Identification and characterization of microRNA expression in Ginkgo biloba L. leaves

Ginkgo biloba L., an extant primitive gymnosperm species, occupies an important position in the evolution of the plant kingdom. Furthermore, its leaves contain a large number of active, medicinally valuable compounds; therefore, it is also an important tree economically. MicroRNAs (miRNAs) are important regulators of gene expression implicated in controlling plant development and environmental interaction. Extensive studies of miRNAs have been performed in angiosperms, but no reports have focused on small RNAs in the basal gymnosperm G. biloba. Here, we used Illumina next-generation sequencing HiSeq™ 2000 platform to identify and quantitatively profile small RNAs in G. biloba leaves. A total of 13,414,548 and 12,293,511 reads, containing 202 and 201 known miRNAs, belonging to 82 and 78 families, were sequenced from two small RNA libraries constructed from female and male leaves, respectively. We also identified 174/174 novel miRNAs in the female/male leaves of G. biloba. Relative expression levels of the miRNAs and the precursor sequences of several of the known and novel miRNAs were further validated by subcloning and quantitative real-time PCR (qRT-PCR). Target predictions of the 380 and 340 known miRNAs, from female and male leaves, resulted in 2116 and 2002 target genes, while those of the 155 and 158 novel miRNAs resulted in 596 and 793 target genes, respectively. KEGG annotation analysis revealed that high-ranked miRNA-target genes are implicated in plant-pathogen interactions, plant hormone signal transduction, and flavonoid biosynthesis. Taken together, we identified a large number of miRNAs in mature female and male G. biloba leaves for the first time. We ascertained that the regulatory networks of the miRNAs are involved in many different primary biological processes based on potential target designations.