Abstract
Intermittent fertilization intensity (IFI) is closely related to higher fertilization in chicken hens. Recent studies have identified genes influencing sperm-oocyte interactions and immune regulation that impact the fertilization process in chickens. This research aims to identify key candidate genes and variants regulating IFI through transcriptomic analysis and dual luciferase assays. Our study's transcriptomic analysis of 12 individuals exhibiting extreme IFI revealed several key candidate genes. Validation using quantitative PCR highlighted PRSS12, DNER, WIF1, and NRXN1 as potential contributors to variations in IFI. Notably, we observed significant differences in the expression of LMO4, located 247.2 kb downstream of IFI-associated genomic regions. To explore variants potentially involved in the regulation of LMO4, we conducted short variant annotation and SV-GWAS, but found no significant associations with IFI. Further motif analysis and dual luciferase validation uncovered three regulatory elements within the associated region that exhibited enhanced promoter or enhancer activity following significant SNP mutations. In conclusion, our findings indicate that LMO4, PRSS12, DNER, WIF1, and NRXN1 serve as primary candidate genes for regulating IFI. Additionally, three regulatory elements significantly associated with IFI were identified upstream of LMO4. These variants hold promise for use in selecting low-IFI lines.