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Small RNA sequencing in grapevine berries from two varieties (Cabernet Sauvignon and Sangiovese) grown in three different environments and sampled at four developmental stages

ABSTRACT: Purpose: The goal of this study is to detect the environmental influence on small RNAs population (miRNAs and other smallRNAs) in berries from two different grapevine varieties, evaluating Genotype x Environment interactions. Methods: The grapevine berries were collected in liquid nitrogen and their small RNA fractions were isolated from total RNA for library construction and subsequent sequencing on an Illumina HiSeq 2000. Results: Reads were mapped to the reference Vitis vinifera L. genomic sequence V1 (PN40024, (Jaillon et al., 2007) using Bowtie and retaining reads perfectly aligned to the genome. The static clustering analysis was carried out, using a proximity-based pipeline built with custom Perl and database scripts and MySQL database queries, to group and quantify clusters of small RNAs. The identification of annotated (conserved or known) and novel (or specie-specific) miRNAs was carried out applying a conservative and robust pipeline. Conclusions: Our results show that the distribution of small RNA-producing loci is variable between the two cultivars, and that the level of variation depends on the vineyard. Differently, the profile of miRNA accumulation mainly depends on the developmental stage. One of the environment, Riccione, maximizes the differences between the varieties, promoting the production of more than one thousand specific small RNA loci and modulating their expression depending on the cultivar and the maturation stage.

ORGANISM(S): Vitis vinifera

PROVIDER: GSE85611 | GEO | 2016/08/16

SECONDARY ACCESSION(S): PRJNA338946

REPOSITORIES: GEO

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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: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.

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Small RNA sequencing in grapevine berries from two varieties (Cabernet Sauvignon and Sangiovese) grown in three different environments and sampled at four developmental stages

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