Computational predictions and expression patterns of conserved microRNAs in loblolly pine (Pinus taeda)

MiRNAs are mostly known for their expressions in sporophytic (diploid) tissues from various angiosperms, and only limited information is available on those expressed in gametophytic (haploid) tissues. Even more limited information is available for gymnosperms, particularly those that are expressed in gametophytic tissues. Homology search of the loblolly pine expressed sequence tag (EST) database using known miRNAs from Arabidopsis thaliana and Picea abies revealed 12 miRNAs previously unreported and/or uncharacterized in loblolly pine (Pinus taeda). Their precursor and mature sequences and secondary structures were obtained using computational approaches. PCR was used to confirm their expressions using samples that represent the sporophyte (i.e., needles) and gametophyte (i.e., mature and germinated pollen) phases of the loblolly pine life cycle. Our results showed that all 12 miRNAs were expressed in the needles, three of which were detected only in the needles, two were expressed only in the needles and germinated pollen, and seven were expressed in all of the three tissues examined. None was expressed only in the mature and germinated pollen, but nine had expressions in both or either of these samples. Target predictions suggest that these newly identified and characterized loblolly pine miRNAs regulate messenger RNAs (mRNAs) that are involved in gene regulation, metabolism, and signal transduction. This study demonstrates the usefulness of computational approach in identifying conserved miRNAs in species with unsequenced genomes like loblolly pine and reveals that conserved miRNAs can be used to differentiate sporophytic and gametophytic tissues and between stages of the male gametophyte.