MicroRNA nomenclature and the need for a revised naming prescription.

A central environment and interface for microRNA (miRNA) registry and repository and a general standardized framework for their systematic annotation was established over a decade ago. However, the numbers of experimentally and computationally identified miRNAs are swiftly accumulating, and new aspects of miRNA-mediated gene regulation are being revealed. Currently, it is of great significance that the annotation framework should be redefined to include newly discovered miRNA species such as the variants of mature miRNAs (isomiRNAs), and organellar miRNAs: cipomiRNAs and mitomiRNAs. It is also of great importance that key terminology referring to the novelty, evolutionary history or biogenesis of miRNAs, as well as the confidence of their identification are standardized in the literature and disseminated in a central miRNA registry. Here, we review the status of miRNA nomenclature, curation and critical points of need for a revision of miRNA nomenclature and terminology.

Root precursors of microRNAs in wild emmer and modern wheats show major differences in response to drought stress.

MicroRNAs, small regulatory molecules with significant impacts on the transcriptional network of all living organisms, have been the focus of several studies conducted mostly on modern wheat cultivars. In this study, we investigated miRNA repertoires of modern durum wheat and its wild relatives, with differing degrees of drought tolerance, to identify miRNA candidates and their targets involved in drought stress response. Root transcriptomes of Triticum turgidum ssp. durum variety Kiziltan and two Triticum turgidum ssp. dicoccoides genotypes TR39477 and TTD-22 under control and drought conditions were assembled from individual RNA-Seq reads and used for in silico identification of miRNAs. A total of 66 miRNAs were identified from all species, across all conditions, of which 46 and 38 of the miRNAs identified from modern durum wheat and wild genotypes, respectively, had not been previously reported. Genotype- and/or stress-specific miRNAs provide insights into our understanding of the complex drought response. Particularly, miR1435, miR5024, and miR7714, identified only from drought-stress roots of drought-tolerant genotype TR39477, can be candidates for future studies to explore and exploit the drought response to develop tolerant varieties.

Harnessing NGS and Big Data Optimally: Comparison of miRNA Prediction from Assembled versus Non-assembled Sequencing Data--The Case of the Grass Aegilops tauschii Complex Genome.

MicroRNAs (miRNAs) are small, endogenous, non-coding RNA molecules that regulate gene expression at the post-transcriptional level. As high-throughput next generation sequencing (NGS) and Big Data rapidly accumulate for various species, efforts for in silico identification of miRNAs intensify. Surprisingly, the effect of the input genomics sequence on the robustness of miRNA prediction was not evaluated in detail to date. In the present study, we performed a homology-based miRNA and isomiRNA prediction of the 5D chromosome of bread wheat progenitor, Aegilops tauschii, using two distinct sequence data sets as input: (1) raw sequence reads obtained from 454-GS FLX Titanium sequencing platform and (2) an assembly constructed from these reads. We also compared this method with a number of available plant sequence datasets. We report here the identification of 62 and 22 miRNAs from raw reads and the assembly, respectively, of which 16 were predicted with high confidence from both datasets. While raw reads promoted sensitivity with the high number of miRNAs predicted, 55% (12 out of 22) of the assembly-based predictions were supported by previous observations, bringing specificity forward compared to the read-based predictions, of which only 37% were supported. Importantly, raw reads could identify several repeat-related miRNAs that could not be detected with the assembly. However, raw reads could not capture 6 miRNAs, for which the stem-loops could only be covered by the relatively longer sequences from the assembly. In summary, the comparison of miRNA datasets obtained by these two strategies revealed that utilization of raw reads, as well as assemblies for in silico prediction, have distinct advantages and disadvantages. Consideration of these important nuances can benefit future miRNA identification efforts in the current age of NGS and Big Data driven life sciences innovation.

Stress responsive miRNAs and isomiRs in cereals.

Abiotic and biotic stress conditions are vital determinants in the production of cereals, the major caloric source in human nutrition. Small RNAs, miRNAs and isomiRs are central to post-transcriptional regulation of gene expression in a variety of cellular processes including development and stress responses. Several miRNAs have been identified using new technologies and have roles in stress responses in plants, including cereals. The overall knowledge about the cereal miRNA repertoire, as well as an understanding of complex miRNA mediated mechanisms of target regulation in response to stress conditions, is far from complete. Ongoing efforts that add to our understanding of complex miRNA machinery have implications in plant response to stress conditions. Additionally, sequence variants of miRNAs (isomiRNAs or isomiRs), regulation of their expression through dissection of upstream regulatory elements, the role of Processing-bodies (P-bodies) in miRNA exerted gene regulation and yet unveiled organellar plant miRNAs are newly emerging topics, which will contribute to the elucidation of the miRNA machinery and its role in cereal tolerance against abiotic and biotic stresses.

