Identification and Analysis of Key miRNA for Sex Differentiation in Hermaphroditic Juglans mandshurica Maxim

Juglans mandshurica Maxim. is heterodichogamous. Protogyny and protandry are its types. However, the mechanisms of sex differentiation and heterodichogamous development in Juglans mandshurica are unknown. The study used high-throughput sequencing to sequence male and female Juglans mandshurica flower buds for microRNA. Twelve small RNA libraries identified 310 conserved and 278 novel miRNA. A total of 129 miRNAs with significant differential expression were discovered using DESeq2. psRobots predicted 638 miRNA target genes. After that, GO and KEGG enrichment analysis examined these target genes. The target genes were largely enriched in metabolic pathways, including reproduction, floral development, and phytohormone signaling pathways. Therefore, miR159c-3p, miR156d-3p, miR172d, miR171b, miR164a, miR167a, miR319a, miR399a, and miR396t may be the key miRNAs affecting the sex differentiation of Juglans mandshurica, with miR164a, miR166c, and miR171c-5p affecting heterodichogamous differentiation. These key miRNAs and their target genes constituted a regulatory network. Real-time fluorescence quantitative PCR identified nine randomly selected miRNAs. After analyzing these miRNAs and transcriptome sequencing data, a major co-expression regulatory network of differentially expressed mRNAs and key miRNAs was created. These miRNAs are involved in sex differentiation in Juglans mandshurica, according to this study. The integration of transcriptome data allows a multi-omics study of Juglans mandshurica sex differentiation regulatory network. The results of this study showed that these mi RNAs regulated the expression of target genes in Juglans mandshurica. This regulatory mechanism subsequently influences hormone secretion, thereby impacting sex differentiation and the development of heteromorphism, This study lays the framework for understanding Juglans mandshurica molecular sex differentiation and heterodichogamy.