miRNAome in Salvia miltiorrhiza: High-Throughput Sequencing, Characterization and Biogenetic Pathway Analysis

Salvia miltiorrhiza is a well-known plant with great economic and medicinal value. Its root has been widely used in traditional Chinese medicine (TCM) for thousands of years and is known to possess a variety of pharmacological effects, such as antibacterial, antioxidant, anti-inflammatory, and antineoplastic activities. S. miltiorrhiza is being developed to be a model medicinal plant. A working draft of the S. miltiorrhiza genome has been obtained recently and genes involved in the major lipophilic bioactive components, diterpenoid tanshinones, have been identified at a genome-wide scale. However, there is no information available for miRNAs, a class of small endogenous non-coding RNAs playing vital regulatory roles in many organisms. Here, we used Solexa deep sequencing technology to sequence five small RNA (sRNA) libraries prepared from S. miltiorrhiza roots at different developmental stages. We obtained, for each library, 10,811,910, 12,739,642, 10,812,659, 11,774,166, and 9,391,557 high quality clean reads, respectively. Comprehensive analysis of the sRNAs obtained allowed us to identify a number of conserved and novel miRNAs. Many siRNAs associated with root development were also revealed. In order to understand the biogenetic pathways of sRNAs, we performed genome-wide analysis of three key gene families (DCL, AGO and RDR) involved in sRNA biogenesis and action based on the working draft of the S. miltiorrhiza genome. A total of five DCLs, eleven AGOs and five RDRs were first computationally predicted and then experimentally cloned. Gene structures, phylogenetic relationships and protein conserved motifs of these genes were analyzed. Using the quantitative real-time PCR method, we found that the genes involved in sRNA biogenesis showed differential expression patterns in various tissues of S. miltiorrhiza with or without PEG treatment. Our results provide the first clue for elucidating the function and biogenetic pathway of sRNAs in S. miltiorrhiza.