Evolving dynamics of insect frass fertilizer for sustainable nematode management and potato production.

Potato production faces major challenges from inadequate soil fertility, and nematode infestation, yet synthetic fertilizers and nematicides are costly and harmful to the environment. This study explored the potential of chitin-fortified black soldier fly-composted organic fertilizer (BSFCOF) as a multipurpose organic fertilizer amendment for enhancing potato yield and suppressing potato cyst nematodes (PCN). The BSFCOF was applied at a rate equivalent to 150 kg N ha-1 and fortified with chitin from black soldier fly pupal exuviae at inclusion rates equivalent to 0.5, 1, 2, 3, 4 and 5% chitin. Data were collected on potato growth characteristics, PCN population densities, and soil chemical properties for two growing cycles. Results showed that chitin fortified BSFCOF significantly improved potato growth parameters, chlorophyll concentration, marketable tuber yield and number of marketable tubers. The marketable tuber yield achieved using chitin-fortified BSFCOF was 70 - 362%, and 69 - 238% higher than the values achieved using unfertilized soil during the first and second growing cycles, respectively. Soil amendment with chitin-fortified BSFCOF significantly reduced the number of cysts per 200 g soil-1, number of eggs and J2 per cyst-1, eggs g-1 soil and reproduction rate by 32 - 87%, 9 - 92%, 31- 98% and 31 - 98%, respectively. The PCN suppression increased with chitin inclusion rates. There were significantly higher values for soil pH, ammonium nitrogen, nitrate nitrogen, available phosphorus, calcium, magnesium, potassium, and cation exchange capacity in soil amended with BSFCOF compared to unamended soil. This study demonstrates that BSFCOF fortified with 5% chitin is an effective soil enhancer with multiple benefits, including improved soil fertility, potato performance, and effective management of potato cyst nematodes.

In situ nitrogen mineralization and nutrient release by soil amended with black soldier fly frass fertilizer.

Although black soldier fly frass fertilizer (BSFFF) is effective on crop performance, information on nitrogen (N) mineralization and nutrient release capacity of soils amended with BSFFF is lacking. This study utilized field incubation experiments to investigate the ammonification, nitrification, microbial populations, and quantities of nutrients released by soils amended with BSFFF and commercial organic fertilizer (SAFI) for a period equivalent to two maize cropping seasons. For the control treatment, no BSFFF or SAFI was added. Results indicated that most of the N in BSFFF amended soils was available in the ammonium form, while soils treated with SAFI had higher nitrate concentration. The BSFFF amended soils experienced shorter net immobilization periods of N (30-60 days) compared to SAFI treated soils (60-95 days). Increased rates of mineralization (3-10 times) and nitrification (2-4 times) were observed in soils treated with BSFFF during the second season of application. The BSFFF treated soils showed significantly higher N, phosphorus, and magnesium release than the control. Repeated application of BSFFF led to increased N release by three-folds in the soil. Furthermore, soil amendment with BSFFF increased the populations of bacteria and fungi, reduced soil acidity, and increased phosphorus (two-folds) and magnesium (two-four-folds) release than SAFI treated soils. Our findings highlight the crucial role of BSFFF in improving soil health by addressing the challenges of soil acidity, phosphorus fixation and nutrient mining, which is characteristic of most tropical soils.

Black Soldier Fly-Composted Organic Fertilizer Enhances Growth, Yield, and Nutrient Quality of Three Key Vegetable Crops in Sub-Saharan Africa.

