Project description:MicroRNAs (miRNAs) play a important part in post-transcriptional gene regulation and have been shown to control many genes involved in various biological and metabolic processes. There have been extensive studies to discover miRNAs and analyze their functions in model plant species, such as Arabidopsis and rice and other plants. However, the number of miRNAs discovered in grape is relatively low and little is known about miRNAs responded gibberellin during fruit germination. In this study, a small RNA library from gibberellin grape fruits was sequenced by the high throughput sequencing technology. A total of 16,033,273 reads were obtained. 812,099 total reads representing 1726 unique sRNAs matched to known grape miRNAs. Further analysis confirmed a total of 149 conserved grapevine miRNA (Vv-miRNA) belonging to 27 Vv-miRNA families were validated, and 74 novel potential grapevine-specific miRNAs and 23 corresponding novel miRNAs* were discovered. Twenty-seven (36.5%) of the novel miRNAs exhibited differential QRT-PCR expression profiles in different development gibberellin-treated grapevine berries that could further confirm their existence in grapevine. QRT-PCR analysis on transcript abundance of 27 conserved miRNA family and the new candidate miRNAs revealed that most of them were differentially regulated by the gibberellin, with most conserved miRNA family and 26 miRNAs being specifically induced by gibberellin exposure. All novel sequences had not been earlier described in other plant species. In addition, 117 target genes for 29 novel miRNAs were successfully predicted. Our results indicated that miRNA-mediated gene expression regulation is present in gibberellin-treated grape berries. This study led to the confirmation of 101 known miRNAs and the discovery of 74 novel miRNAs in grapevine. Identification of miRNAs resulted in significant enrichment of the gibberellin of grapevine miRNAs and provided insights into miRNA regulation of genes expressed in grape berries. GSM604831 is the control for the gibberellin-treated sample. The mixture samples of young berries (one week after flowering) large berries (five week after flowering after flowering), and old berries (nine week after flowering) treated with gibberellin, respectively, were generated by deep sequencing, in triplicate, using Illumina 1G Genome Analyzer.

Project description: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 revelation of novel miRNAs. Although there are two recent reports on high throughput sequencing analysis of small RNA libraries from different organs of two grapevine wine varieties, there were 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. Here, we initially constructed a small RNA library of flower and fruit tissues of a table grapevine cultivar M-bM-^@M-^XSummer BlackM-bM-^@M-^Y and performed sequencing and analysis of sRNAs using the Illumina Solexa platform, expecting to discover more miRNAs related to the development of grapevine flowers and berries and the formation of dessert quality in grapevine berries. Totally, 130 conserved grapevine miRNA (Vv-miRNA) belonging to 28 Vv-miRNA families were validated, and 92 novel potential grapevine-specific ones representing 80 unique ones were first discovered. Forty-two (48.84%) of the novel miRNAs possessed differential semi-quantitative PCR expression profiles in various grapevine tissues that could further confirm their existence in the grapevine, among which twenty were expressed only in grapevine berries, indicating some fruit-specificity. 130 target genes for 46 novel 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. Size fractionated small RNAs (16-30 bp) from total RNA extracts was ligated to 5' and 3' adapters, and reverse transcribed. After PCR amplification the sample was subjected to Solexa sequencing. The resultant 35nt sequence data were filtered according to base quality value. The remained sequences were used to trim 5' and 3' adaptors. The clean tags were used for further analysis.

Project description:MicroRNA (miRNA) is a class of functional non-coding small RNA with 19-25 nucleotides in length. Amur grape (Vitis amurensis Rupr.) is an important wild fruit crop with the strongest cold resistance in the Vitis genus and is used as an excellent breeding parent for grapevine, and with growing interest in terms of wine production. To date, there is a relatively large number of grapevine miRNAs (vv-miRNAs) from cultivated grapevine varieties such as Vitis vinifera L. and hybrids of V. vinifera and V. labrusca, but there is no report on miRNAs from Vitis amurensis Rupr, a wild grapevine species. In this study, a small RNA library from Amur grapes was constructed and Solexa technology used to perform deep sequencing of the library followed by subsequent bioinformatics analysis to identify new miRNAs. In total, 126 conserved miRNA belonging to 27 miRNA families were identified, and 34 known but non-conserved miRNAs were also found. Significantly, 72 new potential Amur grapevine-specific miRNAs were discovered. The sequences of these new potential va-miRNAs were further validated through miR-RACE, accumulation of 18 new va-miRNAs in seven tissues of grapevines were also confirmed by real time RT-PCR (qRT-PCR) analysis, and expression levels of va-miRNAs in flowers and berries were basically consistent in identity to those from deep sequenced sRNAs libraries of independent corresponding tissues. We also describe the conservation and variation of va-miRNAs using miR-SNPs and miR-LDs during plant evolution based on comparison of orthologous sequences, and revealed the number and sites of miR-SNP of diverse miRNA families exhibited distinct divergence. Finally, 346 target genes for the new miRNAs were predicted and they include a number of Amur grapevine stress tolerance genes and many genes regulating anthocyanin systhesis and sugar metabolism. Deep sequencing of short RNAs from Amur grapes flowers and fruits identified 72 new potential miRNAs and 34 known but non-conserved miRNAs, indicating that specific miRNAs exist in Amur grapes. These results show that a number of regulatory miRNAs exist in Amur grapes and play an important role in Amur grape growth, development, and response to abiotic or biotic stress. High throughput sequencing was employed to identify miRNAs in Amur grapevine and try to describe their functions in Amur grapevine growth and development.

Project description:MicroRNA (miRNA) is a class of functional non-coding small RNA with 19-25 nucleotides in length. Amur grape (Vitis amurensis Rupr.) is an important wild fruit crop with the strongest cold resistance in the Vitis genus and is used as an excellent breeding parent for grapevine, and with growing interest in terms of wine production. To date, there is a relatively large number of grapevine miRNAs (vv-miRNAs) from cultivated grapevine varieties such as Vitis vinifera L. and hybrids of V. vinifera and V. labrusca, but there is no report on miRNAs from Vitis amurensis Rupr, a wild grapevine species. In this study, a small RNA library from Amur grapes was constructed and Solexa technology used to perform deep sequencing of the library followed by subsequent bioinformatics analysis to identify new miRNAs. In total, 126 conserved miRNA belonging to 27 miRNA families were identified, and 34 known but non-conserved miRNAs were also found. Significantly, 72 new potential Amur grapevine-specific miRNAs were discovered. The sequences of these new potential va-miRNAs were further validated through miR-RACE, accumulation of 18 new va-miRNAs in seven tissues of grapevines were also confirmed by real time RT-PCR (qRT-PCR) analysis, and expression levels of va-miRNAs in flowers and berries were basically consistent in identity to those from deep sequenced sRNAs libraries of independent corresponding tissues. We also describe the conservation and variation of va-miRNAs using miR-SNPs and miR-LDs during plant evolution based on comparison of orthologous sequences, and revealed the number and sites of miR-SNP of diverse miRNA families exhibited distinct divergence. Finally, 346 target genes for the new miRNAs were predicted and they include a number of Amur grapevine stress tolerance genes and many genes regulating anthocyanin systhesis and sugar metabolism. Deep sequencing of short RNAs from Amur grapes flowers and fruits identified 72 new potential miRNAs and 34 known but non-conserved miRNAs, indicating that specific miRNAs exist in Amur grapes. These results show that a number of regulatory miRNAs exist in Amur grapes and play an important role in Amur grape growth, development, and response to abiotic or biotic stress.

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Genome-wide identiﬁcation of Vitis vinifera microRNAs reveals their differential regulation by gibberellin

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