Identification and verification of microRNA in wheat (Triticum aestivum)

MicroRNAs (miRNAs) are small, endogenous RNAs that regulate gene expression in both plants and animals. A large number of miRNAs has been identified from various animals and model plant species such as Arabidopsis thaliana and rice (Oryza sativa); however, characteristics of wheat (Triticum aestivum) miRNAs are poorly understood. Here, computational identification of miRNAs from wheat EST sequences was preformed by using the in-house program GenomicSVM, a prediction model for miRNAs. This study resulted in the discovery of 79 miRNA candidates. Nine out of 22 miRNA representatives randomly selected from the 79 candidates were experimentally validated with Northern blotting, indicating that prediction accuracy is about 40%. For the 9 validated miRNAs, 59 wheat ESTs were predicted as their putative targets. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Weibo Jin and Nannan Li contributed equally to the work.     

The construction of Arabidopsis expressed sequence tag assemblies. A new resource to facilitate gene identification.

The generation of large numbers of partial cDNA sequences, or expressed sequence tags (ESTs), has provided a method with which to sample a large number of genes from an organism. More than 25,000 Arabidopsis thaliana ESTs have been deposited in public databases, producing the largest collection of ESTs for any plant species. We describe here the application of a method of reducing redundancy and increasing information content in this collection by grouping overlapping ESTs representing the same gene into a "contig" or assembly. The increased information content of these assemblies allows more putative identifications to be assigned based on the results of similarity searches with nucleotide and protein databases. The results of this analysis indicate that sequence information is available for approximately 12,600 nonoverlapping ESTs from Arabidopsis. Comparison of the assemblies with 953 Arabidopsis coding sequences indicates that up to 57% of all Arabidopsis genes are represented by an EST. Clustering analysis of these sequences suggests that between 300 and 700 gene families are represented by between 700 and 2000 sequences in the EST database. A database of the assembled sequences, their putative identifications, and cellular roles is available through the World Wide Web.     

Macro effects of microRNAs in plants

MicroRNAs (miRNAs) are 20- to 22-nucleotide fragments that regulate expression of mRNAs that have complementary sequences. They are numerous and widespread among eukaryotes, being conserved throughout evolution. The few miRNAs that have been fully characterized were found in Caenorhabditis elegans and are required for development. Recently, a study of miRNAs isolated from Arabidopsis showed that here also developmental genes are putative regulatory targets. A role for miRNAs have in plant development is supported by the developmental phenotypes of mutations in the genes required for miRNA processing.     

Initial assessment of gene diversity for the oomycete pathogen Phytophthora infestans based on expressed sequences

A total of 1000 expressed sequence tags (ESTs) corresponding to 760 unique sequence sets were identified using random sequencing of clones from a cDNA library constructed from mycelial RNA of Phytophthora infestans. A number of software programs, represented by a relational database and an analysis pipeline, were developed for the automated analysis and storage of the EST sequence data. A set of 419 nonredundant sequences, which correspond to a total of 632 ESTs (63.2%), were identified as showing significant matches to sequences deposited in public databases. A putative cellular identity and role was assigned to all 419 sequences. All major functional categories were represented by at least several ESTs. Four novel cDNAs containing sequences related to elicitins, a family of structurally related proteins that induce the hypersensitive response and condition avirulence of P. infestans on Nicotiana plants, were among the most notable genes identified. Two of these elicitin-like cDNAs were among the most abundant cDNAs examined. The set also contained several ESTs with high sequence similarity to unique plant genes.     

Analysis of FMRFamide-like peptide (FLP) diversity in phylum Nematoda

This study reports a series of systematic BLAST searches of nematode ESTs on the Genbank database, using search strings derived from known nematode FLPs (those encoded by Caenorhabditis elegans flp genes as well as those isolated from other nematodes including Ascaris suum), as well as query sequences representative of theoretical FLPs. Over 1000 putative FLP-encoding ESTs were identified from multiple nematode species. A total of 969 ESTs representing sequelogs of the 23 known C. elegans flp genes were identified in 32 species, from clades I, III, IV and V. Numerical analysis of EST numbers suggests that flp-1, flp-11 and flp-14 are amongst the most highly expressed flp genes. Speculative BLAST searches were performed using theoretical FLP C-termini as queries, in an attempt to identify putative novel FLP sequences in the EST database. These searches yielded eight multi-species sequelogs encoding FLPs with novel signatures that are believed to identify distinct flp genes. These novel genes encode 25 distinct previously unidentified FLPs, and raise the current total of known nematode flp genes to 31. Additionally, software-based analyses of the presence of signal peptides were performed, with signal peptides being identified on at least one member of each group of flp ESTs, further confirming their status as secreted peptides. The data reveal that nematode FLPs encompass the most complex neuropeptide family known within the metazoa. Moreover, individual FLPs and FLP motifs are highly conserved across the nematodes with little evidence for inter-clade or inter-lifestyle variation, supporting their fundamental role in free-living and parasitic species.     