Worldwide, French beans (Phaseolus vulgaris L.), tomato (Solanum lycopersicum L.), and kales (Brassica oleracea L. var. acephala) are considered economically important food crops. There is a rapid decline in their yield due to severe soil degradation. Thus, high commercial fertilizer inputs are crucial, though they remain expensive and inaccessible to resource poor farmers. We investigated the comparative performance of composted black soldier fly frass fertilizer (BSFFF), conventionally composted brewer's spent grain (BSG), commercial organic fertilizer (Evergrow), and mineral [nitrogen, phosphorus, and potassium (NPK)] fertilizer on growth, yield, N use efficiency, and nutritional quality (crude protein, crude fiber, crude fats, ash, and carbohydrate concentrations) of tomatoes, kales, and French beans under greenhouse and open-field conditions for two seasons. The fertilizers were applied at rates equivalent to 371 kg of N ha-1. For each crop, the plots were treated with sole rates of BSFFF, BSG, Evergrow, and NPK to supply 100% of the N required. Additional treatments included a combination of BSFFF and NPK, and BSG and NPK so that each fertilizer supplies 50% of the N required. The control treatment consisted of unfertilized soil. Results show that vegetable yields achieved using a combination of BSFFF and NPK were 4.5, 2.4, and 5.4-folds higher than the yield from the control treatment for tomatoes, kales, and French beans, respectively. The combined application of BSFFF and NPK produced 22-135%, 20-27%, and 38-50% higher yields than sole NPK for tomatoes, kales, and French beans, respectively, under both greenhouse and open-field conditions. The highest agronomic N use efficiency was achieved in sole BSFFF-treated plots compared to sole BSG and Evergrow. The N taken up by the vegetables was significantly higher when BSFFF and NPK were integrated. Vegetables grown using a combination of BSFFF and NPK had the highest crude protein and ash concentrations. Our findings demonstrate that the integration of BSFFF and NPK in vegetable cropping systems at the recommended rate of 1.24 t ha-1 BSFFF and 322 kg ha-1 NPK would improve soil health, boost yield, and nutritional quality of vegetable crops.

Low-cost technology for recycling agro-industrial waste into nutrient-rich organic fertilizer using black soldier fly.

Efforts to recycle organic waste using black soldier fly (BSF) larvae into high-quality alternative protein ingredients in animal feeds and organic fertilizers have gained momentum worldwide. However, there is limited information on waste manipulation to increase nutrient retention for enhanced larval performance and frass fertilizer quality. In the present study, brewer's spent grain with a carbon to nitrogen (C/N) ratio of 11 (control) was amended with sawdust to obtain substrates with C/N ratios of 15, 20, 25 and 30. The effects of substrate C/N ratios on BSF larval yield, waste degradation, biomass conversion efficiency, compost maturity and nutrient levels of frass fertilizer were evaluated. Substrates amended with sawdust did not significantly affect waste degradation efficiency and biomass conversion rates of BSF larvae. The wet and dried larval yields were significantly higher for substrates with C/N ratio of 15 compared to the other amended substrates. An amended substrate with C/N ratio of 15 enhanced nutrients uptake by BSF larvae, and increased nitrogen (N) and phosphorus retention in frass compost by 21 and 15%, respectively. Compost maturation time was shortened to five weeks, as indicated by the stable C/N ratios and high seed germination indices. This study has demonstrated that the amendment of the substrate with sawdust to C/N ratio of 15 could generate compost with desirable nutrients for use as high-quality fertilizer for organic farming.

Exploring Black Soldier Fly Frass as Novel Fertilizer for Improved Growth, Yield, and Nitrogen Use Efficiency of Maize Under Field Conditions.

Black soldier fly frass fertilizer (BSFFF) is increasingly gaining momentum worldwide as organic fertilizer. However, research on its performance on crop production remains largely unknown. Here, we evaluate the comparative performance of BSFFF and commercial organic fertilizer (SAFI) on maize (H513) production. Both fertilizers were applied at the rates of 0, 2.5, 5, and 7.5 t ha-1, and 0, 30, 60, and 100 kg nitrogen (N) ha-1. Mineral fertilizer (urea) was also applied at 0, 30, 60 and 100 kg N ha-1 to establish the N fertilizer equivalence (NFE) of the organic fertilizers. Maize grown in plots treated with BSFFF had the tallest plants and highest chlorophyll concentrations. Plots treated with 7.5 t ha-1 of BSFFF had 14% higher grain yields than plots treated with a similar rate of SAFI. There was a 27% and 7% increase in grain yields in plots treated with 100 kg N ha-1 of BSFFF compared to those treated with equivalent rates of SAFI and urea fertilizers, respectively. Application of BSFFF at 7.5 t ha-1 significantly increased N uptake by up to 23% compared to the equivalent rate of SAFI. Likewise, application of BSFFF at 100 kg N ha-1 increased maize N uptake by 76% and 29% compared to SAFI and urea, respectively. Maize treated with BSFFF at 2.5 t ha-1 and 30 kg N ha-1 had higher nitrogen recovery efficiencies compared to equivalent rates of SAFI. The agronomic N use efficiency (AEN) of maize treated with 2.5 t ha-1 of BSFFF was 2.4 times higher than the value achieved using an equivalent rate of SAFI. Also, the AEN of maize grown using 30 kg N ha-1 was 27% and 116% higher than the values obtained using equivalent rates of SAFI and urea fertilizers, respectively. The NFE of BSFFF (108%) was 2.5 times higher than that of SAFI. Application rates of 2.5 t ha-1 and 30 kg N ha-1 of BSFFF were found to be effective in improving maize yield, while double rates of SAFI were required. Our findings demonstrate that BSFFF is a promising and sustainable alternative to commercial fertilizers for increased maize production.

