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1.  Historical Introgression of the Downy Mildew Resistance Gene Rpv12 from the Asian Species Vitis amurensis into Grapevine Varieties 
PLoS ONE  2013;8(4):e61228.
The Amur grape (Vitis amurensis Rupr.) thrives naturally in cool climates of Northeast Asia. Resistance against the introduced pathogen Plasmopara viticola is common among wild ecotypes that were propagated from Manchuria into Chinese vineyards or collected by Soviet botanists in Siberia, and used for the introgression of resistance into wine grapes (Vitis vinifera L.). A QTL analysis revealed a dominant gene Rpv12 that explained 79% of the phenotypic variance for downy mildew resistance and was inherited independently of other resistance genes. A Mendelian component of resistance–a hypersensitive response in leaves challenged with P. viticola–was mapped in an interval of 0.2 cM containing an array of coiled-coil NB-LRR genes on chromosome 14. We sequenced 10-kb genic regions in the Rpv12+ haplotype and identified polymorphisms in 12 varieties of V. vinifera using next-generation sequencing. The combination of two SNPs in single-copy genes flanking the NB-LRR cluster distinguished the resistant haplotype from all others found in 200 accessions of V. vinifera, V. amurensis, and V. amurensis x V. vinifera crosses. The Rpv12+ haplotype is shared by 15 varieties, the most ancestral of which are the century-old ‘Zarja severa’ and ‘Michurinets’. Before this knowledge, the chromosome segment around Rpv12+ became introgressed, shortened, and pyramided with another downy mildew resistance gene from North American grapevines (Rpv3) only by phenotypic selection. Rpv12+ has an additive effect with Rpv3+ to protect vines against natural infections, and confers foliar resistance to strains that are virulent on Rpv3+ plants.
PMCID: PMC3625174  PMID: 23593440
2.  Grapevine microRNAs responsive to exogenous gibberellin 
BMC Genomics  2014;15:111.
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).
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.
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.
PMCID: PMC3937062  PMID: 24507455
Grapevine; Berry; microRNAs; Exogenous gibberellin; High throughput sequencing
3.  Characterization of microRNAs Identified in a Table Grapevine Cultivar with Validation of Computationally Predicted Grapevine miRNAs by miR-RACE 
PLoS ONE  2011;6(7):e21259.
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.
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.
PMCID: PMC3145640  PMID: 21829435
4.  Identification and Functional Analysis of Flowering Related microRNAs in Common Wild Rice (Oryza rufipogon Griff.) 
PLoS ONE  2013;8(12):e82844.
MicroRNAs (miRNAs) is a class of non-coding RNAs involved in post- transcriptional control of gene expression, via degradation and/or translational inhibition. Six-hundred sixty-one rice miRNAs are known that are important in plant development. However, flowering-related miRNAs have not been characterized in Oryza rufipogon Griff. It was approved by supervision department of Guangdong wild rice protection. We analyzed flowering-related miRNAs in O. rufipogon using high-throughput sequencing (deep sequencing) to understand the changes that occurred during rice domestication, and to elucidate their functions in flowering.
Three O. rufipogon sRNA libraries, two vegetative stage (CWR-V1 and CWR-V2) and one flowering stage (CWR-F2) were sequenced using Illumina deep sequencing. A total of 20,156,098, 21,531,511 and 20,995,942 high quality sRNA reads were obtained from CWR-V1, CWR-V2 and CWR-F2, respectively, of which 3,448,185, 4,265,048 and 2,833,527 reads matched known miRNAs. We identified 512 known rice miRNAs in 214 miRNA families and predicted 290 new miRNAs. Targeted functional annotation, GO and KEGG pathway analyses predicted that 187 miRNAs regulate expression of flowering-related genes. Differential expression analysis of flowering-related miRNAs showed that: expression of 95 miRNAs varied significantly between the libraries, 66 are flowering-related miRNAs, such as oru-miR97, oru-miR117, oru-miR135, oru-miR137, et al. 17 are early-flowering -related miRNAs, including osa-miR160f, osa-miR164d, osa-miR167d, osa-miR169a, osa-miR172b, oru-miR4, et al., induced during the floral transition. Real-time PCR revealed the same expression patterns as deep sequencing. miRNAs targets were confirmed for cleavage by 5′-RACE in vivo, and were negatively regulated by miRNAs.
This is the first investigation of flowering miRNAs in wild rice. The result indicates that variation in miRNAs occurred during rice domestication and lays a foundation for further study of phase change and flowering in O. rufipogon. Complicated regulatory networks mediated by multiple miRNAs regulate the expression of flowering genes that control the induction of flowering.
