Deep sequencing and microarray hybridization identify conserved and species-specific microRNAs during somatic embryogenesis in hybrid yellow poplar

Abstract

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.

Figure 1. Hybrid yellow poplar (L. tulipifera×L. chinense) plantlets and somatic embryos.

Plantlets generated by somatic embryogenesis (A), and somatic embryos at sequential developmental stages (B). PEM, pre-embryonic mass; E, embryo stage.

Figure 2. Sequence length distribution in hybrid yellow poplar.

Length distribution and abundance of redundant (A) and unique (C) sequences; (B) the ratio of redundant to unique sequences at each length; (D) Length distribution of the conserved and species-specific miRNAs.

Figure 3. Conservation of hybrid yellow poplar miRNAs.

The miRNA conservation was studied by aligning of pre-miRNAs of P. trichocarpa, Arabidopsis, Oryza, V. vinifera and liriodendron, using Weblogo software. (A) miRNA156; (B) miRNA397. The mature sequences are highlighted in a rectangle box.

Figure 4. Number of identical miRNA members in each family in hybrid yellow poplar.

Figure 5. Paralogs per conserved miRNA family in hybrid yellow poplar, Arabidopsis, P.trichocarpa, and Oryza.

The number of family members in Arabidopsis, P. trichocarpa, and Oryza miRBase and the number of family members in hybrid yellow poplar from deep-sequencing results in this study are shown.

Figure 6. Predicted secondary structures of identified miRNAs in hybrid yellow poplar.

The uppercase letters refer to mature sequences. The secondary structures are generated from the Liriodendron genome and EST sequences.

Figure 7. Relationship between the miRNA microarray signals and deep-sequencing reads.

(A) Layout diagram (left) and color map (right) of the miRNA microarray. The empty control, spike-calibration control and biosequence control are colored in purple, bright blue and yellow respectively. The probes ordered according to the sequencing result are colored in green. (B) Comparison of microarray signals (blue) and sequencing reads (red) for the same predicted conserved miRNAs. (C) Analysis of the relationship between microarray signals and sequencing reads under natural logarithm in 24 conserved miRNAs (left; miRNAs are ordered as in B) and 26 species-specific miRNAs (right).

Figure 8. Putative molecular functions and biological processes of predicted conserved and species-specific miRNA targets.

The miRNA targets in hybrid yellow poplar were predicted by carrying out BLAST search of A. thaliana and Liriodendron EST databases with psRNATarget tool and then analyzed using Gene Ontology.

Figure 9. qRT-PCR expression analysis of ltu-miR397b and its predicted target gene in hybrid yellow poplar.

(A) ltu-miR397 and the middle region of ltuLAC-like gene were detected in PEM, E1, E2, E3, E5 and E9 tissues by qRT-PCR. Both 5S rRNA and 18S rRNA were used as internal controls. The product of each sample was separated on a 2% (w/v) agarose gel. (B) Relative expression level of ltu-miR397b and ltuLAC-like gene in SEs. Error bars indicate one standard deviation of three different biological replicates (n = 3). All relative quantitative values were plotted on a logarithmic scale.