Identification and expression profiling of Vigna mungo microRNAs from leaf small RNA transcriptome by deep sequencing.

MicroRNAs (miRNAs) represent a class of small non-coding RNA molecules that play a crucial role in post-transcriptional gene regulation. Several conserved and species-specific miRNAs have been characterized to date, predominantly from the plant species whose genome is well characterized. However, information on the variability of these regulatory RNAs in economically important but genetically less characterized crop species are limited. Vigna mungo is an important grain legume, which is grown primarily for its protein-rich edible seeds. miRNAs from this species have not been identified to date due to lack of genome sequence information. To identify miRNAs from V. mungo, a small RNA library was constructed from young leaves. High-throughput Illumina sequencing technology and bioinformatic analysis of the small RNA reads led to the identification of 66 miRNA loci represented by 45 conserved miRNAs belonging to 19 families and eight non-conserved miRNAs belonging to seven families. Besides, 13 novel miRNA candidates in V. mungo were also identified. Expression patterns of selected conserved, non-conserved, and novel miRNA candidates have been demonstrated in leaf, stem, and root tissues by quantitative polymerase chain reaction, and potential target genes were predicted for most of the conserved miRNAs. This information offers genomic resources for better understanding of miRNA mediated post-transcriptional gene regulation.

Paul S ，Kundu A ，Pal A 《-》

Identification of Taxus microRNAs and their targets with high-throughput sequencing and degradome analysis.

Plant microRNAs (miRNAs) have an impact in the regulation of several biological processes such as development, growth and metabolism by negatively controlling gene expression at the post-transcriptional level. However, the role of these small molecules in the medicinal gymnosperm species Taxus remained elusive. To elucidate the role of miRNAs in Taxus we used a deep sequencing approach to analyze the small RNA and degradome sequence tags of Taxus mairei leaves. For miRNAs, the sequencing library generated 14.9 million short sequences, resulting in 13.1 million clean reads. The library contains predominantly small RNAs with 21 nucleotide length, followed by 19-nt and 20-nt small RNAs. Around 29% of total small RNAs are matched to the T. mairei transcriptome. By sequence alignment, we identified 871 mature miRNAs, 15 miRNA* and 869 miRNA precursors representing known plant miRNA families. There are 547 unique small RNA matching the miRNA precursors. We predict 37 candidate novel miRNAs from the unannotated small RNAs that could be mapped to the reference transcriptome. The expression of the selected candidates was for the first time quantified by real-time reverse transcription polymerase chain reaction. The novel miRNA m0034 turns out to be from the intron sequence of the paclitaxel biosynthetic gene taxadiene synthase. The 21 potential targets of nine novel miRNAs are also predicted. Additionally, 56 targets for known miRNA families and 15 targets for novel candidate miRNA families were identified by high-throughput degradome-sequencing approach. It is found that two paclitaxel biosynthetic genes, taxane 13α hydroxylase and taxane 2α-O-benzoyltransferase, are the cleavage targets of miR164 and miR171, respectively. This study represents the first transcriptome-based analysis of miRNAs and degradome in gymnosperms.

Hao DC ，Yang L ，Xiao PG ，Liu M ... -  《-》

High throughput sequencing reveals modulation of microRNAs in Vigna mungo upon Mungbean Yellow Mosaic India Virus inoculation highlighting stress regulation.

MicroRNAs (miRNAs) are 20-24 nucleotides long non-coding RNAs known to play important regulatory roles during biotic and abiotic stresses by controlling gene expression. Blackgram (Vigna mungo), an economically important grain legume is highly susceptible to pathogenic begomovirus Mungbean Yellow Mosaic India Virus (MYMIV) and resulting in high yield loss. In this study two different leaf-small-RNA libraries were prepared from the pooled RNA at three different time points of resistant V. mungo inbred line VM84 inoculated either with viruliferous or non-viruliferous whiteflies carrying MYMIV and performed high-throughput Illumina sequencing. Sequencing followed by bioinformatics analysis of the small RNA reads indicated that the expression patterns of most of the known and novel miRNAs were altered in resistant line over mock-inoculated sample during the plant virus incompatible interaction. Highly altered miRNAs belong to the families of miR156, miR159, miR160, miR166, miR398, miR1511, miR1514, miR2118 and novel vmu-miRn7, vmu-miRn8, vmu-miRn13 and vmu-miRn14. These results were validated using qPCR, and most of the miRNAs showed similar pattern of expression like that of Illumina reads. The expression patterns of some selected known and novel miRNAs were also compared between the infected MYMIV-resistant and -susceptible genotypes and most of these were modulated after MYMIV-inoculation. Target transcripts like NB-LRR, NAC, MYB, Zinc finger, CCAAT-box transcription factor, fructose 2-6 bisphosphate, HDZIP protein that confers immune response were predicted as targets amongst identified miRNAs using psRNATarget server. Some selected target transcripts including NB-LRR, ARF, SOD, SPB, Basic blue copper protein were validated and their differential expression were demonstrated between MYMIV-resistant and -susceptible V. mungo by qPCR data analyses. In the present study we have identified miRNAs that implicate in the regulation of MYMIV-induced stress response in V. mungo; and generated genomic resources for a non-model legume with the aid of bioinformatics tools supplemented by experimental validation.