PMCID: PMC3875430  PMID: 24386120
5.  Identification and characterization of miRNAome in root, stem, leaf and tuber developmental stages of potato (Solanum tuberosum L.) by high-throughput sequencing 
BMC Plant Biology  2014;14:6.
MicroRNAs (miRNAs) are ubiquitous components of endogenous plant transcriptome. miRNAs are small, single-stranded and ~21 nt long RNAs which regulate gene expression at the post-transcriptional level and are known to play essential roles in various aspects of plant development and growth. Previously, a number of miRNAs have been identified in potato through in silico analysis and deep sequencing approach. However, identification of miRNAs through deep sequencing approach was limited to a few tissue types and developmental stages. This study reports the identification and characterization of potato miRNAs in three different vegetative tissues and four stages of tuber development by high throughput sequencing.
Small RNA libraries were constructed from leaf, stem, root and four early developmental stages of tuberization and subjected to deep sequencing, followed by bioinformatics analysis. A total of 89 conserved miRNAs (belonging to 33 families), 147 potato-specific miRNAs (with star sequence) and 112 candidate potato-specific miRNAs (without star sequence) were identified. The digital expression profiling based on TPM (Transcripts Per Million) and qRT-PCR analysis of conserved and potato-specific miRNAs revealed that some of the miRNAs showed tissue specific expression (leaf, stem and root) while a few demonstrated tuberization stage-specific expressions. Targets were predicted for identified conserved and potato-specific miRNAs, and predicted targets of four conserved miRNAs, miR160, miR164, miR172 and miR171, which are ARF16 (Auxin Response Factor 16), NAM (NO APICAL MERISTEM), RAP1 (Relative to APETALA2 1) and HAM (HAIRY MERISTEM) respectively, were experimentally validated using 5′ RLM-RACE (RNA ligase mediated rapid amplification of cDNA ends). Gene ontology (GO) analysis for potato-specific miRNAs was also performed to predict their potential biological functions.
We report a comprehensive study of potato miRNAs at genome-wide level by high-throughput sequencing and demonstrate that these miRNAs have tissue and/or developmental stage-specific expression profile. Also, predicted targets of conserved miRNAs were experimentally confirmed for the first time in potato. Our findings indicate the existence of extensive and complex small RNA population in this crop and suggest their important role in pathways involved in diverse biological processes, including tuber development.
PMCID: PMC3913621  PMID: 24397411
Potato; Tuberization; microRNA; High-throughput sequencing; Digital expression profiling; qRT-PCR; Target; Gene ontology; RLM-RACE
6.  Cloning and characterization of microRNAs from wheat (Triticum aestivum L.) 
Genome Biology  2007;8(6):R96.
A small RNA library was used to identify 58 miRNAs from 43 miRNA families from wheat (Triticum aestivum L.), and 46 potential targets were predicted.
MicroRNAs (miRNAs) are a class of small, non-coding regulatory RNAs that regulate gene expression by guiding target mRNA cleavage or translational inhibition. So far, identification of miRNAs has been limited to a few model plant species, such as Arabidopsis, rice and Populus, whose genomes have been sequenced. Wheat is one of the most important cereal crops worldwide. To date, only a few conserved miRNAs have been predicted in wheat and the computational identification of wheat miRNAs requires the genome sequence, which is unknown.
To identify novel as well as conserved miRNAs in wheat (Triticum aestivum L.), we constructed a small RNA library. High throughput sequencing of the library and subsequent analysis revealed the identification of 58 miRNAs, comprising 43 miRNA families. Of these, 35 miRNAs belong to 20 conserved miRNA families. The remaining 23 miRNAs are novel and form 23 miRNA families in wheat; more importantly, 4 of these new miRNAs (miR506, miR510, miR514 and miR516) appear to be monocot-specific. Northern blot analysis indicated that some of the new miRNAs are preferentially expressed in certain tissues. Based on sequence homology, we predicted 46 potential targets. Thus, we have identified a large number of monocot-specific and wheat-specific miRNAs. These results indicate that both conserved and wheat-specific miRNAs play important roles in wheat growth and development, stress responses and other physiological processes.
This study led to the discovery of 58 wheat miRNAs comprising 43 miRNA families; 20 of these families are conserved and 23 are novel in wheat. It provides a first large scale cloning and characterization of wheat miRNAs and their predicted targets.
PMCID: PMC2394755  PMID: 17543110
7.  Genome Wide Transcriptional Profile Analysis of Vitis amurensis and Vitis vinifera in Response to Cold Stress 
PLoS ONE  2013;8(3):e58740.