Identification of MiRNA from Eggplant (Solanum melongena L.) by Small RNA Deep Sequencing and Their Response to Verticillium dahliae Infection

MiRNAs are a class of non-coding small RNAs that play important roles in the regulation of gene expression. Although plant miRNAs have been extensively studied in model systems, less is known in other plants with limited genome sequence data, including eggplant (Solanum melongena L.). To identify miRNAs in eggplant and their response to Verticillium dahliae infection, a fungal pathogen for which clear understanding of infection mechanisms and effective cure methods are currently lacking, we deep-sequenced two small RNA (sRNA) libraries prepared from mock-infected and infected seedlings of eggplants. Specifically, 30,830,792 reads produced 7,716,328 unique miRNAs representing 99 known miRNA families that have been identified in other plant species. Two novel putative miRNAs were predicted with eggplant ESTs. The potential targets of the identified known and novel miRNAs were also predicted based on sequence homology search. It was observed that the length distribution of obtained sRNAs and the expression of 6 miRNA families were obviously different between the two libraries. These results provide a framework for further analysis of miRNAs and their role in regulating plant response to fungal infection and Verticillium wilt in particular.     

Frequency, type, distribution and annotation of simple sequence repeats in Rosaceae ESTs

Genomic resources for peach, a model species for Rosaceae, are being developed to accelerate gene discovery in other Rosaceae species by comparative mapping. Simple sequence repeats (SSRs) are an important tool for comparative mapping because of their high polymorphism and transportability. To accelerate the development of SSR markers, we analyzed publicly available Rosaceae expressed sequence tags (ESTs) for SSRs. A total of 17,284 ESTs from almond, peach and rose were assembled into putatively non-redundant EST sets. For comparison, 179,099 ESTs from Arabidopsis were also used in the analysis. About 4% of the assembled ESTs contained SSRs in Rosaceae, which was higher than the 2.4% found in Arabidopsis. About half of the SSRs were found in the putative UTR, and the estimated average distance between SSRs in the UTR was 5.5 kb in rose, 5.1 kb in almond, 7 kb in peach and 13 kb in Arabidopsis. In the putative coding region, the estimated average distance was two to four times longer than in the UTR. Rosaceae ESTs containing SSRs were functionally annotated using the GenBank nr database and further classified using the gene ontology terms associated with the matching sequences in the SwissProt database. The detailed data including the sequences and annotation results are available from .     

ProSplicer: a database of putative alternative splicing information derived from protein,mRNA and expressed sequence tag sequence data

ProSplicer is a database of putative alternative splicing information derived from the alignment of proteins, mRNA sequences and expressed sequence tags (ESTs) against human genomic DNA sequences. Proteins, mRNA and ESTs provide valuable evidence that can reveal splice variants of genes. The alternative splicing information in the database can help users investigate the alternative splicing and tissue-specific expression of genes.     

Identification of miRNAs and their targets from Brassica napus by high-throughput sequencing and degradome analysis

ABSTRACT: BACKGROUND: MicroRNAs (miRNAs) are endogenous regulators of a broad range of physiological processes and act by either degrading mRNA or blocking its translation. Oilseed rape (Brassica napus) is one of the most important crops in China, Europe and other Asian countries with publicly available expressed sequence tags (ESTs) and genomic survey sequence (GSS) databases, but little is known about its miRNAs and their targets. To date, only 46 miRNAs have been identified in B. napus. RESULTS: Forty-one conserved and 62 brassica-specific candidate B. napus miRNAs, including 20 miRNA* sequences, were identified using Solexa sequencing technology. Furthermore, 33 non-redundant mRNA targets of conserved brassica miRNAs and 19 new non-redundant mRNA targets of novel brassica-specific miRNAs were identified by genome-scale sequencing of mRNA degradome. CONCLUSIONS: This study describes large scale cloning and characterization of B. napus miRNAs and their potential targets, providing the foundation for further characterization of miRNA function in the regulation of diverse physiological processes in B. napus.     

Isolation and identification of miRNAs in Jatropha curcas

MicroRNAs (miRNAs) are small noncoding RNAs that play crucial regulatory roles by targeting mRNAs for silencing. To identify miRNAs in Jatropha curcas L, a bioenergy crop, cDNA clones from two small RNA libraries of leaves and seeds were sequenced and analyzed using bioinformatic tools. Fifty-two putative miRNAs were found from the two libraries, among them six were identical to known miRNAs and 46 were novel. Differential expression patterns of 15 miRNAs in root, stem, leave, fruit and seed were detected using quantitative real-time PCR. Ten miRNAs were highly expressed in fruit or seed, implying that they may be involved in seed development or fatty acids synthesis in seed. Moreover, 28 targets of the isolated miRNAs were predicted from a jatropha cDNA library database. The miRNA target genes were predicted to encode a broad range of proteins. Sixteen targets had clear BLASTX hits to the Uniprot database and were associated with genes belonging to the three major gene ontology categories of biological process, cellular component, and molecular function. Four targets were identified for JcumiR004. By silencing JcumiR004 primary miRNA, expressions of the four target genes were up-regulated and oil composition were modulated significantly, indicating diverse functions of JcumiR004.