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. 2011 Oct 11:12:500.
doi: 10.1186/1471-2164-12-500.

Analysis of conserved microRNAs in floral tissues of sexual and apomictic Boechera species

Samuel Amiteye  1 , José M Corral, Heiko Vogel, Timothy F Sharbel
Affiliations

Analysis of conserved microRNAs in floral tissues of sexual and apomictic Boechera species

Samuel Amiteye et al. BMC Genomics. .

Abstract

Background: Apomixis or asexual seed formation represents a potentially important agronomic trait whose introduction into crop plants could be an effective way to fix and perpetuate a desirable genotype through successive seed generations. However, the gene regulatory pathways underlying apomixis remain unknown. In particular, the potential function of microRNAs, which are known to play crucial roles in many aspects of plant growth and development, remains to be determined with regards to the switch from sexual to apomictic reproduction.

Results: Using bioinformatics and microarray validation procedures, 51 miRNA families conserved among angiosperms were identified in Boechera. Microarray assay confirmed 15 of the miRNA families that were identified by bioinformatics techniques. 30 cDNA sequences representing 26 miRNAs could fold back into stable pre-miRNAs. 19 of these pre-miRNAs had miRNAs with Boechera-specific nucleotide substitutions (NSs). Analysis of the Gibbs free energy (ΔG) of these pre-miRNA stem-loops with NSs showed that the Boechera-specific miRNA NSs significantly (p ≤ 0.05) enhance the stability of stem-loops. Furthermore, six transcription factors, the Squamosa promoter binding protein like SPL6, SPL11 and SPL15, Myb domain protein 120 (MYB120), RELATED TO AP2.7 DNA binding (RAP2.7, TOE1 RAP2.7) and TCP family transcription factor 10 (TCP10) were found to be expressed in sexual or apomictic ovules. However, only SPL11 showed differential expression with significant (p ≤ 0.05) up-regulation at the megaspore mother cell (MMC) stage of ovule development in apomictic genotypes.

Conclusions: This study constitutes the first extensive insight into the conservation and expression of microRNAs in Boechera sexual and apomictic species. The miR156/157 target squamosa promoter binding protein-like 11 (SPL11) was found differentially expressed with significant (p ≤ 0.05) up-regulation at the MMC stage of ovule development in apomictic genotypes. The results also demonstrate that nucleotide changes in mature miRNAs significantly (p ≤ 0.05) enhance the thermodynamic stability of pre-miRNA stem-loops.

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Figures

Figure 1
Figure 1
Scheme for search of conserved miRNAs in Boechera species.
Figure 2
Figure 2
Percentage of conserved miRNAs between Boechera and other plant species (species with < 1% not included). Ath, Arabidopsis thaliana; Ptc, Populus trichocarpa; Osa, Oryza sativa; Zma, Zea mays; Mtr, Medicago truncatula; Gma, Glycine max; Sbi, Sorghum bicolor; Vvi, Vitis vinifera; Ppt, Physcomitrella patens; Rco, Ricinus communis; Tae, Triticum aestivum; Ghr, Gossypium hirsutum; Sly, Solanum lycopersicum; Ahy, Arachis hypogaea; Sof, Saccharum officinarum.
Figure 3
Figure 3
Abundance (based on total number of transcripts) of conserved miRNAs in Boechera species.
Figure 4
Figure 4
Predicted pre-miRNA stem-loops of miR156/157 families with nucleotide substitutions in Boechera species. Shaded red letters correspond to the sequence of the mature miRNA. Nucleotide substitutions of conserved miRNAs in other plant species compared with the corresponding miRNAs in Boechera species are shown as italicized, bold and underlined blue letters. MiRNA precursors could be slightly longer than the sequences shown in this figure.
Figure 5
Figure 5
Predicted pre-miRNA stem-loops of miRNA families with nucleotide substitutions in Boechera species. Shaded red letters correspond to the sequence of the mature miRNA. Nucleotide substitutions of conserved miRNAs in other plant species compared with the corresponding miRNAs in Boechera species are shown as italicized, bold and underlined blue letters. MiRNA precursors could be slightly longer than the sequences shown in this figure.
Figure 6
Figure 6
Predicted pre-miRNA stem-loops of miR414 family with nucleotide substitutions in Boechera species. Shaded red letters correspond to the sequence of the mature miRNA. Nucleotide substitutions of conserved miRNAs in other plant species compared with the corresponding miRNAs in Boechera species are shown as italicized, bold and underlined blue letters. MiRNA precursors could be slightly longer than the sequences shown in this figure.
Figure 7
Figure 7
Predicted pre-miRNA stem-loops of miRNA families with nucleotide substitutions in Boechera species. Shaded red letters correspond to the sequence of the mature miRNA. Nucleotide substitutions of conserved miRNAs in other plant species compared with the corresponding miRNAs in Boechera species are shown as italicized, bold and underlined blue letters. MiRNA precursors could be slightly longer than the sequences shown in this figure.
Figure 8
Figure 8
Frequency of nucleotide substitutions related to Arabidopsis thaliana in mature Boechera miRNAs.
Figure 9
Figure 9
Expression of transcription factors across Boechera sexual and apomictic ovule developmental stages. Refer to Sharbel et al. [40] for descriptions of ovule stages.
Figure 10
Figure 10
Relative differential expression of SPL11 transcription factor in stage two of apomictic and sexual ovules.
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