Deep Sequencing and Microarray Hybridization Identify Conserved and Species-Specific MicroRNAs during Somatic Embryogenesis in Hybrid Yellow Poplar

Background

To date, several studies have indicated a major role for microRNAs (miRNAs) in regulating plant development, but miRNA-mediated regulation of the developing somatic embryo is poorly understood, especially during early stages of somatic embryogenesis in hardwood plants. In this study, Solexa sequencing and miRNA microfluidic chips were used to discover conserved and species-specific miRNAs during somatic embryogenesis of hybrid yellow poplar (Liriodendron tulipifera×L. chinense).

Methodology/Principal Findings

A total of 17,214,153 reads representing 7,421,623 distinct sequences were obtained from a short RNA library generated from small RNAs extracted from all stages of somatic embryos. Through a combination of deep sequencing and bioinformatic analyses, we discovered 83 sequences with perfect matches to known miRNAs from 33 conserved miRNA families and 273 species-specific candidate miRNAs. MicroRNA microarray results demonstrated that many conserved and species-specific miRNAs were expressed in hybrid yellow poplar embryos. In addition, the microarray also detected another 149 potential miRNAs, belonging to 29 conserved families, which were not discovered by deep sequencing analysis. The biological processes and molecular functions of the targets of these miRNAs were predicted by carrying out BLAST search against Arabidopsis thaliana GenBank sequences and then analyzing the results with Gene Ontology.

Conclusions

Solexa sequencing and microarray hybridization were used to discover 232 candidate conserved miRNAs from 61 miRNA families and 273 candidate species-specific miRNAs in hybrid yellow poplar. In these predicted miRNAs, 64 conserved miRNAs and 177 species-specific miRNAs were detected by both sequencing and microarray hybridization. Our results suggest that miRNAs have wide-ranging characteristics and important roles during all stages of somatic embryogenesis in this economically important species.     

Identification and differential expression of microRNAs during metamorphosis of the Japanese flounder (Paralichthys olivaceus)

Background

MicroRNAs (miRNAs) are a class of endogenous small non-coding RNAs of 20–25 nucleotides that play a key role in diverse biological processes. Japanese flounder undergo dramatic metamorphosis in their early development. The metamorphosis is characterized by morphological transformation from a bilaterally symmetrical to an asymmetrical body shape concomitant with extensive morphological and physiological remodeling of organs. So far, only a few miRNAs have been identified in fish and there are very few reports about the Japanese flounder miRNA.

Methodology/Principal Findings

Solexa sequencing technology was used to perform high throughput sequencing of the small RNA library from the metamorphic period of Japanese flounder. Subsequently, aligning these sequencing data with metazoan known miRNAs, we characterized 140 conserved miRNAs and 57 miRNA: miRNA* pairs from the small RNA library. Among these 57 miRNA: miRNA* pairs, twenty flounder miRNA precursors were amplified from genomic DNA. We also demonstrated evolutionary conservation of Japanese flounder miRNAs and miRNA* in the animal evolution process. Using miRNA microarrays, we identified 66 differentially expressed miRNAs at two metamorphic stages (17 and 29 days post hatching) of Japanese flounder. The results show that miRNAs might play a key role in regulating gene expression during Japanese flounder metamorphosis.

Conclusions/Significance

We identified a large number of miRNAs during flounder metamorphosis, some of which are differentially expressed at two different metamorphic stages. The study provides an opportunity for further understanding of miRNA function in the regulation of flounder metamorphosis and gives us clues for further studies of the mechanisms of metamorphosis in Japanese flounder.     

Deep sequencing discovery and profiling of conserved and novel miRNAs in the ovule of <Emphasis Type="Italic">Ginkgo biloba</Emphasis>

Key message

High-throughput sequencing and subsequent analysis identified multiple miRNAs closely related to ovule, indicating that miRNAs are important in Ginkgo biloba ovule.

Abstract

MicroRNAs (miRNAs) are small, noncoding, regulatory RNAs that play crucial regulatory roles in the process of plant growth and development. However, limited information regarding their functions in gymnosperm reproduction is available. Here, we used high-throughput sequencing combined with computational analysis to identify and characterize miRNAs from ovules of G. biloba, and identified 34 conserved miRNA families and 99 novel miRNAs. The precursor sequences of several of the conserved and novel miRNAs were further validated by RT-PCR and sequencing. Furthermore, we found that some target genes, e.g. MYB, homeodomain-leucine zipper (HD-ZIPIII) and auxin response factor (ARF), may be involved in ovule development, and that the significantly enriched pathways of some miRNA targets were related to plant–pathogen interactions and the biosynthesis of secondary metabolites. Twenty-six conserved miRNA families were found to be expressed in both leaves and ovules, while miRNA156, miRNA164, miRNA167, miRNA169, miRNA172 and miRNA390 were up-regulated in ovules. Thus, multiple miRNAs closely related to G. biloba ovule development were identified, resulting in a greater understanding of the important regulatory functions of miRNAs in plant ovules.

     

Characterization of Evolutionarily Conserved MicroRNAs in Amphioxus

Amphioxus is an extant species closest to the ancestry of vertebrates.Observation of microRNA(miRNA)distribution of amphioxus would lend some hints for evolutionary research of vertebrates.In this study,using the publicly available scaffold data of the Florida amphioxus(Branchiostoma floridae)genome,we screened and characterized homologs of miRNAs that had been identified in other species.In total,68 pieces of such homologs were obtained and classified into 33 families.Most of these miRNAs were distributed as clusters in genome.Inter-species comparison showed that many miRNAs,which had been thought as vertebrate-or mammal-specific before,were also present in amphioxus,while some miRNAs that had been considered as protostome-specific before also existed in amphioxus.Compared with ciona,amphioxus had an apparent miRNA gene expansion,but phylogenetic analysis showed that the duplicated miRNAs or clusters of amphioxus had a higher homology level than those duplicated ones in vertebrates.     

