Transcriptome profiling of granulosa cells during follicular development identifies RMDN2 polymorphisms associated with reproductive traits in chickens.

Egg-laying performance in chickens is primarily dependent on the normal development and maturation of follicles, which are regulated by various functional genes. However, the functional genes that regulate follicular development are not fully known. Here, we utilized RNA sequencing (RNA-seq) technology and bioinformatics analysis to identify the potential genes involved in the modulation of granulosa cells (GCs) from small yellow follicles (SYFG, 4-8 mm in diameter), the smallest hierarchical follicle (F6G, 9-12 mm in diameter) and the largest hierarchical follicle (F1G, ∼40 mm in diameter). Furthermore, association analysis was performed between polymorphisms of the identified candidate RMDN2 and egg production performance. Transcriptome analysis revealed 284 and 1026 differentially expressed genes (DEGs) in SYFG.vs.F6G and F6G.vs.F1G under the condition of |log2 fold change| > 1 and q-value < 0.01, respectively. Among these, 107 DEGs were consistently detected in both SYFG.vs.F6G and F6G.vs.F1G comparisons. These 107 DEGs were primarily enriched in the PPAR signalling pathway, thyroid hormone synthesis, and steroid biosynthesis pathways. Notably, 41 of these DEGs have been strongly linked to reproductive processes in previous studies. qRT-PCR validation validated that 13 genes (UTRN, LOC422926, MYH11, CD9, TIGAR, RARA, PLBD1, PPP1CB, RMDN2, SORCS2, SLC9A5, PDLIM4 and PPP4R4) exhibited similar expression trends in GCs across three stages in G1 layer line. Correlation analysis uncovered that RMND2 expression was negatively associated with the differentiation markers of FSHR and AMH, as well as the proliferation markers of CDK2, but positively correlated with the steroidogenesis markers of CYP11A1, STAR, and ESR1. Analysis of polymorphisms in the RMDN2 gene indicated that c.250G > A (SNP1) and c.270G > C (SNP2) were significantly associated with the egg number at 40-week-old (EN40, p < 0.05) in G1 layer line hens. The haplotype combination H1H2 exhibited the highest EN40. In summary, this study identified functional genes associated with GC development in chickens and highlighted RMDN2 polymorphisms as potential markers for laying traits selection in chickens.