Global identification of Ginkgo biloba microRNAs and insight into their role in metabolism regulatory network of terpene trilactones by high-throughput sequencing and degradome analysis

•High-throughput sequencing could provide a comprehensive data for miRNA in Ginkgo.•Identified 9 miRNA participate TTL metabolism through predicting 12 structure genes.•24 miRNAs regulated TFs, and participate TTL metabolism by mediating structural gene.•Revealing miRNA-mRNA regulation network associated with TTL metabolism in G. biloba. Terpene trilactones (TTLs) are important secondary metabolites and healthy components of Ginkgo biloba. They are essential for the quality of G. biloba and have been widely used in food, medicine and natural health products. However, there are no reports of miRNA associated with the biosynthesis of TTLs. To screen and identify the potential miRNA that participates in TTL metabolism in G. biloba, we performed high-throughput sequencing and bioinformatics methods of microRNAs and the degradome in 10 libraries of G. biloba. In total, 3,314 miRNAs (1,197 conserved miRNAs and 2,117 novel miRNAs) were identified among five organs, which were used to test those miRNAs by northern blot, quantitative real-time PCR (qRT-PCR), RLM-RACE, and degradome sequencing. In addition, we evaluated the correlation among the miRNA and mRNA expression level and TTL content in different organs of G. biloba. The results showed that four conserved miRNAs and five novel miRNAs might participate in TTL biosynthesis pathways by predicting the targets of those miRNAs to 12 TTL biosynthesis structure genes. Furthermore, 25 miRNAs were identified to potentially play vital role in TTL accumulation by targeting transcript factor genes, bHLH, WRKY, and AP2, respectively, which are candidate regulatory genes in the pathway of TTL biosynthesis. qRT-PCR analysis verified the expression patterns of 31 miRNAs and corresponding 27 target genes. This study is the first to authenticate miRNAs as well as their potential target genes involved in TTL biosynthesis, thereby providing molecular evidence for revealing the comprehensive miRNA-mRNA regulatory network participated in TTL metabolism of G. biloba.