Whole-transcriptome insights into follicle selection: deciphering key regulatory networks in Luxi gamecock

Abstract

Background: Luxi gamecock is a native Chinese breed recognized for its substantial body size, well-developed musculature, and aggressive behavior. Despite these advantageous traits, the breed's egg production rate remains relatively low, insufficient to meet market demands. Follicle selection plays a crucial role in determining the egg-laying performance of hens, yet research on follicle selection in Luxi gamecock is limited. In this study, RNA sequencing was performed on small yellow follicles (SYFs) and large yellow follicles (LYFs) from Luxi gamecock to identify RNA transcript expression, and subsequent RNA networks were constructed.

Methods: SYFs and LYFs were collected from 15 Luxi gamecocks and divided randomly into three biological groups. RNA was isolated to profile the expression of mRNA, lncRNA, circRNA, and miRNA. The results were validated using qRT-PCR. Functional analysis, including GO and KEGG, was conducted. Competitive endogenous RNA (ceRNA) networks were also constructed.

Results: A total of 1,113 mRNAs, 245 lncRNAs, 264 circRNAs, and 90 miRNAs were differentially expressed between SYFs and LYFs. qRT-PCR validation showed high consistency with the RNA-seq results. Functional enrichment indicated that these differentially expressed RNAs are associated with critical biological processes and involved in several key signaling pathways. To investigate the potential interactions among circRNAs, lncRNAs, and miRNAs, ceRNA networks were constructed.

Conclusion: This study provides a detailed characterization of the transcriptomes in SYFs and LYFs of Luxi gamecock through RNA sequencing. The functional analysis revealed that many RNAs may contribute to follicle selection. Furthermore, ceRNA networks were built to better understand the molecular mechanisms behind follicle selection. These findings shed light on the potential regulatory roles of various RNA molecules in the follicle selection of Luxi gamecock, and also uncover the interactions among them, laying a foundation for improving the breed's egg-laying performance.

Keywords: Luxi gamecock; RNA-seq; ceRNA network; differential expression RNAs; follicle selection.

FIGURE 1

(A) Hens and (B) follicle grades of Luxi gamecock.

FIGURE 2

Heatmap and volcano plot representations of differentially expressed RNAs. The heatmaps display changes in expression for (A) mRNAs, (B) lncRNAs, (C) circRNAs, and (D) miRNAs. Volcano plots depict differential expression for (E) mRNAs, (F) lncRNAs, (G) circRNAs, and (H) miRNAs. In both visualizations, red indicates genes with higher expression levels, while blue represents those with lower expression levels.

FIGURE 3

Validation of mRNA and miRNA expression levels using qRT-PCR based on RNA-seq data. Gene expression was normalized using ACTB and 18S rRNA as references. Results are presented as mean ± SEM (n = 5). The blue bars correspond to RNA-seq results, while the pink bars represent data from qRT-PCR. Statistical significance is denoted as *p < 0.05, **p < 0.01, and ***p < 0.001.

FIGURE 4

Functional characterization of differentially expressed mRNAs. (A) GO analysis of the differentially expressed mRNAs. (B) KEGG pathway analysis of the differentially expressed mRNAs. Enrichment plots for the following pathways: (C) Thyroid hormone synthesis, (D) Notch signaling pathway, (E) AMPK signaling pathway, and (F) Hippo signaling pathway.

FIGURE 5

Functional characterization of targets of differentially expressed lncRNAs. (A) GO functional enrichment analysis of cis-regulatory targets of lncRNAs. (B) KEGG pathway analysis of cis-regulatory lncRNA targets. (C) GO functional enrichment analysis of trans-regulatory lncRNA targets. (D) KEGG pathway analysis of trans-regulatory lncRNA targets.

FIGURE 6

Functional enrichment analysis of genes associated with differentially expressed circRNAs and miRNAs. (A) GO enrichment analysis of genes originating from the parental sources of differentially expressed circRNAs. (B) KEGG pathway analysis of the parental genes of differentially expressed circRNAs. (C) GO enrichment analysis of genes regulated by differentially expressed miRNAs. (D) KEGG pathway analysis of genes targeted by differentially expressed miRNAs.

FIGURE 7

ceRNA network construction and functional analysis. (A) ceRNA network associated with circRNAs. (B) ceRNA network associated with lncRNAs. (C) GO functional enrichment analysis of the target mRNAs. (D) KEGG pathway analysis of the target mRNAs.

FIGURE 8

Visualization of crucial genes in the ceRNA network. (A) The ceRNA subnetworks of differentially expressed mRNAs in Ras signaling pathway. (B) The ceRNA subnetwork of GRIN2B. (C) The ceRNA subnetwork of MET.