Grape is one of the most important fruit crops worldwide. The suitable geographical locations and productivity of grapes are largely limited by temperature. Vitis amurensis is a wild grapevine species with remarkable cold-tolerance, exceeding that of Vitis vinifera, the dominant cultivated species of grapevine. However, the molecular mechanisms that contribute to the enhanced freezing tolerance of V. amurensis remain unknown. Here we used deep sequencing data from restriction endonuclease-generated cDNA fragments to evaluate the whole genome wide modification of transcriptome of V. amurensis under cold treatment. Vitis vinifera cv. Muscat of Hamburg was used as control to help investigate the distinctive features of V. amruensis in responding to cold stress. Approximately 9 million tags were sequenced from non-cold treatment (NCT) and cold treatment (CT) cDNA libraries in each species of grapevine sampled from shoot apices. Alignment of tags into V. vinifera cv. Pinot noir (PN40024) annotated genome identified over 15,000 transcripts in each library in V. amruensis and more than 16,000 in Muscat of Hamburg. Comparative analysis between NCT and CT libraries indicate that V. amurensis has fewer differential expressed genes (DEGs, 1314 transcripts) than Muscat of Hamburg (2307 transcripts) when exposed to cold stress. Common DEGs (408 transcripts) suggest that some genes provide fundamental roles during cold stress in grapes. The most robust DEGs (more than 20-fold change) also demonstrated significant differences between two kinds of grapevine, indicating that cold stress may trigger species specific pathways in V. amurensis. Functional categories of DEGs indicated that the proportion of up-regulated transcripts related to metabolism, transport, signal transduction and transcription were more abundant in V. amurensis. Several highly expressed transcripts that were found uniquely accumulated in V. amurensis are discussed in detail. This subset of unique candidate transcripts may contribute to the excellent cold-hardiness of V. amurensis.
PMCID: PMC3596283  PMID: 23516547
8.  Identification of cold-inducible microRNAs in grapevine 
Low temperature is one of the most important environmental factors that limits the geographical distribution and productivity of grapevine. However, the molecular mechanisms on how grapevine responds to cold stress remains to be elucidated. MicroRNAs (miRNAs) are a class of endogenous small non-coding RNAs that play an essential role during plant development and stress responses. Although miRNAs and their targets have been identified in several Vitis species, their participation during cold accumulation in grapevine remains unknown. In this study, two small RNA libraries were generated from micropropagated ‘Muscat Hamburg’ (V. vinifera) plantlets under normal and low temperatures (4°C). A total of 163 known miRNAs and 67 putative novel miRNAs were detected from two small RNA libraries by Solexa sequencing. Forty-four cold-inducible miRNAs were identified through differentially expressed miRNAs (DEMs) analysis; among which, 13 belonged to upregulated DEMs while 31 belonged downregulated DEMs. The expression patterns of the 13 DEMs were verified by real-time RT-PCR analysis. The prediction of the target genes for DEMs indicated that miRNA may regulate transcription factors, including AP2, SBP, MYB, bHLH, GRAS, and bZIP under cold stress. The 5′-RLM RACE were conducted to verify the cleavage site of predicted targets. Seven predicted target genes for four known and three novel vvi-miRNAs showed specific cleavage sites corresponding to their miRNA complementary sequences. The expression pattern of these seven target genes revealed negative correlation with the expression level of the corresponding vvi-miRNAs. Our results indicated that a diverse set of miRNAs in V. vinifera are cold-inducible and may play an important role in cold stress response.
PMCID: PMC4523783  PMID: 26300896
Vitis vinifera; microRNA; cold-inducible; abiotic stress; Solexa
9.  Genomic Organization, Phylogenetic Comparison and Differential Expression of the SBP-Box Family Genes in Grape 
PLoS ONE  2013;8(3):e59358.
The SBP-box gene family is specific to plants and encodes a class of zinc finger-containing transcription factors with a broad range of functions. Although SBP-box genes have been identified in numerous plants including green algae, moss, silver birch, snapdragon, Arabidopsis, rice and maize, there is little information concerning SBP-box genes, or the corresponding miR156/157, function in grapevine.
Methodology/Principal Findings
Eighteen SBP-box gene family members were identified in Vitis vinifera, twelve of which bore sequences that were complementary to miRNA156/157. Phylogenetic reconstruction demonstrated that plant SBP-domain proteins could be classified into seven subgroups, with the V. vinifera SBP-domain proteins being more closely related to SBP-domain proteins from dicotyledonous angiosperms than those from monocotyledonous angiosperms. In addition, synteny analysis between grape and Arabidopsis demonstrated that homologs of several grape SBP genes were found in corresponding syntenic blocks of Arabidopsis. Expression analysis of the grape SBP-box genes in various organs and at different stages of fruit development in V. quinquangularis ‘Shang-24’ revealed distinct spatiotemporal patterns. While the majority of the grape SBP-box genes lacking a miR156/157 target site were expressed ubiquitously and constitutively, most genes bearing a miR156/157 target site exhibited distinct expression patterns, possibly due to the inhibitory role of the microRNA. Furthermore, microarray data mining and quantitative real-time RT-PCR analysis identified several grape SBP-box genes that are potentially involved in the defense against biotic and abiotic stresses.
