Comparative transcriptome profiling of leg muscle development from the embryonic to growth stages in Huanglang chickens

The leg muscles of poultry are an important source of high-quality protein and key trace elements. Most of the research on muscle development are focused on the embryonic or growth stage, there is relatively little research on the muscle development of chicken from embryonic stage to growth stages. Therefore, in this study, transcriptome sequencing was performed on leg muscle samples from female Huanglang chickens across embryonic (E13, E17) to growth stages (D1, D14, D35). A total of 7,691 differentially expressed genes (DEGs) were identified by comparison in pairs of the ten groups, and the highest number of DEGs was observed between E13 and D35, with 4,645 DEGs. Time-series expression analysis using STEM revealed three distinct expression clusters: cluster I (2,582 genes, downregulated genes), cluster II (1,666 genes, upregulated genes), and cluster III (614 genes, transient upregulation peaking at D1). GO and KEGG enrichment analyses showed that the DEGs were enriched in pathways such as cell cycle and DNA replication (cluster I), metabolic and muscle structure-related pathways (cluster II), oxidative phosphorylation and mitochondrial function (cluster III). Protein-protein interaction network analysis identified hub genes, including CDC45/MCM complex members in cluster I, mitochondrial genes such as CYTB and ND1 in cluster II, and oxidative phosphorylation genes including NDUFS3 and COX5A in cluster III. Transcription factor prediction highlighted KDM5A, KDM5B, PHF8, and SAP30 as key regulators. The RT-qPCR results of seven genes ( ACTN2, PDK4, MYOG, MYOD1, MYH1F, MYH1B , and MYF6 ) were consistent with the RNA sequencing (RNA-seq) analysis. This study is the first to systematically analyzed the leg muscle development mechanisms of local chicken breeds from embryonic stage to early growth stage. It provides a comprehensive transcriptomic resource for understanding leg muscle development in chickens.