Simultaneous extraction and preliminary purification of polyphenols from grape pomace using an aqueous two-phase system exposed to ultrasound irradiation: Process characterization and simulation.

In this study, an ultrasound-assisted aqueous two-phase (ATP) extraction method was used for the extraction and purification of phenolic compounds from grape pomace. The effect of acoustic energy densities (AED, 41.1, 63.5, 96.1, 111.2 W/L) and temperatures (20, 30, 40°C) on the yield of phenolics was investigated. An artificial neural network (ANN) was successfully used to correlate the extraction parameters with phenolic yield. Then, a diffusion model based on Fick's second law was used to model the mass transfer process during ultrasound-assisted ATP extraction and evaluate the effective diffusion coefficient of phenolics. The results revealed the increase in AED, and the temperature increased the effective diffusivity of phenolics. The HPLC analysis of anthocyanins and flavonols showed that ultrasound significantly increased the extraction yield of anthocyanins compared with the traditional method. High amounts of rutin and myricetin were recovered using the ATPS systems. Sugars were mainly distributed in the bottom phase, whereas phenolics were located in the top phase. Conclusively, ultrasound-assisted aqueous two-phase (ATP) extraction can be used as an effective method to achieve the simultaneous separation and preliminary purification of phenolics from grape pomace.

Unlocking the Secret of Bio-additive Components in Rubber Compounding in Processing Quality Nitrile Glove.

The sustainability of nitrile glove production process is essential both in the financial and energy perspective. Nitrile glove has the lowest material cost with positive mechanical and chemical performance quality for the disposable glove market. Nitrile glove also holds a major market in disposable gloves sector, and nitrile rubber compounds may contribute to the huge reduction of the capital cost for a pair of surgical gloves due to the inexpensive raw material compares with other synthetic polyisoprene or neoprene. Hence, blending of bio-additive into the nitrile latex might support the 3 pillars of sustainability for environmental, societal, and financial sector. Bio-additives helps increase the degradation rate of gloves under natural conditions. Bio-based substances could be derived from food waste, natural plants, and aquatic plants like micro- and macro algae. Furthermore, antimicrobial agent (e.g. brilliant green and cyclohexadiene) is the trend in surgical glove for coated as protecting layer, due to the capability to remove pathogens or bacterial on the surgeon hands during operation period. Besides, the section in energy recovery is a proposing gateway for reducing the financial cost and makes the process sustainable.

A comparative morphological and transcriptomic study on autotetraploid Stevia rebaudiana (bertoni) and its diploid.

Stevia rebaudiana is an important medical plant for producing steviol glycosides (SGs) or stevioside. Autotetraploids (4x = 44) show an increasing level of morphology, physiology and tolerances comparing to diploids (2x = 22). However, little information regarded on the comparative transcriptome analysis between diploid and autotetraploid S. rebaudiana was found. In this study, synthetic autotetraploid was induced and morphological features were confirmed. A comprehensive transcriptome of stevia leaf, stem and root from the diploids and autotetraploids was constructed based on RNA-seq, yielded 1,000,892,422 raw reads and subsequently assembled into 251,455 transcripts, corresponded to 146,130 genes. Pairwise comparisons of the six leaf libraries between the diploids and autotetraploids revealed 4114 differentially expression genes (DEGs), in which 2105 (51.17%) were up-regulated in autotetraploids and associated with SGs biosynthesis, plant growth and secondary metabolism. Moreover, weighted gene co-expression network analysis showed co-expressed genes of fifteen genes of SG biosynthesis pathway were enriched in photosynthesis, flavonoid and secondary metabolic process, plant growth and morphogenesis. A hundred of DEGs related to plant resistance were identified by interviewing PlantPReS database. This study has highlighted molecular changes related to SGs metabolism of polyploidy, and advanced our understanding in plant resistance responsible for phenotypic change of autotetraploids.

VvmiR160s/VvARFs interaction and their spatio-temporal expression/cleavage products during GA-induced grape parthenocarpy.