The results presented here provide a further understanding of SBP-box gene function in plants, and yields additional insights into the mechanism of stress management in grape, which may have important implications for the future success of this crop.
PMCID: PMC3601960  PMID: 23527172
10.  Direct sequencing and expression analysis of a large number of miRNAs in Aedes aegypti and a multi-species survey of novel mosquito miRNAs 
BMC Genomics  2009;10:581.
MicroRNAs (miRNAs) are a novel class of gene regulators whose biogenesis involves hairpin structures called precursor miRNAs, or pre-miRNAs. A pre-miRNA is processed to make a miRNA:miRNA* duplex, which is then separated to generate a mature miRNA and a miRNA*. The mature miRNAs play key regulatory roles during embryonic development as well as other cellular processes. They are also implicated in control of viral infection as well as innate immunity. Direct experimental evidence for mosquito miRNAs has been recently reported in anopheline mosquitoes based on small-scale cloning efforts.
We obtained approximately 130, 000 small RNA sequences from the yellow fever mosquito, Aedes aegypti, by 454 sequencing of samples that were isolated from mixed-age embryos and midguts from sugar-fed and blood-fed females, respectively. We also performed bioinformatics analysis on the Ae. aegypti genome assembly to identify evidence for additional miRNAs. The combination of these approaches uncovered 98 different pre-miRNAs in Ae. aegypti which could produce 86 distinct miRNAs. Thirteen miRNAs, including eight novel miRNAs identified in this study, are currently only found in mosquitoes. We also identified five potential revisions to previously annotated miRNAs at the miRNA termini, two cases of highly abundant miRNA* sequences, 14 miRNA clusters, and 17 cases where more than one pre-miRNA hairpin produces the same or highly similar mature miRNAs. A number of miRNAs showed higher levels in midgut from blood-fed female than that from sugar-fed female, which was confirmed by northern blots on two of these miRNAs. Northern blots also revealed several miRNAs that showed stage-specific expression. Detailed expression analysis of eight of the 13 mosquito-specific miRNAs in four divergent mosquito genera identified cases of clearly conserved expression patterns and obvious differences. Four of the 13 miRNAs are specific to certain lineage(s) within mosquitoes.
This study provides the first systematic analysis of miRNAs in Ae. aegypti and offers a substantially expanded list of miRNAs for all mosquitoes. New insights were gained on the evolution of conserved and lineage-specific miRNAs in mosquitoes. The expression profiles of a few miRNAs suggest stage-specific functions and functions related to embryonic development or blood feeding. A better understanding of the functions of these miRNAs will offer new insights in mosquito biology and may lead to novel approaches to combat mosquito-borne infectious diseases.
PMCID: PMC2797818  PMID: 19961592
11.  Correction: High throughput approaches reveal splicing of primary microRNA transcripts and tissue specific expression of mature microRNAs in Vitis vinifera 
BMC Genomics  2010;11:109.
The version of this article published in BMC Genomics 2009, 10:558, contains data in Table 1 which are now known to be unreliable, and an illustration, in Figure 1, of unusual miRNA processing events predicted by these unreliable data. In this full-length correction, new data replace those found to be unreliable, leading to a more straightforward interpretation without altering the principle conclusions of the study. Table 1 and associated methods have been corrected, Figure 1 deleted, supplementary file 1 added, and modifications made to the sections "Deep sequencing of small RNAs from grapevine leaf tissue" and "Microarray analysis of miRNA expression". The editors and authors regret the inconvenience caused to readers by premature publication of the original paper.
MicroRNAs are short (~21 base) single stranded RNAs that, in plants, are generally coded by specific genes and cleaved specifically from hairpin precursors. MicroRNAs are critical for the regulation of multiple developmental, stress related and other physiological processes in plants. The recent annotation of the genome of the grapevine (Vitis vinifera L.) allowed the identification of many putative conserved microRNA precursors, grouped into multiple gene families.
Here we use oligonucleotide arrays to provide the first indication that many of these microRNAs show differential expression patterns between tissues and during the maturation of fruit in the grapevine. Furthermore we demonstrate that whole transcriptome sequencing and deep-sequencing of small RNA fractions can be used both to identify which microRNA precursors are expressed in different tissues and to estimate genomic coordinates and patterns of splicing and alternative splicing for many primary miRNA transcripts.
