Unraveling the Multilayered Regulatory Networks of miRNAs and PhasiRNAs in Ginkgo biloba

Small RNAs (sRNAs) are pivotal in regulating gene expression and are involved in a diverse array of biological processes. Among these, microRNAs (miRNAs) and phased small interfering RNAs (phasiRNAs) have been extensively investigated over the past decades. We conducted an in-depth analysis of deep sequencing data from the gymnosperm <i>Ginkgo biloba</i>, encompassing sRNA, transcriptome, and degradome libraries. Our analysis identified a total of 746 miRNAs and 654 phasiRNA precursor (PHAS) loci, with 526 (80%) of the PHAS loci predicted to be triggered by 515 miRNAs (69%). Several miRNA-PHAS modules, particularly the miR159/miR319-PHAS module, were found to potentially regulate reproductive development by targeting <i>GAMYB</i> genes and triggering phasiRNA biogenesis. The miR390-PHAS module appears to be involved in flavonoid biosynthesis by targeting key enzyme genes such as chalcone synthase (<i>CHS</i>) and anthocyanin synthase (<i>ANS</i>). Through target gene identification and coexpression analysis, we uncovered two distinct models of complex regulatory networks: growth-related factors like <i>ARF</i> and <i>GRF</i> seem to be regulated exclusively by miRNAs (Model 1), while certain disease resistance-related genes are predicted to be regulated by both miRNAs and phasiRNAs (Model 2), indicating diverse regulatory mechanisms across different biological processes. Overall, our study provides a comprehensive annotation of miRNA and PHAS loci in <i>G. biloba</i> and elucidates a post-transcriptional regulatory network, offering novel insights into sRNA research in gymnosperms.