BACKGROUND: Grape (Vitis vinifera) is highly sensitive to gibberellin (GA), which effectively induce grape parthenocarpy. Studies showed that miR160s and their target AUXIN RESPONSIVE FACTOR (ARF) responding hormones are indispensable for various aspects of plant growth and development, but their functions in GA-induced grape parthenocarpy remain elusive. RESULTS: In this study, the morphological changes during flower development in response to GA treatments were examined in the 'Rosario Bianco' cultivar. The precise sequences of VvmiR160a/b/c/d/e and their VvARF10/16/17 target genes were cloned, sequenced and characterized. The phylogenetic relationship and intron-exon structure of VvARFs and other ARF family members derived from different species were investigated. All VvmiR160s (except VvmiR160b) and VvARF10/16/17 had the common cis-elements responsive to GA, which support their function in GA-mediated grape parthenocarpy. The cleavage role of VvmiR160s-mediated VvARF10/16/17 was verified in grape flowers. Moreover, spatio-temporal expression analysis demonstrated that among VvmiR160 family, VvmiR160a/b/c highly expressed at late stage of flower/berry development, while VvARF10/16/17showed a reverse expression trend. Interestingly, GA exhibited a long-term effect through inducing the expression of VvmiR160a/b/c/e to increase their cleavage product accumulations from 5 to 9 days after treatment, but GA enhanced the expressions of VvARF10/16/17 only at short term. Pearson correlation analysis based on expression data revealed a negative correlation between VvmiR160a/b/c and VvARF10/16/17 in flowers not berries during GA-induced grape parthenocarpy. CONCLUSIONS: This work demonstrated that the negative regulation of VvARF10/16/17 expression by VvmiR160a/b/c as key regulatory factors is critical for GA-mediated grape parthenocarpy, and provide significant implications for molecular breeding of high-quality seedless berry.

Determination of the precise sequences of computationally predicted miRNAs in Citrus reticulata by miR-RACE and characterization of the related target genes using RLM-RACE.

MicroRNAs play vital roles in various biological and metabolic processes by regulating the expression of their target genes in model plants. Since there are limited reports on miRNAs in Citrus reticulata (Crt-miRNAs), the determination of precise sequences of miRNAs is essential to further analyze the functions of miRNAs in Citrus reticulata. Here, miR-RACE, a recently developed technique for determination of the potential miRNAs computationally, was employed to identify the precise sequences of Crt-miRNAs. Tissue- and development-specific expression of nine miRNAs were identified by quantitative RT-PCR in the leaves, stems, flowers and fruits Subsequently, 10 potential target genes were predicated for the eight Crt-miRNAs, most of which were transcription factors and disease resistance proteins. Four target genes were experimentally validated by Poly (A) polymerase-mediated 3' rapid amplification of cDNA ends and RNA ligase-mediated 5' rapid amplification of cDNA ends (PPM-RACE and RLM-RACE). Our findings showed that regulatory miRNAs in C. reticulata may play a key role in regulating growth, development, and response to disease. Future work is required to study the functions of miRNAs and their targets of C. reticulata.

Identification and characterization of cold-responsive microRNAs in tea plant (Camellia sinensis) and their targets using high-throughput sequencing and degradome analysis.