Our results show that many microRNAs are differentially expressed in different tissues and during fruit maturation in the grapevine. Furthermore, the demonstration that whole transcriptome sequencing can be used to identify candidate splicing events and approximate primary microRNA transcript coordinates represents a significant step towards the large-scale elucidation of mechanisms regulating the expression of microRNAs at the transcriptional and post-transcriptional levels.
PMCID: PMC2831844  PMID: 20152027
12.  Identification and characterization of microRNAs in Phaseolus vulgaris by high-throughput sequencing 
BMC Genomics  2012;13:83.
MicroRNAs (miRNAs) are endogenously encoded small RNAs that post-transcriptionally regulate gene expression. MiRNAs play essential roles in almost all plant biological processes. Currently, few miRNAs have been identified in the model food legume Phaseolus vulgaris (common bean). Recent advances in next generation sequencing technologies have allowed the identification of conserved and novel miRNAs in many plant species. Here, we used Illumina's sequencing by synthesis (SBS) technology to identify and characterize the miRNA population of Phaseolus vulgaris.
Small RNA libraries were generated from roots, flowers, leaves, and seedlings of P. vulgaris. Based on similarity to previously reported plant miRNAs,114 miRNAs belonging to 33 conserved miRNA families were identified. Stem-loop precursors and target gene sequences for several conserved common bean miRNAs were determined from publicly available databases. Less conserved miRNA families and species-specific common bean miRNA isoforms were also characterized. Moreover, novel miRNAs based on the small RNAs were found and their potential precursors were predicted. In addition, new target candidates for novel and conserved miRNAs were proposed. Finally, we studied organ-specific miRNA family expression levels through miRNA read frequencies.
This work represents the first massive-scale RNA sequencing study performed in Phaseolus vulgaris to identify and characterize its miRNA population. It significantly increases the number of miRNAs, precursors, and targets identified in this agronomically important species. The miRNA expression analysis provides a foundation for understanding common bean miRNA organ-specific expression patterns. The present study offers an expanded picture of P. vulgaris miRNAs in relation to those of other legumes.
PMCID: PMC3359237  PMID: 22394504
13.  A Leafhopper-Transmissible DNA Virus with Novel Evolutionary Lineage in the Family Geminiviridae Implicated in Grapevine Redleaf Disease by Next-Generation Sequencing 
PLoS ONE  2013;8(6):e64194.
A graft-transmissible disease displaying red veins, red blotches and total reddening of leaves in red-berried wine grape (Vitis vinifera L.) cultivars was observed in commercial vineyards. Next-generation sequencing technology was used to identify etiological agent(s) associated with this emerging disease, designated as grapevine redleaf disease (GRD). High quality RNA extracted from leaves of grape cultivars Merlot and Cabernet Franc with and without GRD symptoms was used to prepare cDNA libraries. Assembly of highly informative sequence reads generated from Illumina sequencing of cDNA libraries, followed by bioinformatic analyses of sequence contigs resulted in specific identification of taxonomically disparate viruses and viroids in samples with and without GRD symptoms. A single-stranded DNA virus, tentatively named Grapevine redleaf-associated virus (GRLaV), and Grapevine fanleaf virus were detected only in grapevines showing GRD symptoms. In contrast, Grapevine rupestris stem pitting-associated virus, Hop stunt viroid, Grapevine yellow speckle viroid 1, Citrus exocortis viroid and Citrus exocortis Yucatan viroid were present in both symptomatic and non-symptomatic grapevines. GRLaV was transmitted by the Virginia creeper leafhopper (Erythroneura ziczac Walsh) from grapevine-to-grapevine under greenhouse conditions. Molecular and phylogenetic analyses indicated that GRLaV, almost identical to recently reported Grapevine Cabernet Franc-associated virus from New York and Grapevine red blotch-associated virus from California, represents an evolutionarily distinct lineage in the family Geminiviridae with genome characteristics distinct from other leafhopper-transmitted geminiviruses. GRD significantly reduced fruit yield and affected berry quality parameters demonstrating negative impacts of the disease. Higher quantities of carbohydrates were present in symptomatic leaves suggesting their possible role in the expression of redleaf symptoms.
PMCID: PMC3673993  PMID: 23755117
14.  MicroRNAs and their putative targets in Brassica napus seed maturation 
BMC Genomics  2013;14:140.
MicroRNAs (miRNAs) are 20–21 nucleotide RNA molecules that suppress the transcription of target genes and may also inhibit translation. Despite the thousands of miRNAs identified and validated in numerous plant species, only small numbers have been identified from the oilseed crop plant Brassica napus (canola) – especially in seeds.
