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.

Plant variety and cultivar identification is one of the most important aspects in agricultural systems. The large number of varieties or landraces among crop plants has made it difficult to identify and characterize varieties solely on the basis of morphological characters because they are non stable and originate due to environmental and climatic conditions, and therefore phenotypic plasticity is an outcome of adaptation. To mitigate this, scientists have developed and employed molecular markers, statistical tests and software to identify and characterize the required plant cultivars or varieties for cultivation, breeding programs as well as for cultivar-right-protection. The establishment of genome and transcriptome sequencing projects for many crops has led to generation of a huge wealth of sequence information that could find much use in identification of plants and their varieties. We review the current status of plant variety and cultivar identification, where an attempt has been made to describe the different strategies available for plant identification. We have found that despite the availability of methods and suitable markers for a wide range of crops, there is dearth of simple ways of making both morphological descriptors and molecular markers easy, referable and practical to use although there are ongoing attempts at making this possible. Certain limitations present a number of challenges for the development and utilization of modern scientific methods in variety or cultivar identification in many important crops.

Fruit skin coloration is a unique phase in the life cycle of fruiting plants and is mainly attributed to anthocyanin pigments. Anthocyanins are the largest and most diverse group of plant pigments derived from the phenyl propanoid pathway. They are water-soluble phenolic compounds that form part of a large and common group of plant flavonoids. Coloration encompasses several physiological and biochemical changes that happen through differential expression of various developmentally regulated genes. Due to research importance and economic value, Arabidopsis thaliana (chromosome no. = 5) and Vitis vinifera (chromosome no. = 19) have been used for investigations of the structural genes involved in anthocyanin biosynthesis. Thus for this review, V. vinifera is used as a model crop. In anthocyanin biosynthesis, a wide range of constructive genes including phenylalanine ammonia lyase, chalcone synthase and anthocyanidin synthase that are regulated by MYB transcription factors are involved. These genes are coordinately expressed and their levels of expression are positively related to the anthocyanin concentrations. Expression or suppression of the constructive genes contributes to a variety of changes that make fruits visually attractive and edible. Transgenic approaches also have discovered a strong relationship between phenyl propanoid/flavonoid gene expressions for fruit skin coloration. In this study, various developments that have taken place in the last decade with respect to identifying and altering the function of color-related genes are described.

MicroRNAs (miRNAs) play critical regulatory roles mainly through cleaving their target mRNAs or repressing gene translation during plant development. Grapevines are among the most economically important fruit crops with available whole genome sequences. Studies on grapevine miRNAs (Vv-miRNAs) are also widely available. However, studies on the regulation mode of Vv-miRNAs on their target mRNAs during grapevine development have not been studied well, especially at the transcriptome-wide level. Here, six small RNA and mRNA libraries from various grapevine tissues were constructed for Illumina and Degradome sequencing. Subsequently, we systematically analyzed the spatiotemporal variations in the regulation of the target genes of regulation of Vv-miRNAs. In total, 242 known and 132 novel Vv-miRNAs and 193 target mRNAs were identified, including 103 target mRNAs for known and 90 target mRNAs for novel miRNAs, were validated in one or more of the tissues examined. More than 50 % of novel miRNAs were expressed exclusively in the flowers and berries, where they cleaved their target genes in a tissue-specific manner, especially, the breadth of their cleavage sites in flower tissues. Moreover, six novel miRNAs in berries responded to exogenous gibberellin and/or ethylene under a quantitative real time RT-PCR analysis, which confirmed their regulatory functions during berry development. Up to 93.6 % of the known miRNAs were highly conserved in various tissues, where their expression levels exhibited dynamic variations during grapevine development. Significantly, some Vv-miRNA families had one key member that acted as the main regulator of their target genes during grapevine development.

MicroRNAs (miRNAs) are an extensive class of newly identified small RNAs that regulate gene expression at post-transcription level by mRNA cleavage or translation. In our study, we used qRT-PCR and found that Vv-miR164 is expression in grapevine leaves, stems, tendrils, inflorescences, flowers and fruits. In addition, two potential target genes for Vv-miR164 were also found and verified by PPM-RACE and RLM-RACE. The results not only maps the cleavage site of the target mRNA but allowed for detection the expression pattern of cleaved fragments that can indicate the regulatory function of this miRNA on its target genes. These target genes were explored by qRT-PCR where some exhibited different expression patterns from their corresponding miRNA, indicating the cleavage mode of the miRNA on its target genes. The efficient and powerful approach used in this study can help in further understanding of how miRNAs cleaved their target mRNAs. Results from this study prove the importance of Vv-miR164 in regulating development and growth of grapes, and adds to the existing knowledge of small RNAmediated regulation in grapes.