MicroRNAs (miRNAs) are 20 to 24 nucleotides short RNAs involved in posttranscriptional regulation in plants and animals. MiRNAs are processed from larger precursors with extensive secondary structure. In plants, a total of 286 miRNA genes in Arabidopsis, rice and maize had been identified by March 2005, clustered in 43 families.

Here, we report the bioinformatic identification of 200 members of the 43 miRNA families in the genomes of maize, sorghum, medick and poplar. Furthermore, we report evidence for expression of 37 miRNA precursors that are present in EST collections of soybean and sugarcane. We have used the enlarged data set to systematically analyze several parameters of the plant precursors including stem length, conservation of the precursors and variation in the secondary structure of the miRNA along the precursor.

Based on this 83% increase in available miRNA precursor sequences, we present an improved view of phylogenetic distribution, positional nucleotide preference, structural features and conservation of miRNA genes. Our results suggest that there are two different classes of plant miRNA precursors. The most abundant class includes precursors that have only two strongly conserved regions, corresponding to the mature miRNA and its complementary sequence. A less frequent class, which includes the miRNA families miR159/319 and miR394, displays two additional conserved sequence blocks. These precursors have larger stems with more extensive secondary structure.