Abstract
MicroRNAs are post-transcriptional regulators of gene expression, taking roles in a variety of fundamental biological processes. Hence, their identification, annotation and characterization are of great significance, especially in bread wheat, one of the main food sources for humans. The recent availability of 5× coverage Triticum aestivum L. whole-genome sequence provided us with the opportunity to perform a systematic prediction of a complete catalogue of wheat microRNAs. Using an in silico homology-based approach, stem-loop coding regions were derived from two assemblies, constructed from wheat 454 reads. To avoid the presence of pseudo-microRNAs in the final data set, transposable element related stem-loops were eliminated by repeat analysis. Overall, 52 putative wheat microRNAs were predicted, including seven, which have not been previously published. Moreover, with distinct analysis of the two different assemblies, both variety and representation of putative microRNA-coding stem-loops were found to be predominant in the intergenic regions. By searching available expressed sequences and small RNA library databases, expression evidence for 39 (out of 52) putative wheat microRNAs was provided. Expression of three of the predicted microRNAs (miR166, miR396 and miR528) was also comparatively quantified with real-time quantitative reverse transcription PCR. This is the first report on in silico prediction of a whole repertoire of bread wheat microRNAs, supported by the wet-lab validation.
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Abbreviations
- miRNA:
-
MicroRNA
- pri-miRNA:
-
Primary miRNA
- pre-miRNA:
-
Precursor miRNA
- RISC:
-
RNA-induced silencing complex
- SVM:
-
Support vector machine
- EST:
-
Expressed sequence tag
- LCG:
-
Low copy number assembly
- TREP:
-
Triticeae repeat database
- OG:
-
Orthologous group assembly
- OGR:
-
Orthologous group representatives
- cDNA:
-
Complementary DNA
- MFE:
-
Minimal folding free energy
- MITE:
-
Miniature inverted terminal repeat element
- TE-MIR:
-
Transposable element-related miRNA group
- GEO:
-
Gene expression omnibus
- TIR:
-
Terminal inverted repeat
- LTR:
-
Long terminal repeat
- siRNA:
-
Small interfering RNA
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Acknowledgments
The authors are grateful to Dr. Rachel Brenchley and her collegues (Centre for Genome Research, University of Liverpool, UK) for use of the 5× coverage wheat genome assemblies and the transcriptome assembly.
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The authors declare that they have no conflict of interest.
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Kuaybe Yucebilgili Kurtoglu and Melda Kantar equally contributed to this research.
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Table S1
(.xls) Primer Sequences Used in This Study List of all primers used in this study (PDF 87.6 kb)
Table S2
(.xls) Repeat Elements Found in Putative miRNA-Coding Regions List of repeat elements found to be present in stem-loop coding regions (RepeatMasker data) (PDF 129 kb)
Table S3
(.xls) miRNAs and miRNA*s Expressed by Evidence of Small RNA Library Blast Search Results of in silico small RNA library search of predicted miRNA-coding stem-loops (PDF 24.7 KB)
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Kurtoglu, K.Y., Kantar, M. & Budak, H. New wheat microRNA using whole-genome sequence. Funct Integr Genomics 14, 363–379 (2014). https://doi.org/10.1007/s10142-013-0357-9
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DOI: https://doi.org/10.1007/s10142-013-0357-9