History and current status of wheat miRNAs using next-generation sequencing and their roles in development and stress.

As small molecules that aid in posttranscriptional silencing, microRNA (miRNA) discovery and characterization have vastly benefited from the recent development and widespread application of next-generation sequencing (NGS) technologies. Several miRNAs were identified through sequencing of constructed small RNA libraries, whereas others were predicted by in silico methods using the recently accumulating sequence data. NGS was a major breakthrough in efforts to sequence and dissect the genomes of plants, including bread wheat and its progenitors, which have large, repetitive and complex genomes. Availability of survey sequences of wheat whole genome and its individual chromosomes enabled researchers to predict and assess wheat miRNAs both in the subgenomic and whole genome levels. Moreover, small RNA construction and sequencing-based studies identified several putative development- and stress-related wheat miRNAs, revealing their differential expression patterns in specific developmental stages and/or in response to stress conditions. With the vast amount of wheat miRNAs identified in recent years, we are approaching to an overall knowledge on the wheat miRNA repertoire. In the following years, more comprehensive research in relation to miRNA conservation or divergence across wheat and its close relatives or progenitors should be performed. Results may serve valuable in understanding both the significant roles of species-specific miRNAs and also provide us information in relation to the dynamics between miRNAs and evolution in wheat. Furthermore, putative development- or stress-related miRNAs identified should be subjected to further functional analysis, which may be valuable in efforts to develop wheat with better resistance and/or yield.

New wheat microRNA using whole-genome sequence.

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.

Unique and conserved microRNAs in wheat chromosome 5D revealed by next-generation sequencing.

MicroRNAs are a class of short, non-coding, single-stranded RNAs that act as post-transcriptional regulators in gene expression. miRNA analysis of Triticum aestivum chromosome 5D was performed on 454 GS FLX Titanium sequences of flow-sorted chromosome 5D with a total of 3,208,630 good quality reads representing 1.34x and 1.61x coverage of the short (5DS) and long (5DL) arms of the chromosome respectively. In silico and structural analyses revealed a total of 55 miRNAs; 48 and 42 miRNAs were found to be present on 5DL and 5DS respectively, of which 35 were common to both chromosome arms, while 13 miRNAs were specific to 5DL and 7 miRNAs were specific to 5DS. In total, 14 of the predicted miRNAs were identified in wheat for the first time. Representation (the copy number of each miRNA) was also found to be higher in 5DL (1,949) compared to 5DS (1,191). Targets were predicted for each miRNA, while expression analysis gave evidence of expression for 6 out of 55 miRNAs. Occurrences of the same miRNAs were also found in Brachypodium distachyon and Oryza sativa genome sequences to identify syntenic miRNA coding sequences. Based on this analysis, two other miRNAs: miR1133 and miR167 were detected in B. distachyon syntenic region of wheat 5DS. Five of the predicted miRNA coding regions (miR6220, miR5070, miR169, miR5085, miR2118) were experimentally verified to be located to the 5D chromosome and three of them : miR2118, miR169 and miR5085, were shown to be 5D specific. Furthermore miR2118 was shown to be expressed in Chinese Spring adult leaves. miRNA genes identified in this study will expand our understanding of gene regulation in bread wheat.

Drought tolerance in modern and wild wheat.

The genus Triticum includes bread (Triticum aestivum) and durum wheat (Triticum durum) and constitutes a major source for human food consumption. Drought is currently the leading threat on world's food supply, limiting crop yield, and is complicated since drought tolerance is a quantitative trait with a complex phenotype affected by the plant's developmental stage. Drought tolerance is crucial to stabilize and increase food production since domestication has limited the genetic diversity of crops including wild wheat, leading to cultivated species, adapted to artificial environments, and lost tolerance to drought stress. Improvement for drought tolerance can be achieved by the introduction of drought-grelated genes and QTLs to modern wheat cultivars. Therefore, identification of candidate molecules or loci involved in drought tolerance is necessary, which is undertaken by "omics" studies and QTL mapping. In this sense, wild counterparts of modern varieties, specifically wild emmer wheat (T. dicoccoides), which are highly tolerant to drought, hold a great potential. Prior to their introgression to modern wheat cultivars, drought related candidate genes are first characterized at the molecular level, and their function is confirmed via transgenic studies. After integration of the tolerance loci, specific environment targeted field trials are performed coupled with extensive analysis of morphological and physiological characteristics of developed cultivars, to assess their performance under drought conditions and their possible contributions to yield in certain regions. This paper focuses on recent advances on drought related gene/QTL identification, studies on drought related molecular pathways, and current efforts on improvement of wheat cultivars for drought tolerance.

Physical mapping integrated with syntenic analysis to characterize the gene space of the long arm of wheat chromosome 1A.

