Project description:Small RNAs (21-24 nt) are pivotal regulators of gene expression that guide both transcriptional and post-transcriptional silencing mechanisms in diverse eukaryotes, including most if not all plants. MicroRNAs (miRNAs) and short interfering RNAs (siRNAs) are the two major types, both of which have a demonstrated and important role in plant development, stress responses and pathogen resistance. In this work, we used a deep sequencing approach (Sequencing-By-Synthesis, or SBS) to develop sequence resources of small RNAs from Mimulus guttatus tissues (including leaves, flowers and roots). The high depth of the resulting datasets enabled us to examine in detail critical small RNA features as size distribution, tissue-specific regulation and sequence conservation between different organs in this species. We also developed database resources and a dedicated website (http://smallrna.udel.edu/) with computational tools for allowing other users to identify new miRNAs or siRNAs involved in specific regulatory pathways, verify the degree of conservation of these sequences in other plant species and map small RNAs on genes or larger regions of the maize genome under study. Small RNA libraries were derived from leaves, flowers and roots of Mimulus guttatus, strain âIM62â. Total RNA was isolated using the Plant RNA Purification Reagent (Invitrogen), and submitted to Illumina (Hayward, CA, http://www.illumina.com) for small RNA library construction using approaches described in (Lu et al., 2007) with minor modifications. The small RNA libraries were sequenced with the Sequencing-By-Synthesis (SBS) technology by Illumina. PERL scripts were designed to remove the adapter sequences and determine the abundance of each distinct small RNA. We thank John Willis for providing the plant material as well as Kan Nobuta and Gayathri Mahalingam for assistance with the computational methods.
Project description:Small RNAs (21-24 nt) are pivotal regulators of gene expression that guide both transcriptional and post-transcriptional silencing mechanisms in diverse eukaryotes, including most if not all plants. MicroRNAs (miRNAs) and short interfering RNAs (siRNAs) are the two major types, both of which have a demonstrated and important role in plant development, stress responses and pathogen resistance. In this work, we used a deep sequencing approach (Sequencing-By-Synthesis, or SBS) to develop sequence resources of small RNAs from Mimulus guttatus tissues (including leaves, flowers and roots). The high depth of the resulting datasets enabled us to examine in detail critical small RNA features as size distribution, tissue-specific regulation and sequence conservation between different organs in this species. We also developed database resources and a dedicated website (http://smallrna.udel.edu/) with computational tools for allowing other users to identify new miRNAs or siRNAs involved in specific regulatory pathways, verify the degree of conservation of these sequences in other plant species and map small RNAs on genes or larger regions of the maize genome under study.
Project description:The seeds of angiosperms are comprised of three main tissues--the seed coat, embryo, and endosperm--whose coordinated development will enable the next generation to disperse with sufficient maternal resources and germinate when conditions are favorable. We use high-throughput RNA sequencing technology to characterize the developmental dynamics of gene expression in whole seeds resulting from a compatible cross between two species of the Mimulus guttatus complex.We find that development in ovules and seeds involves the activation of the majority of annotated genes in M. guttatus (64-67%), and the differential expression over time of 6,691 genes, including 424 transcription factors. Most of the genes we detected (69%) were expressed at all stages, from ovules to heart-stage embryo seeds. We also detected and validated four genes exhibiting paternally-biased expression (MDB13, ATXR5, DnaJ and BGAL11). Intriguingly, three of our validated PEGs are have imprinted homologues in other plant species, suggesting that these proteins perform a shared role in endosperm development among distantly related plant taxa. Additionally, the overlap in gene expression profiles between stages of development suggests that most genes fulfill multiple developmental and biological roles. Future analyses should examine whether different regions of the seed (embryo, endosperm, and seed coat) have unique patterns of gene expression, and the extent to which spatial coordination and regulation of gene expression may play a role in regulating seed development.