Identification of molecular pathways and candidate genes associated with cocks' comb size trait by genome-wide transcriptome analysis

Abstract

The comb of the male is an important secondary sexual characteristic. Although quantitative trait loci (QTLs) related to comb size have been identified, molecular mechanisms underlying this trait remain mostly unknown. In this study, RNA sequencing (RNA-seq) was employed to compare whole transcriptomic differences between two groups of Partridge Shank chickens that are divergent in comb sizes. A total of 563 differentially expressed genes (DEGs) were identified, including 277 up-regulated and 286 down-regulated DEGs. According to the animal QTL database, eight DEGs including BMP2 and CHADL matching the reported QTLs were associated with the comb size. Functional annotation analysis revealed that DEGs were involved in cell communication and calcium signaling. Protein-protein interaction network analysis showed that STK32A, PIK3R1, EDN1, HSPA5, and HSPA8 have an impact on comb growth. Moreover, potential alternative splicing events and single nucleotide polymorphisms were also identified. Our data provide a source for identifying genes and pathways with functions critical to comb size and accelerate studies involving molecular mechanisms of this sexual ornament.

Figure 1

Distribution of 12 types of alternative splicing (AS) events in six samples. The six bars of different colors indicate the numbers of AS events identified in the big comb size (BC) and small comb size (SC) libraries.

Figure 2

Distribution of SNPs identified in six samples. The lengths of the bars indicate the numbers of SNPs identified in the big comb size (BC) and small comb size (SC) libraries.

Figure 3

Hierarchical clustering of the differentially expressed genes between the big comb size (BC) and small comb size (SC) groups. Each column represents a sample, and each row represents a gene. The red and blue gradients indicate an increase and a decrease in gene expression abundance, respectively.

Figure 4

Illustrating of the qPCR confirmation results for the 10 selected differentially expressed genes. (A) The X-axis represents the selected 10 genes and the Y-axis represents the log₂(foldchange) values derived from RNA-seq and qPCR. (B) Regression analysis of the log₂(foldchange) values between RNA-seq and qPCR.

Figure 5

GO analysis of the differentially expressed genes between the big comb size (BC) and small comb size (SC) groups. Significantly enriched GO terms of three types are used (P-value ≤ 0.05). The lengths of the bars indicate the corresponding numbers of genes for each GO term.

Figure 6

KEGG pathway analysis of the differentially expressed genes (DEGs) between the big comb size (BC) and small comb size (SC) groups. Rich factor = Amount of DEGs enriched in the pathway/Amount of all genes in the background gene set. The size and color of each bubble represent the amount of DEGs enriched in the pathway and enrichment significance, respectively.

Figure 7

Protein-protein interaction network for the differentially expressed genes (DEGs). Node represents protein, edge represents interaction between proteins. The size of a node is proportional to degree of this node (degree of the node defined as the amount of proteins that interact with this node), and the color of the node represents the expression regulation type of DEGs (Red means up-regulation and blue means down-regulation in the BC group).