Using next-generation sequencing technologies, we performed a comprehensive analysis of miRNAs during seed maturation at 9 time points from 10 days after flowering (DAF) to 50 DAF using whole seeds and included separate analyses of radicle, hypocotyl, cotyledon, embryo, endosperm and seed coat tissues at 4 selected time points. We identified more than 500 conserved miRNA or variant unique sequences with >300 sequence reads and also found 10 novel miRNAs. Only 27 of the conserved miRNA sequences had been previously identified in B. napus (miRBase Release 18). More than 180 MIRNA loci were identified/annotated using the B. rapa genome as a surrogate for the B.napus A genome. Numerous miRNAs were expressed in a stage- or tissue-specific manner suggesting that they have specific functions related to the fine tuning of transcript abundance during seed development. miRNA targets in B. napus were predicted and their expression patterns profiled using microarray analyses. Global correlation analysis of the expression patterns of miRNAs and their targets revealed complex miRNA-target gene regulatory networks during seed development. The miR156 family was the most abundant and the majority of the family members were primarily expressed in the embryo.
Large numbers of miRNAs with diverse expression patterns, multiple-targeting and co-targeting of many miRNAs, and complex relationships between expression of miRNAs and targets were identified in this study. Several key miRNA-target expression patterns were identified and new roles of miRNAs in regulating seed development are suggested. miR156, miR159, miR172, miR167, miR158 and miR166 are the major contributors to the network controlling seed development and maturation through their pivotal roles in plant development. miR156 may regulate the developmental transition to germination.
PMCID: PMC3602245  PMID: 23448243
Seed development; Embryo; Next generation sequencing
15.  miRVine: a microRNA expression atlas of grapevine based on small RNA sequencing 
BMC Genomics  2015;16(1):393.
miRNAs are the most abundant class of small non-coding RNAs, and they are involved in post-transcriptional regulations, playing a crucial role in the refinement of genetic programming during plant development. Here we present a comprehensive picture of miRNA regulation in Vitis vinifera L. plant during its complete life cycle. Furthering our knowledge about the post-transcriptional regulation of plant development is fundamental to understand the biology of such an important crop.
We analyzed 70 small RNA libraries, prepared from berries, inflorescences, tendrils, buds, carpels, stamens and other samples at different developmental stages. One-hundred and ten known and 175 novel miRNAs have been identified and a wide grapevine expression atlas has been described. The distribution of miRNA abundance reveals that 22 novel miRNAs are specific to stamen, and two of them are, interestingly, involved in ethylene biosynthesis, while only few miRNAs are highly specific to other organs. Thirty-eight miRNAs are present in all our samples, suggesting a role in key regulatory circuit. On the basis of miRNAs abundance and distribution across samples and on the estimated correlation, we suggest that miRNA expression define organ identity. We performed target prediction analysis and focused on miRNA expression analysis in berries and inflorescence during their development, providing an initial functional description of the identified miRNAs.
Our findings represent a very extensive miRNA expression atlas in grapevine, allowing the definition of how the spatio-temporal distribution of miRNAs defines organ identity. We describe miRNAs abundance in specific tissues not previously described in grapevine and contribute to future targeted functional analyses. Finally, we present a deep characterization of miRNA involvement in berry and inflorescence development, suggesting a role for miRNA-driven hormonal regulation.
Electronic supplementary material
The online version of this article (doi:10.1186/s12864-015-1610-5) contains supplementary material, which is available to authorized users.
PMCID: PMC4434875  PMID: 25981679
Grapevine; microRNAs; Deep Sequencing; RT-qPCR; Berries; Inflorescences; Plant development; Ethylene biosynthesis
16.  Identification of miRNAs and their target genes in developing soybean seeds by deep sequencing 
BMC Plant Biology  2011;11:5.
MicroRNAs (miRNAs) regulate gene expression by mediating gene silencing at transcriptional and post-transcriptional levels in higher plants. miRNAs and related target genes have been widely studied in model plants such as Arabidopsis and rice; however, the number of identified miRNAs in soybean (Glycine max) is limited, and global identification of the related miRNA targets has not been reported in previous research.
In our study, a small RNA library and a degradome library were constructed from developing soybean seeds for deep sequencing. We identified 26 new miRNAs in soybean by bioinformatic analysis and further confirmed their expression by stem-loop RT-PCR. The miRNA star sequences of 38 known miRNAs and 8 new miRNAs were also discovered, providing additional evidence for the existence of miRNAs. Through degradome sequencing, 145 and 25 genes were identified as targets of annotated miRNAs and new miRNAs, respectively. GO analysis indicated that many of the identified miRNA targets may function in soybean seed development. Additionally, a soybean homolog of Arabidopsis SUPPRESSOR OF GENE SLIENCING 3 (AtSGS3) was detected as a target of the newly identified miRNA Soy_25, suggesting the presence of feedback control of miRNA biogenesis.