Kundu A ，Paul S ，Dey A ，Pal A ... -  《-》

High-throughput sequencing and degradome analysis reveal neutral evolution of Cercis gigantea microRNAs and their targets.

High-throughput sequencing and degradome analysis for Cercis gigantea identified 194 known miRNAs and 23 novel miRNAs with 61 targets. The comparison results of highly conserved miRNAs and non-conserved miRNAs implied that C. gigantea miRNAs were subjected to the neutral evolution. MicroRNAs play a key role in post-transcriptionally regulating gene expression during plant growth, development and other various biological processes. Although numerous miRNAs have been identified and documented, to date, there are no reports on Cercis gigantea (C. gigantea) miRNAs. In this study, C. gigantea miRNAs and their target genes were investigated by extracting RNA from young roots, tender stems, young leaves, and flower buds of C. gigantea to establish a small RNA and a degradome library to further sequence. This study identified 194 known miRNAs belonging to 52 miRNA families and 23 novel miRNAs. Among these, 158 miRNAs from 27 miRNA families were highly conserved and existed in a plurality of plants. In addition, 60 different targets for 30 known families and one target for novel miRNA were identified by high-throughput sequencing and degradome analysis in C. gigantea. The comparison results revealed that highly conserved miRNAs have higher expression levels, more family members and more targets than non-conserved miRNAs, indicating that C. gigantea miRNAs were subjected to the neutral evolution. Meanwhile, these conserved miRNAs were also found to be involved in auxin signal transduction, regulation of transcription, and other developmental processes, which will help further understanding regulatory mechanisms of C. gigantea miRNAs.

Guo W ，Zhang Y ，Wang Q ，Zhan Y ，Zhu G ，Yu Q ，Zhu L ... -  《-》

Deep sequencing of grapevine flower and berry short RNA library for discovery of novel microRNAs and validation of precise sequences of grapevine microRNAs deposited in miRBase.

MicroRNAs (miRNAs) are a class of non-coding RNA molecules which have significant gene regulation roles in organisms. The advent of new high-throughput sequencing technologies has enabled the discovery of novel miRNAs. Although there are two recent reports on high-throughput sequencing analysis of small RNA libraries from different organs of two wine grapevine varieties, there was a significant divergence in the number and kinds of miRNAs sequenced in these studies. More sequencing of small RNA libraries is still important for the discovery of novel miRNAs in grapevine. In this study, a total of 130 conserved grapevine Vitis vinifera miRNA (Vv-miRNA) belonging to 28 Vv-miRNA families were validated, other 80 unconserved Vv-miRNAs including 72 novel potential and 8 known but unconserved ones were found. Fifty-two (52.5%) of these 80 unconserved Vv-miRNAs exhibited differential poly(A)-tailed reverse transcriptase-polymerase chain reaction expression profiles in various grapevine tissues that could further confirm their existence in grapevine, among which 20 were expressed only in grapevine berries, indicating a degree of fruit-specificity. One hundred thirty target genes for 56 unconserved miRNAs could be predicted. The locations of these potential target genes on grapevine chromosomes and their complementary levels with the corresponding miRNAs were also analyzed. These results point to a regulatory role of miRNAs in grapevine berry development and response to various environments.

Wang C ，Wang X ，Kibet NK ，Song C ，Zhang C ，Li X ，Han J ，Fang J ... -  《-》