BACKGROUND: MicroRNAs (miRNAs) are approximately 19 ~ 21 nucleotide noncoding RNAs produced by Dicer-catalyzed excision from stem-loop precursors. Many plant miRNAs have critical functions in development, nutrient homeostasis, abiotic stress responses, and pathogen responses via interaction with specific target mRNAs. Camellia sinensis is one of the most important commercial beverage crops in the world. However, miRNAs associated with cold stress tolerance in C. sinensis remains unexplored. The use of high-throughput sequencing can provide a much deeper understanding of miRNAs. To obtain more insight into the function of miRNAs in cold stress tolerance, Illumina sequencing of C. sinensis sRNA was conducted. RESULT: Solexa sequencing technology was used for high-throughput sequencing of the small RNA library from the cold treatment of tea leaves. To align the sequencing data with known plant miRNAs, we characterized 106 conserved C. sinensis miRNAs. In addition, 215 potential candidate miRNAs were found, among, which 98 candidates with star sequences were chosen as novel miRNAs. Both congruously and differentially regulated miRNAs were obtained, and cultivar-specific miRNAs were identified by microarray-based hybridization in response to cold stress. The results were also confirmed by quantitative real-time polymerase chain reaction. To confirm the targets of miRNAs, two degradome libraries from two treatments were constructed. According to degradome sequencing, 455 and 591 genes were identified as cleavage targets of miRNAs from cold treatments and control libraries, respectively, and 283 targets were present in both libraries. Functional analysis of these miRNA targets indicated their involvement in important activities, such as development, regulation of transcription, and stress response. CONCLUSIONS: We discovered 31 up-regulated miRNAs and 43 down-regulated miRNAs in 'Yingshuang', and 46 up-regulated miRNA and 45 down-regulated miRNAs in 'Baiye 1' in response to cold stress, respectively. A total of 763 related target genes were detected by degradome sequencing. The RLM-5'RACE procedure was successfully used to map the cleavage sites in six target genes of C. sinensis. These findings reveal important information about the regulatory mechanism of miRNAs in C. sinensis, and promote the understanding of miRNA functions during the cold response. The miRNA genotype-specific expression model might explain the distinct cold sensitivities between tea lines.

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.

Grapevine microRNAs responsive to exogenous gibberellin.

BACKGROUND: MicroRNAs (miRNAs), involving in various biological and metabolic processes, have been discovered and analyzed in quite a number of plants species, such as Arabidopsis, rice and other plants. However, there have been few reports about grapevine miRNAs in response to gibberelline (GA3). RESULTS: Solexa technology was used to sequence small RNA libraries constructed from grapevine berries treated with GA3 and the control. A total of 122 known and 90 novel grapevine miRNAs (Vvi-miRNAs) were identified. Totally, 137 ones were found to be clearly responsive to GA3, among which 58 were down-regulated, 51 were up-regulated, 21 could only be detected in the control, and seven were only detected in the treatment. Subsequently, we found that 28 of them were differentially regulated by GA3, with 12 conserved and 16 novel Vvi-miRNAs, based on the analysis of qRT-PCR essays. There existed some consistency in expression levels of GA3-responsive Vvi-miRNAs between high throughput sequencing and qRT-PCR essays. In addition, 117 target genes for 29 novel miRNAs were predicted. CONCLUSIONS: Deep sequencing of short RNAs from grapevine berries treated with GA3 and the control identified 137 GA3-responsive miRNAs, among which 28 exhibited different expression profiles of response to GA3 in the diverse developmental stages of grapevine berries. These identified Vvi-miRNAs might be involved in the grapevine berry development and response to environmental stresses.

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.

Validation and characterization of Citrus sinensis microRNAs and their target genes.

BACKGROUND: MicroRNAs play vital role in plant growth and development by changeable expression of their target genes with most plant microRNAs having perfect or near-perfect complementarities with their target genes but miRNAs in Citrus sinensis (csi-miRNAs) and their function have not been widely studied. FINDINGS: In this study, 15 potential microRNAs in Citrus sinensis (csi-miRNAs) were identified and bioinformatically validated using miR-RACE, a newly developed method for determination of miRNAs prediction computationally. The expression of these fifteen C. sinensis miRNAs can be detected in leaves, stems, flowers and fruits of C. sinensis by QRT-PCR with some of them showed tissue-specific expression. Six potential target genes were identified for six csi-miRNAs and also experimentally verified by Poly (A) polymerase -mediated 3' rapid amplification of cDNA ends (PPM-RACE) and RNA ligase-mediated 5' rapid amplification of cDNA ends (RLM-RACE) which mapped the cleavage site of target mRNAs and detected expression patterns of cleaved fragments that indicate the regulatory function of the miRNAs on their target genes. CONCLUSIONS: Our results confirm that small RNA-mediated regulation whereby all csi-miRNAs regulate their target genes by degradation.

