Identification of Known and Novel microRNAs and Their Targets in Peach (Prunus persica) Fruit by High-Throughput Sequencing.

MicroRNAs (miRNAs) are a group of non-coding RNAs that have functions in post-transcriptional gene regulation in plants. Although the most important economic component of peach trees (Prunus persica) is the fruit, not much is known about miRNAs in this organ. In this study, miRNAs and their targets were identified and characterized from libraries of small RNAs of peach fruit through Solexa based-sequencing and bioinformatics approaches. A total of 557 known peach miRNAs belonging to 34 miRNA families were identified, and some of these miRNAs were found to be highly conserved in at least four other plant species. Using the most current criteria for miRNA annotation, 275 putative novel miRNAs were predicted, and the sequencing frequencies of these novel miRNAs were less than those of the conserved miRNAs. In total, 3959 and 1614 target genes for 349 known and 193 novel miRNAs, respectively, were predicted with the criteria that a single target gene can be targeted by different miRNAs and that a single miRNA can also have a large number of target genes. Three targets were even found to be targeted by 13 novel miRNAs that contained the same complete miRNA sequence at different locations and had different scaffolds. The proteins predicted to be targeted by the miRNAs identified in this study encompass a wide range of transcription factors and are involved in many biological processes and pathways, including development, metabolism, stress responses and signal transduction. A total of 115 and 101 target genes were identified to be cleaved by 60 known miRNAs and 27 novel miRNAs through degradome sequencing, respectively. These miRNAs induce cleavage of their targets precisely at the position between nucleotides 10 and 11 of the miRNA sequences from the 5' to the 3' end. Thirty conserved miRNAs and 19 novel miRNAs exhibited differential expression profiles in the peach, and the expression patterns of some miRNAs appeared to be tissue- or developmental stage-specific. The findings of this study provide an important basis for the analysis of miRNAs, their targets and the functions of these targets in peach fruit.

Advances in identification and validation of plant microRNAs and their target genes.

Developments in the field of molecular biology and genetics, such as microarray, gene transfer and discovery of small regulatory RNAs, have led to significant advances in plant biotechnology. Among the small RNAs, microRNAs (miRNAs) have elicited much interest as key post-transcriptional regulators in eukaryotic gene expression. Advances in genome and transcriptome sequencing of plants have facilitated the generation of a huge wealth of sequence information that can find much use in the discovery of novel miRNAs and their target genes. In this review, we present an overview of the developments in the strategies and methods used to identify and study miRNAs, their target genes and the mechanisms by which these miRNAs interact with their target genes since the discovery of the first miRNA. The approaches discussed include both reverse and forward genetics. We observed that despite the availability of advanced methods, certain limitations ranging from the cost of materials, equipment and personnel to the availability of genome sequences for many plant species present a number of challenges for the development and utilization of modern scientific methods for the elucidation and development of miRNAs in many important plant species.

Computational identification of microRNAs in the strawberry (Fragaria x ananassa) genome sequence and validation of their precise sequences by miR-RACE.

In plants, microRNAs (miRNAs) play significant roles in post-transcriptional gene regulation and have been found to control many genes involved in different biological and metabolic processes. Extensive studies were carried out to discover miRNAs and analyze their functions in model plant species, such as in Arabidopsis and rice that have been reported. In this research, we used bioinformatics to predict microRNAs in an important strawberry rootstock cultivar to discover and validate precise sequences of microRNAs in strawberry. By adopting a range of filtering criteria, we obtained 59 potential miRNAs belonging to 40 miRNA families from the Fragaria vesca genome. Using two specific 5' and 3' miRNA RACE PCR reactions and a sequence-directed cloning method, we accurately determined 34 precise sequences of candidate miRNAs, while six other sequences exhibited some minor divergence in their termini nucleotides, and 19 miRNAs that could not be cloned owing to expression abundance may be too low or these mirRNAs predicted could not be existing in strawberry. Potential target genes were further predicted for the miRNAs above. The expression of the 16 miRNAs unreported and having exact sequences and their targets by experiment could be detected in different tissues of strawberry ranging from roots, stems, leaves, flowers and fruits by qRT-PCR and some of them showed differential expression in various tissues. The functional analysis of 16 miRNAs and their targets was carried out. Finally, we conclude that there are 34 mirRNAs in strawberry and their targets play vital roles not only in growth and development, but also in diverse physiological processes. These results show that regulatory miRNAs exist in agronomically important strawberry and might have an important function in strawberry growth and development.

Characterization of target mRNAs for grapevine microRNAs with an integrated strategy of modified RLM-RACE, newly developed PPM-RACE and qPCRs.

MicroRNAs (miRNAs) regulate target gene expression by mediating target gene cleavage or inhibition of translation at transcriptional and post-transcriptional levels in higher plants. Until now, many grapevine microRNAs (Vv-miRNAs) have been identified and quite a number of miRNA target genes were also verified by various analysis. However, global interaction of miRNAs with their target genes still remained to perform more research. We reported experimental validation of a number of miRNA target genes in table grapevine that had been previously identified by bioinformatics in our earlier studies. To verify more predicted target genes of Vv-miRNAs and elucidate the modes by which these Vv-miRNAs work on their target genes, 31 unverified potential target genes for 18 Vv-miRNAs were experimentally verified by a new integrated strategy employing a modified 5'-RLM-RACE (RNA ligase-mediated 5' rapid amplification of cDNA ends), 3'-PPM-RACE (poly(A) polymerase-mediated 3' rapid amplification of cDNA ends) and qRT-PCRs of cleavage products. The results showed that these Vv-miRNAs negatively regulated expression of their target messenger RNAs (mRNAs) through guiding corresponding target mRNA cleavage, of which about 94.4% Vv-miRNAs cleaved their target mRNAs mainly at the tenth nucleotide of 5'-end of miRNAs. Expression levels of both miRNAs and their target mRNAs in eight tissues exhibited inverse relationships, and expressions both of cleaved targets and miRNAs indicated a cleavage mode of Vv-miRNAs on their target genes. Our results confirm the importance of Vv-miRNAs in grapevine growth and development, and suggest more study on Vv-miRNAs and targets can enrich the knowledge of miRNA mediated-regulation in grapevine.

Characterization and expression profiling of selected microRNAs in tomato (Solanum lycopersicon) 'Jiangshu14'.

Presence of selected tomato (Solanum lycopersicon) microRNAs (sly-miRNAs) was validated and their expression profiles established in roots, stems, leaves, flowers and fruits of tomato variety Jiangshu14 by quantitative RT-PCR (qRT-PCR). In addition conservation characteristics these sly-miRNAs were analyzed and target genes predicted bioinformatically. Results indicate that some of these miRNAs are specific to tomato while most are conserved in other plant species. Predicted sly-miRNA targets genes were shown to be targeted by either by a single or more miRNAs and are involved in diverse processes in tomato plant growth and development. All the 36 miRNAs were present in the cDNA of mixed tissues and qRT-PCR revealed that some of these sly-miRNAs are ubiquitous in tomato while others have tissue-specific expression. The experimental validation and expression profiling as well target gene prediction of these miRNAs in tomato as done in this study can add to the knowledge on the important roles played by these sly-miRNAs in the growth and development, environmental stress tolerance as well as pest and disease resistance in tomatoes and related species. In addition these findings broaden the knowledge of small RNA-mediated regulation in S. lycopersicon. It is recommended that experimental validation of the target genes be done so as to give a much more comprehensive information package on these miRNAs in tomato and specifically in the selected variety.

Whole genome identification and analysis of FK506-binding protein family genes in grapevine (Vitis vinifera L.).

In plant and animal species FK506-binding protein (FKBP) family genes are important conserved genes and it is defined as the receptors of FK506 and rapamycin, where they work as PPIase and protein folding chaperones. FKBP have been isolated from Arabidopsis thaliana, Oryza sativa, and Zea mays. In grape, twenty-three genes containing the FK506-binding domain (FKBP_C) were first time identified by HMMER and blast research, they were classified into three groups and 17 out of the 23 genes were located on 11 chromosomes (Chr1, 3, 5, 7, 8, 14, 15, 16, 17, 18, and 19). The predicted gene expression pattern and semi-quantitative RT-PCR results revealed that five VvFKBPs were expressed in all tissues, while seven VvFKBPs were expressed only in some of the tissues, and the remaining VvFKBPs were not expressed in leaf, stem, inflorescences, flowers, and a mixture of fruit tissues (small, medium and big-sized fruits). Most of the VvFKBPs in grapevine 'Summer Black' were similar to those predicted one in 'Pinot Noir' except for VvFKBP16-4 and VvFKBPa. VvFKBP12, FaFKBP12 and PpFKBP12 were cloned from 'Summer Black', 'Sweet Charlie' and 'Xiahui 6'. Protein structure analysis confirmed that homologous genes have some differences during the process of protein structure construction. In this study, we characterized and verified 23 FKBP family genes in grapevine (Vitis vinifera L.) as well as their sub-cellular and chromosome location. The successful cloning of CDS regions and protein structural analysis of VvFKBP12, FaFKBP12, and PpFKBP12 can provide useful information for further study.

Plant variety and cultivar identification: advances and prospects.

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.

Microarray analysis of differentially expressed genes engaged in fruit development between Prunus mume and Prunus armeniaca.

Microarray analysis is a technique that can be employed to provide expression profiles of single genes and new insights to elucidate the biological mechanisms responsible for fruit development. To evaluate expression of genes mostly engaged in fruit development between Prunus mume and Prunus armeniaca, we first identified differentially expressed transcripts along the entire fruit life cycle by using microarrays spotted with 10,641 ESTs collected from P. mume and other Prunus EST sequences. A total of 1418 ESTs were selected after quality control of microarray spots and analysis for differential gene expression patterns during fruit development of P. mume and P. Armeniaca. From these, 707 up-regulated and 711 down-regulated genes showing more than two-fold differences in expression level were annotated by GO based on biological processes, molecular functions and cellular components. These differentially expressed genes were found to be involved in several important pathways of carbohydrate, galactose, and starch and sucrose metabolism as well as in biosynthesis of other secondary metabolites via KEGG. This could provide detailed information on the fruit quality differences during development and ripening of these two species. With the results obtained, we provide a practical database for comprehensive understanding of molecular events during fruit development and also lay a theoretical foundation for the cloning of genes regulating in a series of important rate-limiting enzymes involved in vital metabolic pathways during fruit development.

Characterization of grapevine microR164 and its target genes.

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 RNA-mediated regulation in grapes.

Deep sequencing discovery of novel and conserved microRNAs in trifoliate orange (Citrus trifoliata).

BACKGROUND: MicroRNAs (miRNAs) play a critical role 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. Deep sequencing technologies have facilitated identification of species-specific or lowly expressed as well as conserved or highly expressed miRNAs in plants. RESULTS: In this research, we used Solexa sequencing to discover new microRNAs in trifoliate orange (Citrus trifoliata) which is an important rootstock of citrus. A total of 13,106,753 reads representing 4,876,395 distinct sequences were obtained from a short RNA library generated from small RNA extracted from C. trifoliata flower and fruit tissues. Based on sequence similarity and hairpin structure prediction, we found that 156,639 reads representing 63 sequences from 42 highly conserved miRNA families, have perfect matches to known miRNAs. We also identified 10 novel miRNA candidates whose precursors were all potentially generated from citrus ESTs. In addition, five miRNA* sequences were also sequenced. These sequences had not been earlier described in other plant species and accumulation of the 10 novel miRNAs were confirmed by qRT-PCR analysis. Potential target genes were predicted for most conserved and novel miRNAs. Moreover, four target genes including one encoding IRX12 copper ion binding/oxidoreductase and three genes encoding NB-LRR disease resistance protein have been experimentally verified by detection of the miRNA-mediated mRNA cleavage in C. trifoliata. CONCLUSION: Deep sequencing of short RNAs from C. trifoliata flowers and fruits identified 10 new potential miRNAs and 42 highly conserved miRNA families, indicating that specific miRNAs exist in C. trifoliata. These results show that regulatory miRNAs exist in agronomically important trifoliate orange and may play an important role in citrus growth, development, and response to disease.