BACKGROUND: Bread wheat (Triticum aestivum L.) is one of the most important crops worldwide and its production faces pressing challenges, the solution of which demands genome information. However, the large, highly repetitive hexaploid wheat genome has been considered intractable to standard sequencing approaches. Therefore the International Wheat Genome Sequencing Consortium (IWGSC) proposes to map and sequence the genome on a chromosome-by-chromosome basis. METHODOLOGY/PRINCIPAL FINDINGS: We have constructed a physical map of the long arm of bread wheat chromosome 1A using chromosome-specific BAC libraries by High Information Content Fingerprinting (HICF). Two alternative methods (FPC and LTC) were used to assemble the fingerprints into a high-resolution physical map of the chromosome arm. A total of 365 molecular markers were added to the map, in addition to 1122 putative unique transcripts that were identified by microarray hybridization. The final map consists of 1180 FPC-based or 583 LTC-based contigs. CONCLUSIONS/SIGNIFICANCE: The physical map presented here marks an important step forward in mapping of hexaploid bread wheat. The map is orders of magnitude more detailed than previously available maps of this chromosome, and the assignment of over a thousand putative expressed gene sequences to specific map locations will greatly assist future functional studies. This map will be an essential tool for future sequencing of and positional cloning within chromosome 1A.

Subgenomic analysis of microRNAs in polyploid wheat.

In this study, a survey of miRNAs using the next-generation sequencing data was performed at subgenomic level. After analyzing shotgun sequences from chromosome 4A of bread wheat (Triticum aestivum L.), a total of 68 different miRNAs were predicted in silico, of which 37 were identified in wheat for the first time. The long arm of the chromosome was found to harbor a higher variety (51) and representation (3,928) of miRNAs compared with the short arm (49; 2,226). Out of the 68 miRNAs, 32 were detected to be common to both arms, revealing the presence of separate miRNA clusters in the two chromosome arms. The differences in degree of representation of the different miRNAs were found to be highly variable, ranging 592-fold, which may have an effect on target regulation. Targets were retrieved for 62 (out of 68) of wheat-specific, newly identified miRNAs indicated that fundamental aspects of plant morphology such as height and flowering were predicted to be affected. In silico expression blast analysis indicated 24 (out of 68) were found to give hits to expressed sequences. This is the first report of species- and chromosome-specific miRNAs.

miRNA expression patterns of Triticum dicoccoides in response to shock drought stress.

Drought is a major environmental stress factor that affects plant growth and development worldwide. Wild emmer wheat (Triticum turgidum ssp. dicoccoides), the ancestor of domesticated durum wheat (Triticum turgidum ssp. durum), has great potential for improving the understanding of the wheat drought response. MicroRNAs (miRNAs) are a recently discovered class of gene expression regulators that have also been linked to several plant stress responses; however, this relationship is just beginning to be understood. miRNA expression patterns of drought-resistant wild emmer wheat in response to drought stress were investigated using a plant miRNA microarray platform. Expression was detected to be 205 miRNAs in control and 438 miRNAs in drought-stressed leaf and root tissues. Of these miRNAs, the following 13 were differentially regulated in response to drought: miR1867, miR896, miR398, miR528, miR474, miR1450, miR396, miR1881, miR894, miR156, miR1432, miR166 and miR171. Regulation of miRNAs upon 4 and 8 h drought stress applications observed by qRT-PCR. Target transcripts of differentially regulated miRNAs were computationally predicted. In addition to miRNA microarray study, five new conserved T. turgidum miRNAs were identified through a homology-based approach, and their secondary structures and putative targets were predicted. These findings both computationally and experimentally highlight the presence of miRNAs in T. dicoccoides and further extend the role of miRNAs under shock drought stress conditions.

Regulation of barley miRNAs upon dehydration stress correlated with target gene expression.

We aim to identify conserved and dehydration responsive microRNAs (miRNAs) in Hordeum vulgare (barley). A total of 28 new barley miRNAs belonging to 18 distinct miRNA families were identified. Detailed nucleotide analyses revealed that barley pre-miRNAs are in the range of 46-114 nucleotides with average of 77.14. Using 28 newly detected miRNAs as queries, 445 potential target mRNAs were predicted. The predicted miRNAs were differentially expressed and some of them behaved similarly in leaf and root tissues upon stress treatment. Hvu-MIR156, Hvu-MIR166, Hvu-MIR171, and Hvu-MIR408 were detected as dehydration stress-responsive barley miRNAs. To discover target transcripts of barley miRNAs a modified 5' RLM-RACE was performed and seven cleaved miRNA transcripts were retrieved from drought stressed leaf samples. In silico analysis indicated 15 potential EST targets. Measurement of expression levels showed a positive correlation between levels of miRNA expression and suppression of their target mRNA transcripts in dehydration-stress-treated barley.