Identification of soybean microRNAs and their targets.

The microRNAs (miRNAs) are a newly identified class of small non-protein-coding regulatory RNA. Using comparative genomics, we identified 69 miRNAs belonging to 33 families in the domesticated soybean (Glycine max) as well as five miRNAs in the soybean wild species Glycine soja and Glycine clandestine. TaqMan((R)) MicroRNA Assay analyses demonstrated that these miRNAs were differentially expressed in soybean tissues, with certain classes expressed preferentially in both a spatiotemporal and a tissue-specific manner. Detailed sequence analyses revealed that soybean pre-miRNAs vary in length from 44 to 259 nt with an average of 106 +/- 45 nt, harbor mature miRNAs that differ in their physical location within the pre-miRNAs, and encode more than a single mature miRNA. Comparative sequence analyses of soybean miRNA sequences showed that uracil is the dominant base in the first position at the 5' end of the mature miRNAs while cytosine is dominant at the 19th position, which is indicative that these two bases may have an important functional role in miRNA biogenesis and/or miRNA-mediated gene regulation. Soybeans were unique among plants in the frequency of occurrence of miRNA clusters. For the first time, antisense miRNAs were identified in plants. The five antisense miRNAs and their sense partners from soybean belonged to three miRNA families (miR-157, miR-162 and miR-396). Antisense miRNAs were also identified in soybean wild species. Mature antisense miRNA products appeared to have 1-3 nucleotide changes compared to their sense partners, which suggests that both strands of a miRNA gene can produce functional mature miRNAs and that antisense transcripts may differ functionally from their sense partners. Based on previously established in silico methods, we predicted 152 miRNA-targeted mRNAs, which included a large percentage of mRNAs that encode transcription factors that regulate plant growth and development as well as a lesser percentage of mRNAs that encode environmental signal transduction proteins and central metabolic processes.