Although several studies on grape miRNAs have recently been reported [20
], the grape materials used in all the studies reported belong to the cultivated grape Vitis vinifera
or hybrids of V. vinifera
and V. labrusca
. Amur grape (Vitis amurensis Rupr
.) is one of the most important wild species in the grape family. Currently, only a few genes involved in resistance to downy mildew have been identified in Amur grape [45
] and there are no known reports on Amur grape miRNAs. The identification of entire sets of miRNAs and their targets will lay a foundation for elucidation of the complex miRNA-mediated regulatory systems which control development and other physiological processes [4
]. Systematic studies on miRNAs in Amur grape will contribute to gaining insight into the mechanisms controlling growth and development in this grape species. The advent of deep sequencing technologies has greatly enhanced the capacity of sRNA exploration, and in turn provided a rapid way to identify non-conserved, low accumulation, species-specific as well as conserved miRNAs on a large scale. In this study, using Solexa sequencing, we provide evidence supporting the existence of 106 new and potential, as well as 126 conserved miRNAs in Amur grape. Use of deep sequencing technology, has led to identification of many new grape miRNAs in Vitis vinifera
] or hybrids of V. vinifera
and V. labrusca
], and ITS employment in our study led to the identification of 72 additional miRNAs that are specific to the Amur grape species. Future large scale experimental approaches in more plants are likely to identify additional species-specific miRNAs.
The aim of this work was to identify miRNAs present in Amur grape including miRNAs evolutionary conserved in other plants. Searches for conserved miRNAs revealed that many va-miRNAs have orthologs in other plants like Arabidopsis
], rice [14
], poplar [15
], Solanum lycopersicum
], peanuts [17
], citrus [43
], wheat [33
] etc. In addition, deep sequencing of the small RNA library allowed for identification of the expression levels of each member of a miRNA family. Sequence analysis reveals that the relative abundance of certain members within the miRNA families varied drastically, from 2 to 372,442 copies. Amongst the conserved miRNAs, va-miR166h topped the list in terms of copy numbers, but the annotation of its function is still not available, consistent with the observation in Summer Black grapevine which is a hybrid of V. vinifera
and V. labrusca
]. Conversely, va-miR171h, va-miR169i and va-miR159a/b appeared to have only less than 10 copies each (Figure ). We also discovered that these conserved miRNAs have their orthologs in nearly 30 other plant species/varieties (Table ), as indicated in previous miRNA reports [48
]. Some miRNAs are conserved in several plant species, an aspect which will provide an opportunity for assessment of evolution of these families across diverse plants. Seventy two new and potential va-miRNAs were specific to Amur grape and were not detected in other vitis species; while another twenty-four were only conserved in Vitis amurensis
Rupr. and Vitis vinifera
L. These open the possibility that these miRNA families could have descended from a common ancestor and diverged or were lost during evolution of vitis plants. It is also possible that regulatory interactions directed by these vitis-specific miRNAs are involved in the adaptation to the diverse ecological environments.
Since the roles of miRNAs in development and biotic or abiotic stress regulation are executed through the cleavage or translation repression of target genes, miRNA target prediction is critical for gaining insight to regulatory functions of miRNAs. Before this report, target genes for conserved miRNAs in grape had been systematically investigated in other vitis species [20
]. Conserved miRNAs have high sequence conservation in closely related plant species and thus their target genes also possess some conservation of functions. For non-conserved and new or potential va-miRNAs, we reveal that a number of disease resistance, stress resistance, anthocyanin synthesis regulatory genes as well as some other genes related to secondary metabolism and sugar metabolism might be targeted by va-miRNAs based on previous reports on functional annotation of orthologous genes in other plants [17
]. Outstandingly, we found 11 important transcript factors and regulators for va-miRNAs, and these might play a wide range of vital roles during development or response to stress in Amur grape. Other target genes were associated with signal transduction, metabolism, transport, growth and development processes, which is similar to studies by Wu et al.
] in the Amur grape DNA library. All these observations indicate that both the new and non-conserved va-miRNAs identified in this study might play extensive regulatory roles, not only in development, but also in stress response and diverse physiological processes.
The availability of a full grape genome and plenty of grape ESTs helped us to identify 72 new, 34 non-conserved va-miRNAs and 346 target genes. It has been reported that target prediction for new miRNAs may yield some false positives [57
]. However, blast search using miRNA sequences against EST databases can also be a good way of identifying potential targets of the new miRNAs, followed by necessary experimental verification. In this work, we employed the 5'-RLM-RACE method to detect miRNA-guided cleavage of target mRNAs of five Va-miRNAs, where the results showed that five potential target genes for the five Va-miRNAs had specific cleavage sites corresponding to their miRNA complementary sequences. This confirms the actual existence of these potential va-miRNA target genes. For a thorough investigation of the real existence of all the potential target genes, systematic research should be planned in the next phase of this work. In addition, there is no current information available regarding the functions of more than 1/4 of the target genes identified, which makes it difficult to determine whether these miRNA targets have functional bias. Furthermore, it was also observed that consistent with previous reports [55
], most va-miRNA targets have a miRNA-complementary site located in their coding regions and occasionally in their 3' or 5'UTRs. Previous reports indicate that plants usually have a lower number of genes targeted by a single miRNA than in animals. For instance, each drosophila miRNA has on average over 50 predicted targets [58
], while most Arabidopsis
miRNAs have six targets or fewer [4
]. In contrast, some va-miRNAs in this study have more target genes than those reported for drosophila and even for Arabidopsis
, for example va-miR006 which has 103 target genes, a finding that might imply that Amur grape-specific miRNAs have more extensive functions. This study pioneers the first large scale cloning and characterization of Amur grape miRNAs and their predicted targets thus laying a foundation for future functional studies.