The Fate of miRNA* Strand through Evolutionary Analysis: Implication for Degradation As Merely Carrier Strand or Potential Regulatory Molecule?

Background

During typical microRNA (miRNA) biogenesis, one strand of a ∼22 nt RNA duplex is preferentially selected for entry into a silencing complex, whereas the other strand, known as the passenger strand or miRNA* strand, is degraded. Recently, some miRNA* sequences were reported as guide miRNAs with abundant expression. Here, we intended to discover evolutionary implication of the fate of miRNA* strand by analyzing miRNA/miRNA* sequences across vertebrates.

Principal Findings

Mature miRNAs based on gene families were well conserved especially for their seed sequences across vertebrates, while their passenger strands always showed various divergence patterns. The divergence mainly resulted from divergence of different animal species, homologous miRNA genes and multicopy miRNA hairpin precursors. Some miRNA* sequences were phylogenetically conserved in seed and anchor sequences similar to mature miRNAs, while others revealed high levels of nucleotide divergence despite some of their partners were highly conserved. Most of those miRNA precursors that could generate abundant miRNAs from both strands always were well conserved in sequences of miR-#-5p and miR-#-3p, especially for their seed sequences.

Conclusions

The final fate of miRNA* strand, either degraded as merely carrier strand or expressed abundantly as potential functional guide miRNA, may be destined across evolution. Well-conserved miRNA* strands, particularly conservation in seed sequences, maybe afford potential opportunities for contributing to regulation network. The study will broaden our understanding of potential functional miRNA* species.     

Integrative analysis of the Pekin duck (<Emphasis Type="Italic">Anas anas</Emphasis>) MicroRNAome during feather follicle development

Background

The quality and yield of duck feathers are very important economic traits that might be controlled by miRNA regulation. The aim of the present study was to investigate the mechanism underlying the crosstalk between individual miRNAs and the activity of signaling pathways that control the growth of duck feathers during different periods. We therefore conducted a comprehensive investigation using Solexa sequencing technology on the Pekin duck microRNAome over six stages of feather development at days 11, 15, and 20 of embryonic development (during the hatching period), and at 1 day and 4 and 10 weeks posthatch.

Results

There were a total of 354 known miRNAs and 129 novel candidate miRNAs found based on comparisons with known miRNAs in the Gallus gallus miRBase. The series of miRNAs related to feather follicle formation as summarized in the present study showed two expression patterns, with primary follicle developed during embryonic stage and secondary follicle developed mainly at early post hatch stage. Analysis of miRNA expression profiles identified 18 highly expressed miRNAs, which might be directly responsible for regulation of feather development. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis suggested that in addition to Wnt and transforming growth factor (TGFβ) signaling pathways, which were widely reported in response to follicle formation, another group of signaling pathways that regulate lipid synthesis and metabolism, such as the phosphatidylinositol signaling system and glycerolipid metabolism and signaling, are also responsible for follicle formation.

Conclusion

The highly expressed miRNAs provide a valuable reference for further investigation into the functional miRNAs important for feather development. Lipid synthesis and metabolism related signaling pathways might be responsible for lipid formation on the surface of feather, and should be paid much more attention for their relation to feather quality.

     

Comparison of phenotypes produced in response to transient expression of genes encoded by four distinct begomoviruses in Nicotiana benthamiana and their correlation with the levels of developmental miRNAs

Background

Whitefly-transmitted geminiviruses (begomoviruses) are a major limiting factor for the production of numerous dicotyledonous crops throughout the world. Begomoviruses differ in the number of components that make up their genomes and association with satellites, and yet they cause strikingly similar phenotypes, such as leaf curling, chlorosis and stunted plant growth. MicroRNAs (miRNAs) are small endogenous RNAs that regulate plant growth and development. The study described here was aimed at investigating the effects of each virus encoded gene on the levels of developmental miRNAs to identify common trends between distinct begomoviruses.

Results

All genes encoded by four distinct begomoviruses (African cassava mosaic virus [ACMV], Cabbage leaf curl virus [CbLCuV], Tomato yellow leaf curl virus [TYLCV] and Cotton leaf curl virus/Cotton leaf curl betasatellite [CLCuV/CLCuMB]) were expressed from a Potato virus X (PVX) vector in Nicotiana benthamiana. Changes in the levels of ten miRNAs in response to the virus genes were determined by northern blotting using specific miRNA probes. For the monopartite begomoviruses (TYLCV and CLCuMV) the V2 gene product was identified as the major symptom determinant while for bipartite begomoviruses (ACMV and CbLCuV) more than one gene appears to contribute to symptoms and this is reflected in changes in miRNA levels. The phenotype induced by expression of the βC1 gene of the betasatellite CLCuMB was the most distinct and consisted of leaf curling, vein swelling, thick green veins and enations and the pattern of changes in miRNA levels was the most distinct.

Conclusions

Our results have identified symptom determinants encoded by begomoviruses and show that developmental abnormalities caused by transient expression of begomovirus genes correlates with altered levels of developmental miRNAs. Additionally, all begomovirus genes were shown to modulate miRNA levels, the first time this has been shown to be the case.