Computational identification of microRNAs in peach expressed sequence tags and validation of their precise sequences by miR-RACE.

Twenty-two potential miRNAs from seven miRNA families were first predicted from more than 80,857 EST sequences of peach (Prunus persica). Using two specific 5' and 3' miRNA RACE (miR-RACE) PCR reactions and sequence-directed cloning, we accurately determined the precise sequences, especially both ends, of eight candidate miRNAs. The sequencing results demonstrated that the ppe-miRNAs were conserved to those that were predicted computationally except ppe-miR171b. We validated the existence of two members (ppe-miR171a and miR171b) of the miR171 family in peach that belonged to different precursors. qRT-PCR was further employed in analyzing expression of the eight miRNAs in peach leaves, flowers, and fruits at different developing stages, where some of the miRNAs showed tissue-specific expression.

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 of grapevine flower and berry short RNA library for discovery of novel microRNAs and validation of precise sequences of grapevine microRNAs deposited in miRBase.

MicroRNAs (miRNAs) are a class of non-coding RNA molecules which have significant gene regulation roles in organisms. The advent of new high-throughput sequencing technologies has enabled the discovery of novel miRNAs. Although there are two recent reports on high-throughput sequencing analysis of small RNA libraries from different organs of two wine grapevine varieties, there was a significant divergence in the number and kinds of miRNAs sequenced in these studies. More sequencing of small RNA libraries is still important for the discovery of novel miRNAs in grapevine. In this study, a total of 130 conserved grapevine Vitis vinifera miRNA (Vv-miRNA) belonging to 28 Vv-miRNA families were validated, other 80 unconserved Vv-miRNAs including 72 novel potential and 8 known but unconserved ones were found. Fifty-two (52.5%) of these 80 unconserved Vv-miRNAs exhibited differential poly(A)-tailed reverse transcriptase-polymerase chain reaction expression profiles in various grapevine tissues that could further confirm their existence in grapevine, among which 20 were expressed only in grapevine berries, indicating a degree of fruit-specificity. One hundred thirty target genes for 56 unconserved miRNAs could be predicted. The locations of these potential target genes on grapevine chromosomes and their complementary levels with the corresponding miRNAs were also analyzed. These results point to a regulatory role of miRNAs in grapevine berry development and response to various environments.

Computational identification of microRNAs in apple expressed sequence tags and validation of their precise sequences by miR-RACE.

Thirty-one potential miRNAs that belong to 16 miRNA families were discovered from more than 324 000 EST sequences of apple (Malus domestica). In addition, precise sequences, especially terminal nucleotides of the 16 apple miRNAs (mdo-miRNAs) in 16 families were validated by miR-RACE, a newly developed method for the determination of the potential miRNAs predicted computationally. The expression of these 16 microRNAs could be detected in apple young leaf, old leaf, young stem, flower bud, flower and developing fruits by quantitative RT-PCR (qRT-PCR) and some of them showed tissue-specific expression. Fifty-six potential targets were identified for the 16 apple miRNAs, most of which were transcription factors that play important roles in apple development. Twelve target genes were experimentally verified by qRT-PCR, with some exhibiting different expression trends from their corresponding microRNAs, indicating the cleavage mode of miRNAs on their target genes.

Analysis of expressed sequence tags from Prunus mume flower and fruit and development of simple sequence repeat markers.

