Integrated multi-omics data to identify the key genes affecting egg-laying performance in late-phase hens

In the whole laying cycle, the late laying phase is the longest segment, and it directly determines total egg production and influences the timing of culling. To investigate the mechanisms underlying the decline in egg-laying performance during this phase, our study selected Wenchang chickens as the research subjects, which are characterized by low egg-laying rates and high phenotypic variation. We investigated egg-laying patterns in 872 Wenchang chickens during the late laying phase and performed mechanistic studies on three high egg-laying (laying rates > 76.0 %) hens and three low egg-laying hens (laying rates < 50.0 %). RNA-seq and ATAC-seq analyses were conducted on ovarian stroma tissues from these hens. RNA-seq analysis identified 800 differentially expressed genes with thresholds of p < 0.05 and |log₂FC| ≥ 1, including 317 upregulated and 483 downregulated genes, which were enriched in biological processes such as immune response, negative regulation of endopeptidase activity, tight junction, and neuroactive ligand-receptor interaction. ATAC-seq revealed 1,065 and 428 candidate differentially accessible regions in the two groups, respectively, with 9.8 % and 5.8 % of these regions located in promoter regions. Integrated analysis identified three key genes potentially influencing the laying performance in the late phase, including MICA, MHCIA6 and MHCIY. These genes all belong to the non-classical MHC-Ⅰ family and are implicated in immune regulation. Additionally, four transcription factors (HOXB6, GATA3, TFAP2A, and WT1) were predicted to regulate these key genes. Collectively, our findings suggest that an imbalance in ovarian immune homeostasis during the late laying phase may be a significant driver of the decline in egg production. This study not only provides mechanistic insights into the maintenance of high egg-laying performance but also offers potential molecular targets for genetic improvement in Wenchang chickens and advances the understanding of reproductive regulation in poultry.