We have identified large numbers of miRNAs and their related target genes through deep sequencing of a small RNA library and a degradome library. Our study provides more information about the regulatory network of miRNAs in soybean and advances our understanding of miRNA functions during seed development.
PMCID: PMC3023735  PMID: 21219599
17.  The miRNAome of globe artichoke: conserved and novel micro RNAs and target analysis 
BMC Genomics  2012;13:41.
Plant microRNAs (miRNAs) are involved in post-transcriptional regulatory mechanisms of several processes, including the response to biotic and abiotic stress, often contributing to the adaptive response of the plant to adverse conditions. In addition to conserved miRNAs, found in a wide range of plant species a number of novel species-specific miRNAs, displaying lower levels of expression can be found. Due to low abundance, non conserved miRNAs are difficult to identify and isolate using conventional approaches. Conversely, deep-sequencing of small RNA (sRNA) libraries can detect even poorly expressed miRNAs.
No miRNAs from globe artichoke have been described to date. We analyzed the miRNAome from artichoke by deep sequencing four sRNA libraries obtained from NaCl stressed and control leaves and roots.
Conserved and novel miRNAs were discovered using accepted criteria. The expression level of selected miRNAs was monitored by quantitative real-time PCR. Targets were predicted and validated for their cleavage site. A total of 122 artichoke miRNAs were identified, 98 (25 families) of which were conserved with other plant species, and 24 were novel. Some miRNAs were differentially expressed according to tissue or condition, magnitude of variation after salt stress being more pronounced in roots. Target function was predicted by comparison to Arabidopsis proteins; the 43 targets (23 for novel miRNAs) identified included transcription factors and other genes, most of which involved in the response to various stresses. An unusual cleaved transcript was detected for miR393 target, transport inhibitor response 1.
The miRNAome from artichoke, including novel miRNAs, was unveiled, providing useful information on the expression in different organs and conditions. New target genes were identified. We suggest that the generation of secondary short-interfering RNAs from miR393 target can be a general rule in the plant kingdom.
PMCID: PMC3285030  PMID: 22272770
18.  Transcriptome-Wide Identification and Characterization of MicroRNAs from Castor Bean (Ricinus communis L.) 
PLoS ONE  2013;8(7):e69995.
MicroRNAs (miRNAs) are endogenously encoded small RNAs that post-transcriptionally regulate gene expression and play essential roles in numerous developmental and physiological processes. Currently, little information on the transcriptome and tissue-specific expression of miRNAs is available in the model non-edible oilseed crop castor bean (Ricinus communis L.), one of the most important non-edible oilseed crops cultivated worldwide. Recent advances in sequencing technologies have allowed the identification of conserved and novel miRNAs in many plant species. Here, we used high-throughput sequencing technologies to identify and characterize the miRNAs in castor bean.
Five small RNA libraries were constructed for deep sequencing from root tips, leaves, developing seeds (at the initial stage, seed1; and at the fast oil accumulation stage, seed2) and endosperms in castor bean. High-throughput sequencing generated a large number of sequence reads of small RNAs in this study. In total, 86 conserved miRNAs were identified, including 63 known and 23 newly identified. Sixteen miRNA isoform variants in length were found from the conserved miRNAs of castor bean. MiRNAs displayed diverse organ-specific expression levels among five libraries. Combined with criteria for miRNA annotation and a RT-PCR approach, 72 novel miRNAs and their potential precursors were annotated and 20 miRNAs newly identified were validated. In addition, new target candidates for miRNAs newly identified in this study were proposed.
The current study presents the first high-throughput small RNA sequencing study performed in castor bean to identify its miRNA population. It characterizes and increases the number of miRNAs and their isoforms identified in castor bean. The miRNA expression analysis provides a foundation for understanding castor bean miRNA organ-specific expression patterns. The present study offers an expanded picture of miRNAs for castor bean and other members in the family Euphorbiaceae.
PMCID: PMC3722108  PMID: 23894571
19.  Deep sequencing discovery of novel and conserved microRNAs in trifoliate orange (Citrus trifoliata) 
BMC Genomics  2010;11:431.
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.
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.
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.
PMCID: PMC2996959  PMID: 20626894
20.  Cloning, characterization, and expression of microRNAs from the Asian malaria mosquito, Anopheles stephensi 
BMC Genomics  2008;9:244.
microRNAs (miRNAs) are non-coding RNAs that are now recognized as a major class of gene-regulating molecules widely distributed in metozoans and plants. miRNAs have been found to play important roles in apoptosis, cancer, development, differentiation, inflammation, longevity, and viral infection. There are a few reports describing miRNAs in the African malaria mosquito, Anopheles gambiae, on the basis of similarity to known miRNAs from other species. An. stephensi is the most important malaria vector in Asia and it is becoming a model Anopheline species for physiological and genetics studies.