BACKGROUND: Expressed Sequence Tag (EST) has been a cost-effective tool in molecular biology and represents an abundant valuable resource for genome annotation, gene expression, and comparative genomics in plants. RESULTS: In this study, we constructed a cDNA library of Prunus mume flower and fruit, sequenced 10,123 clones of the library, and obtained 8,656 expressed sequence tag (EST) sequences with high quality. The ESTs were assembled into 4,473 unigenes composed of 1,492 contigs and 2,981 singletons and that have been deposited in NCBI (accession IDs: GW868575 - GW873047), among which 1,294 unique ESTs were with known or putative functions. Furthermore, we found 1,233 putative simple sequence repeats (SSRs) in the P. mume unigene dataset. We randomly tested 42 pairs of PCR primers flanking potential SSRs, and 14 pairs were identified as true-to-type SSR loci and could amplify polymorphic bands from 20 individual plants of P. mume. We further used the 14 EST-SSR primer pairs to test the transferability on peach and plum. The result showed that nearly 89% of the primer pairs produced target PCR bands in the two species. A high level of marker polymorphism was observed in the plum species (65%) and low in the peach (46%), and the clustering analysis of the three species indicated that these SSR markers were useful in the evaluation of genetic relationships and diversity between and within the Prunus species. CONCLUSIONS: We have constructed the first cDNA library of P. mume flower and fruit, and our data provide sets of molecular biology resources for P. mume and other Prunus species. These resources will be useful for further study such as genome annotation, new gene discovery, gene functional analysis, molecular breeding, evolution and comparative genomics between Prunus species.

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.

MiR-RACE, a new efficient approach to determine the precise sequences of computationally identified trifoliate orange (Poncirus trifoliata) microRNAs.

BACKGROUND: Among the hundreds of genes encoding miRNAs in plants reported, much more were predicted by numerous computational methods. However, unlike protein-coding genes defined by start and stop codons, the ends of miRNA molecules do not have characteristics that can be used to define the mature miRNAs exactly, which made computational miRNA prediction methods often cannot predict the accurate location of the mature miRNA in a precursor with nucleotide-level precision. To our knowledge, there haven't been reports about comprehensive strategies determining the precise sequences, especially two termini, of these miRNAs. METHODS: In this study, we report an efficient method to determine the precise sequences of computationally predicted microRNAs (miRNAs) that combines miRNA-enriched library preparation, two specific 5' and 3' miRNA RACE (miR-RACE) PCR reactions, and sequence-directed cloning, in which the most challenging step is the two specific gene specific primers designed for the two RACE reactions. miRNA-mediated mRNA cleavage by RLM-5' RACE and sequencing were carried out to validate the miRNAs detected. Real-time PCR was used to analyze the expression of each miRNA. RESULTS: The efficiency of this newly developed method was validated using nine trifoliate orange (Poncirus trifoliata) miRNAs predicted computationally. The miRNAs computationally identified were validated by miR-RACE and sequencing. Quantitative analysis showed that they have variable expression. Eight target genes have been experimentally verified by detection of the miRNA-mediated mRNA cleavage in Poncirus trifoliate. CONCLUSION: The efficient and powerful approach developed herein can be successfully used to validate the sequences of miRNAs, especially the termini, which depict the complete miRNA sequence in the computationally predicted precursor.

Identification and characterization of 27 conserved microRNAs in citrus.

MicroRNAs (miRNAs) are a class of non-protein-coding small RNAs. Considering the conservation of many miRNA genes in different plant genomes, the identification of miRNAs from non-model organisms is both practicable and instrumental in addressing miRNA-guided gene regulation. Citrus is an important staple fruit tree, and publicly available expressed sequence tag (EST) database for citrus are increasing. However, until now, little has been known about miRNA in citrus. In this study, 27 known miRNAs from Arabidopsis were searched against citrus EST databases for miRNA precursors, of which 13 searched precursor sequences could form fold-back structures similar with those of Arabidopsis. The ubiquitous expression of those 13 citrus microRNAs and other 13 potential citrus miRNAs could be detected in citrus leaf, young shoot, flower, fruit and root by northern blotting, and some of them showed differential expression in different tissues. Based on the fact that miRNAs exhibit perfect or nearly perfect complementarity with their target sequences, a total of 41 potential targets were identified for 15 citrus miRNAs. The majority of the targets are transcription factors that play important roles in citrus development, including leaf, shoot, and root development. Additionally, some other target genes appear to play roles in diverse physiological processes. Four target genes have been experimentally verified by detection of the miRNA-mediated mRNA cleavage in Poncirus trifoliate. Overall, this study in the identification and characterization of miRNAs in citrus can initiate further study on citrus miRNA regulation mechanisms, and it can help us to know more about the important roles of miRNAs in citrus.