Small RNA and degradome sequencing reveal roles of miRNAs in strobilus development in masson pine (Pinus massoniana)

Abstract

Pinus massoniana (P.massoniana) is a gymnosperm with important economic value. Strobilus is an important organ for the study of morphogenesis and pattern-building mechanisms at the reproductive stage of conifers. miRNA induced post-transcriptional regulation plays a key role in sex differentiation, strobilus development, fertilization and embryogenesis in gymnosperms. In this study, miRNA profiles of strobili (female and male) and vegetative tissues of P. massoniana were constructed and compared by sRNA-seq. By de novo prediction and manual revision, 53 conserved miRNAs and 41 novel miRNAs were identified, including 30 differentially expressed miRNAs. Comprehensive analysis with computational prediction and degradome data provided 349 genes targeted by 102 miRNAs. The target gene enrichment analysis indicated that signal transduction was significantly activated during strobilus (especially male strobilus) development. The miRNA-target network was constructed and its module miRNA (miR156, 529, 396 and Nov23)-SPLs (SQUAMOSA PROMOTER BINDING PROTEIN-LIKE) seemed to be a key regulator in the differentiation of male strobili from female strobili and vegetative tissues. In addition, the precursor and primary transcripts identified with miRNA quantification indicated that the regulatory mechanism of miRNA abundance is complex and needs to be studied further. This study provides important miRNA and target information for the strobilus of P. massoniana. The de novo prediction method was appropriate for miRNA identification in plants without a reference genome. The significantly activated pathways and miRNA-target networks identified here increase the understanding of the posttranscriptional regulatory mechanism of strobilus differentiation and development in the reproductive phase of conifers.