Biochar and gypsum amendment of agro-industrial waste for enhanced black soldier fly larval biomass and quality frass fertilizer.

Black soldier fly (BSF) (Hermetia illucens L.) is one of the most efficient bio-waste recyclers. Although, waste substrate amendments with biochar or gypsum during composting process are known to enhance nutrient retention, their impact on agro-industrial waste have not been documented. Hence, this study focuses on a comparative effect of agro-industrial waste amended with biochar and gypsum on BSF larval performance, waste degradation, and nitrogen (N) and potassium retention in frass fertilizer. Brewery spent grain was amended with biochar or gypsum at 0, 5, 10, 15 and 20% to determine the most effective rates of inclusion. Amending feedstock with 20% biochar significantly increased wet (89%) and dried (86%) larval yields than the control (unamended feedstock). However, amendment with 15% gypsum caused decrease in wet (34%) and dried (30%) larval yields but conserved the highest amount of N in frass. Furthermore, the inclusion of 20% biochar recorded the highest frass fertilizer yield and gave a 21% increase in N retention in frass fertilizer, while biomass conversion rate was increased by 195% compared to the control. Feedstock amendment with 5% biochar had the highest waste degradation efficiency. Potassium content in frass fertilizer was also significantly enhanced with biochar amendment. At maturity, frass compost with more than 10% inclusion rate of biochar had the highest cabbage seed germination indices (>100%). The findings of this study revealed that initial composting of biochar amended feedstocks using BSF larvae can significantly shorten compost maturity time to 5 weeks with enhanced nutrient recycling compared to the conventional composting methods.

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.

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.

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.

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.

Characterization of microRNAs identified in a table grapevine cultivar with validation of computationally predicted grapevine miRNAs by miR-RACE.

BACKGROUND: Alignment analysis of the Vv-miRNAs identified from various grapevine cultivars indicates that over 30% orthologous Vv-miRNAs exhibit a 1-3 nucleotide discrepancy only at their ends, suggesting that this sequence discrepancy is not a random event, but might mainly derive from divergence of cultivars. With advantages of miR-RACE technology in determining precise sequences of potential miRNAs from bioinformatics prediction, the precise sequences of vv-miRNAs predicted computationally can be verified with miR-RACE in a different grapevine cultivar. This presents itself as a new approach for large scale discovery of precise miRNAs in different grapevine varieties. METHODOLOGY/PRINCIPAL FINDINGS: Among 88 unique sequences of Vv-miRNAs from bioinformatics prediction, 83 (96.3%) were successfully validated with MiR-RACE in grapevine cv. 'Summer Black'. All the validated sequences were identical to their corresponding ones obtained from deep sequencing of the small RNA library of 'Summer Black'. Quantitative RT-PCR analysis of the expressions levels of 10 Vv-miRNA/target gene pairs in grapevine tissues showed some negative correlation trends. Finally, comparison of Vv-miRNA sequences with their orthologs in Arabidopsis and study on the influence of divergent bases of the orthologous miRNAs on their targeting patterns in grapevine were also done. CONCLUSION: The validation of precise sequences of potential Vv-miRNAs from computational prediction in a different grapevine cultivar can be a new way to identify the orthologous Vv-miRNAs. Nucleotide discrepancy of orthologous Vv-miRNAs from different grapevine cultivars normally does not change their target genes. However, sequence variations of some orthologous miRNAs in grapevine and Arabidopsis can change their targeting patterns. These precise Vv-miRNAs sequences validated in our study could benefit some further study on grapevine functional genomics.

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.