Integrated small RNA and transcriptome analysis reveals potential miRNA regulatory modules associated with sex-biased reproductive development in Gnetum montanum

Sex differentiation in gymnosperms involves coordinated transcriptional and post-transcriptional regulation, but the contribution of microRNAs (miRNAs) to this process remains poorly understood. Gnetum montanum provides a useful system for investigating sex-biased regulation across vegetative leaves and reproductive strobili. Here, we integrated small RNA sequencing (sRNA-seq) and transcriptome from female leaves (FL), female strobili (FS), male leaves (ML), and male strobili (MS) of G. montanum . A total of 550 miRNAs were identified, including 509 miRNAs detected across all four sample types. Family annotation assigned the identified miRNAs to 329 families, including 24 known and 305 novel families. Sex-biased expression analysis identified 116 differentially expressed miRNAs between FL and ML and 105 between FS and MS. Protein-coding genes (PCGs) showed broader sex-biased transcriptional divergence, with 2,973 differentially expressed genes in leaves and 8,880 in strobili, indicating stronger sex-biased transcriptional differences in reproductive tissues. Integration of miRNA and target gene expression identified 90 inverse miRNA–target pairs in leaves and 316 in strobili. These pairs were associated with signaling, cell communication, metabolic regulation, reproductive development, peptide receptor activity, and phosphatidylcholine-related metabolism. Weighted gene co-expression network analysis (WGCNA) further identified a female-associated honeydew1 module and a male-associated lightpink4 module, both linked to RNA processing, small RNA-related regulation, and developmental processes. Finally, we constructed an integrated network that linked sex-biased miRNAs to sex-associated coexpression modules and protein-protein interaction (PPI) networks, highlighting their potential function in cytoskeleton regulation, reproductive organ development, hormone signaling, metabolism, and protein modification. Our study provides an integrated transcriptional and post-transcriptional view of sex-biased regulation in G. montanum . The results suggest that sex-biased miRNAs may contribute to reproductive differentiation by modulating signaling, RNA processing, developmental, and metabolic pathways. These findings establish a candidate regulatory framework for future functional analyses of sex-biased reproductive development in gymnosperms.