Integrated RNA-seq and ATAC-seq analyses reveal muscle development differences between european meat pigeon and shiqi pigeon during embryonic to post-hatching stages

Figures

• 

  FIGURE 1. Transcriptomic profiling and functional convergence of differential gene expression patterns. (A) Volcano plot of DEGs; (B) Numbers of up- and downregulated DEGs identified in each of the four pairwise comparisons (MME14 vs. SQE14, SQE14 vs. SQP1, MME14 vs. MMP1, and MMP1 vs. SQP1). DEGs were identified separately for each pairwise comparison rather than across all four groups simultaneously; (C) Breed- and stage-specific DEGs overlap between European meat and Shiqi pigeons.

• 

  FIGURE 2. Functional enrichment analysis associated with muscle development dynamics and breed divergence. (A) GO Enrichment Analysis (Developmental stage comparison: E14 vs. P1); (B) KEGG Pathway Enrichment Analysis (Developmental stage comparison: E14 vs. P1); (C) GO Enrichment Analysis (Breed comparison: MM vs. SQ); (D) KEGG Pathway Enrichment Analysis (Breed comparison: MM vs. SQ).

• 

  FIGURE 3. Quality control metrics for ATAC-seq data integrity and reproducibility. (A) Enrichment of ATAC-seq signals around TSS regions; (B) Distribution of functional genomic regions; (C) Correlation heatmap between samples; (D) Principal Component Analysis (PCA) plot; (E) ATAC-seq signal enrichment plot (±2 kb around peak centers); (F) Differential peak analysis.

• 

  FIGURE 4. Functional annotation and chromatin accessibility associations. (A) GO enrichment analysis of top 20 significant biological processes; (B) KEGG pathway analysis of top 15 enriched pathways; (C) Enrichment analysis of genes associated with differentially accessible chromatin regions.

• 

  FIGURE 5. Integrated analysis of ATAC-seq and RNA-seq data. (A) Comparative visualization of RNA-seq (left) and ATAC-seq (right) results; (B) Genomic peak diagram illustrating chromatin accessibility and gene expression relationships (Exon regions represent transcriptome coding sequences); (C) Radar map of the top 18 candidate genes.

• 

  FIGURE 6. Differential gene function and transcriptional regulation. (A) GO enrichment of upregulated DEGs; (B) KEGG enrichment of upregulated DEGs; (C) GO enrichment of downregulated DEGs; (D) KEGG enrichment of downregulated DEGs; (E) Predicted transcription factors and their co-binding sites with star factors; (F) Motif types of transcription factors.

• 

  FIGURE 7. Validation of myogenic DEGs using RT-qPCR. RT-qPCR of Sln, MYOM2, Trim63, Fabp3, ATP1A2, KLF15, IRS2 and RYR3 in SQ and MM during E6-P1. *, P < 0.05; **, P < 0.01. The closer the absolute value of r is to 1, the stronger the linear correlation between the two variables.

• 

  FIGURE 8. Effects of Sln over-expression and interference on proliferation of pigeon embryonic myoblasts. (A) Quantitative experimental validation of over-expression and interference efficiency; (B) EdU staining of pigeon embryonic myoblasts in the over-expression experiment, the upper panels represent the control group (pcDNA3.1-NC), and the lower panels represent the Sln over-expression group (pcDNA3.1-Sln); (C) Statistical analysis of the EdU-positive cell rate in the overexpression experiment; (D) EdU staining of pigeon embryonic myoblasts in the interference experiment; the upper panels represent the control group (siRNA-NC), and the lower panels represent the Sln interference group (siRNA-Sln); (E) Statistical analysis of EdU-positive cells in the interference group; (F) Quantitative analysis of marker gene expression during the proliferation stage; (G) Western blot analysis of proliferation-related proteins. Lanes 1–4 represent the pcDNA3.1-Sln, pcDNA3.1-NC, siRNA-Sln, and siRNA-NC groups, respectively. The pcDNA3.1-NC group served as the empty vector negative control for the Sln overexpression experiment, while the siRNA-NC group served as the non-targeting siRNA negative control…

• 

  FIGURE 9. Effects of Sln over-expression and interference on differentiation of pigeon embryonic myoblasts. (A) Quantitative experimental validation of over-expression and interference efficiency; (B) MyHC immunofluorescence staining of pigeon embryonic myoblasts in the over-expression experiment; the upper panels represent the control group (pcDNA3.1-NC), and the lower panels represent the Sln over-expression group (pcDNA3.1-Sln); (C) Statistical analysis of MyHC fluorescence signals in the over-expression group; (D) MyHC immunofluorescence staining of pigeon embryonic myoblasts in the interference experiment; the upper panels represent the control group (siRNA-NC), and the lower panels represent the Sln interference group (siRNA-Sln); (E) Statistical analysis of MyHC fluorescence signals in the interference group; (F) Quantitative analysis of marker gene expression during the differentiation stage; (G) Western blot analysis of Sln and MyHC protein expression during myogenic differentiation. Lanes 1–4 represent the pcDNA3.1-Sln, pcDNA3.1-NC, siRNA-NC, and siRNA-Sln groups, respectively. GAPDH was used as the loading control to verify equal protein loading among samples; (H) Quantification…

• 

  FIGURE 10. Schematic diagram illustrating the molecular mechanisms regulating breast muscle development in meat pigeons. Based on integrated multi-omics analysis and functional validation, this study identifies key genes, such as Sln, through phenotypic comparison and multi-omics (RNA-seq and ATAC-seq) data screening, and validates their role in promoting myoblast proliferation and differentiation at both gene expression and functional levels, revealing critical molecular pathways affecting pectoral muscle myofiber density in large and small body size pigeon breeds during the embryonic stage.