectively.