Microarray analysis of differentially expressed genes engaged in fruit development between table and wine grape.

Microarray analysis of genes can provide individual gene-expression profiles and new insights for elucidating biological mechanisms responsible for fruit development. To obtain an overall view on expression profiles of metabolism-related genes involved in fruit development of table and wine grapes, a microarray system comprising 15,403 ESTs was used to compare the expressed genes. The expression patterns from the microarray analysis were validated with quantitative real-time polymerase chain reaction analysis of 18 selected genes of interest. During the entire fruit development stage, 2,493 genes exhibited at least 2.0-fold differences in expression levels with 1,244 genes being up-regulated and 1,249 being down-regulated. Following gene ontology analysis, only 929 differentially expressed (including 403 up-regulated and 526 down-regulated) genes were annotated in table and wine grapes. These differentially expressed genes were found to be mainly involved in carbohydrate metabolism, biosynthesis of secondary metabolites as well as energy, lipid and amino acid metabolism via KEGG. Our results provide new insights into the molecular mechanisms and expression profiles of genes in the fruit development stage of table and wine grapes.

Characterization of regulatory mechanism of Poncirus trifoliata microRNAs on their target genes with an integrated strategy of newly developed PPM-RACE and RLM-RACE.

MicroRNAs (miRNAs) play an important role in post-transcriptional gene regulation that involved various biological and metabolic processes. Many extensive studies have been done in model plant species, to discover miRNAs' regulating expression of their target genes and analyze their functions. But, the function of Poncirus trifoliata miRNAs has not been properly investigated. In this study, we employed the RNA ligase-mediated 5' rapid amplification of cDNA ends (RLM-RACE) and the newly developed method called poly (A) polymerase-mediated 3' rapid amplification of cDNA ends (PPM-RACE), which mapped the cleavage site of target mRNAs and detected expression patterns of cleaved fragments that could in turn indicate the regulatory functions of the miRNAs on their target genes. Furthermore, the spatiotemporal expression levels of target genes were analyzed by qRT-PCR, with exhibiting different expression trends from their corresponding miRNAs, thus indicating the cleavage mode of miRNAs on their target genes. The expression patterns of miRNAs, their target mRNAs and cleaved target mRNAs in different organs of juvenile and adult trifoliate orange were studied. The results showed that the expression of miRNAs and their target mRNAs was in a trade-off trend. When the miRNA expression was high, its corresponding target mRNA expression was low, while the cleaved target mRNA expression was high; when the miRNA expression was low, its target mRNA expression was high, while the expression of cleaved target mRNAs follows that of the miRNA. The validation of the cleavage site of target mRNAs and the detection of expression patterns of cleaved fragments can further broaden the knowledge of small RNA-mediated regulation in P. trifoliate.

Transcriptome-wide analysis of dynamic variations in regulation modes of grapevine microRNAs on their target genes during grapevine development.

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.

Evaluation of genome sequencing quality in selected plant species using expressed sequence tags.

BACKGROUND: With the completion of genome sequencing projects for more than 30 plant species, large volumes of genome sequences have been produced and stored in online databases. Advancements in sequencing technologies have reduced the cost and time of whole genome sequencing enabling more and more plants to be subjected to genome sequencing. Despite this, genome sequence qualities of multiple plants have not been evaluated. METHODOLOGY/PRINCIPAL FINDING: Integrity and accuracy were calculated to evaluate the genome sequence quality of 32 plants. The integrity of a genome sequence is presented by the ratio of chromosome size and genome size (or between scaffold size and genome size), which ranged from 55.31% to nearly 100%. The accuracy of genome sequence was presented by the ratio between matched EST and selected ESTs where 52.93% ∼ 98.28% and 89.02% ∼ 98.85% of the randomly selected clean ESTs could be mapped to chromosome and scaffold sequences, respectively. According to the integrity, accuracy and other analysis of each plant species, thirteen plant species were divided into four levels. Arabidopsis thaliana, Oryza sativa and Zea mays had the highest quality, followed by Brachypodium distachyon, Populus trichocarpa, Vitis vinifera and Glycine max, Sorghum bicolor, Solanum lycopersicum and Fragaria vesca, and Lotus japonicus, Medicago truncatula and Malus × domestica in that order. Assembling the scaffold sequences into chromosome sequences should be the primary task for the remaining nineteen species. Low GC content and repeat DNA influences genome sequence assembly. CONCLUSION: The quality of plant genome sequences was found to be lower than envisaged and thus the rapid development of genome sequencing projects as well as research on bioinformatics tools and the algorithms of genome sequence assembly should provide increased processing and correction of genome sequences that have already been published.

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.

Bioinformatics prediction of miRNAs in the Prunus persica genome with validation of their precise sequences by miR-RACE.

We predicted 262 potential MicroRNAs (miRNAs) belonging to 70 miRNA families from the peach (Prunus persica) genome and two specific 5' and 3' miRNA rapid amplification of cDNA ends (miR-RACE) PCR reactions and sequence-directed cloning were employed to accurately validate 61 unique P. persica miRNAs (Ppe-miRNAs) sequences belonging to 61 families comprising 97 Ppe-miRNAs. Validation of the termini nucleotides in particular can define the real sequences of the Ppe-miRNAs on peach genome. Comparison between predicted and validated Ppe-miRNAs through alignment revealed that 43 unique orthologous sequences were identical, while the remaining 18 exhibited some divergences at their termini nucleotides. Quantitative real-time polymerase chain reaction (qRT-PCR) was further employed to analyze the expression of all the 61 miRNAs and 10 putative targets of 8 randomly selected Ppe-miRNAs in peach leaves, flowers and fruits at different stages of development, where both the miRNAs and the putative target genes showed tissue-specific expression.

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.

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

BACKGROUND: MicroRNA (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. RESULTS: A 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. CONCLUSIONS: Deep 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.