Identification of microRNAs from Amur grape (vitis amurensis Rupr.) by deep sequencing and analysis of microRNA variations with bioinformatics

Abstract

BackgroundMicroRNA (miRNA) is a class of functional non-coding small RNA with 19-25 nucleotides in length while Amur grape (Vitis amurensis Rupr.) is an important wild fruit crop with the strongest cold resistance among the Vitis species, is used as an excellent breeding parent for grapevine, and has elicited growing interest in 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.ResultsA small RNA library from Amur grape 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 miRNAs belonging to 27 miRNA families were identified, and 34 known but non-conserved miRNAs were also found. Significantly, 72 new potential Amur grape-specific miRNAs were discovered. The sequences of these new potential va-miRNAs were further validated through miR-RACE, and accumulation of 18 new va-miRNAs in seven tissues of grapevines confirmed by real time RT-PCR (qRT-PCR) analysis. The expression levels of va-miRNAs in flowers and berries were found to be basically consistent in identity to those from deep sequenced sRNAs libraries of combined 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 further reveal that the number and sites of miR-SNP in diverse miRNA families exhibit distinct divergence. Finally, 346 target genes for the new miRNAs were predicted and they include a number of Amur grape stress tolerance genes and many genes regulating anthocyanin synthesis and sugar metabolism.ConclusionsDeep sequencing of short RNAs from Amur grape flowers and berries identified 72 new potential miRNAs and 34 known but non-conserved miRNAs, indicating that specific miRNAs exist in Amur grape. These results show that a number of regulatory miRNAs exist in Amur grape and play an important role in Amur grape growth, development, and response to abiotic or biotic stress.

Identification and Validation of Potential Conserved microRNAs and Their Targets in Peach (Prunus persica)

“…Our results show that two potential target genes for the two ppemiRNAs had specific cleavage sites corresponding to their miRNA complementary sequences. Furthermore, it was also observed that, consistent with previous reports (Debernardi et al, 2012;Song et al, 2010b;Wang et al, 2012), ppe-miRNA targets have an miRNA-complementary site located in their coding regions.…”

“…This allows for the prediction of miRNA targets by computational approaches. Several studies have utilised this fact and identified many target genes in different plant species while allowing 1-3 nucleotide mismatches between the target mRNAs and miRNAs (Song et al, 2009;Wang et al, 2012;Zhang et al, 2008).…”

The Accumulation of miRNAs Differentially Modulated by Drought Stress Is Affected by Grafting in Grapevine

“…The majority (70%) of conserved miRNA sequences fell within the 21-nucleotide category (Fig. 3A), one of the most abundant size classes for known grapevine DCL1-derived products Pantaleo et al, 2010;Wang et al, 2012;Belli Kullan et al, 2015). Among novel candidate miRNAs, two major (each above 30%) size categories were observed at 21 and 22 nucleotides (Fig.…”

“…Previous studies identified conserved and novel miRNAs in grapevine (Carra et al, 2009;Mica et al, 2010;Pantaleo et al, 2010Pantaleo et al, , 2016Wang et al, 2012), and a grapevine miRNA expression atlas was published recently (Belli Kullan et al, 2015). However, no studies specifically addressing the identification of drought stress (DS)-responsive miRNAs have been reported in Vitis spp.…”

MicroRNAs Sequencing for Understanding the Genetic Regulation of Plant Genomes

“…qRT-PCR is one of the most used techniques for detection of miRNA expression due for its high sensitivity to miRNA detection, the capability to identify single nucleotide changes [53]. miR-RACE (PCR-based) is an effective method to determine the precise sequence of miRNA at their 5´ and 3´ ends, which can distinguish between members of a miRNA family, and they can determine expression patterns at different family member levels [53,54].…”