We report the cloning and characterization of 27 distinct miRNAs from 17-day old An. stephensi female mosquitoes. Seventeen of the 27 miRNAs matched previously predicted An. gambiae miRNAs, offering the first experimental verification of miRNAs from mosquito species. Ten of the 27 are miRNAs previously unknown to mosquitoes, four of which did not match any known miRNAs in any organism. Twenty-five of the 27 Anopheles miRNAs had conserved sequences in the genome of a divergent relative, the yellow fever mosquito Aedes aegypti. Two clusters of miRNAs were found within introns of orthologous genes in An. gambiae, Ae. aegypti, and Drosophila melanogaster. Mature miRNAs were detected in An. stephensi for all of the nine selected miRNAs, including the four novel miRNAs (miR-x1- miR-x4), either by northern blot or by Ribonuclease Protection Assay. Expression profile analysis of eight of these miRNAs revealed distinct expression patterns from early embryo to adult stages in An. stephensi. In both An. stephensi and Ae. aegypti, the expression of miR-x2 was restricted to adult females and predominantly in the ovaries. A significant reduction of miR-x2 level was observed 72 hrs after a blood meal. Thus miR-x2 is likely involved in female reproduction and its function may be conserved among divergent mosquitoes. A mosquito homolog of miR-14, a regulator of longevity and apoptosis in D. melanogaster, represented 25% of all sequenced miRNA clones from 17-day old An. stephensi female mosquitoes. An. stephensi miR-14 displayed a relatively strong signal from late embryonic to adult stages. miR-14 expression is consistent during the adult lifespan regardless of age, sex, and blood feeding status. Thus miR-14 is likely important across all mosquito life stages.
This study provides experimental evidence for 23 conserved and four new microRNAs in An. stephensi mosquitoes. Comparisons between miRNA gene clusters in Anopheles and Aedes mosquitoes, and in D. melanogaster suggest the loss or significant change of two miRNA genes in Ae. aegypti. Expression profile analysis of eight miRNAs, including the four new miRNAs, revealed distinct patterns from early embryo to adult stages in An. stephensi. Further analysis showed that miR-x2 is likely involved in female reproduction and its function may be conserved among divergent mosquitoes. Consistent expression of miR-14 suggests that it is likely important across all mosquito life stages from embryos to aged adults. Understanding the functions of mosquito miRNAs will undoubtedly contribute to a better understanding of mosquito biology including longevity, reproduction, and mosquito-pathogen interactions, which are important to disease transmission.
PMCID: PMC2430712  PMID: 18500992
21.  MicroRNAs of Bombyx mori identified by Solexa sequencing 
BMC Genomics  2010;11:148.
MicroRNA (miRNA) and other small regulatory RNAs contribute to the modulation of a large number of cellular processes. We sequenced three small RNA libraries prepared from the whole body, and the anterior-middle and posterior silk glands of Bombyx mori, with a view to expanding the repertoire of silkworm miRNAs and exploring transcriptional differences in miRNAs between segments of the silk gland.
With the aid of large-scale Solexa sequencing technology, we validated 257 unique miRNA genes, including 202 novel and 55 previously reported genes, corresponding to 324 loci in the silkworm genome. Over 30 known silkworm miRNAs were further corrected in their sequence constitutes and length. A number of reads originated from the loop regions of the precursors of two previously reported miRNAs (bmo-miR-1920 and miR-1921). Interestingly, the majority of the newly identified miRNAs were silkworm-specific, 23 unique miRNAs were widely conserved from invertebrates to vertebrates, 13 unique miRNAs were limited to invertebrates, and 32 were confined to insects. We identified 24 closely positioned clusters and 45 paralogs of miRNAs in the silkworm genome. However, sequence tags showed that paralogs or clusters were not prerequisites for coordinated transcription and accumulation. The majority of silkworm-specific miRNAs were located in transposable elements, and displayed significant differences in abundance between the anterior-middle and posterior silk gland.
Conservative analysis revealed that miRNAs can serve as phylogenetic markers and function in evolutionary signaling. The newly identified miRNAs greatly enrich the repertoire of insect miRNAs, and provide insights into miRNA evolution, biogenesis, and expression in insects. The differential expression of miRNAs in the anterior-middle and posterior silk glands supports their involvement as new levels in the regulation of the silkworm silk gland.
PMCID: PMC2838851  PMID: 20199675
22.  Identification and characterization of novel and conserved microRNAs in several tissues of the Chinese rare minnow (Gobiocypris rarus) based on illumina deep sequencing technology 
BMC Genomics  2016;17:283.
MicroRNAs (miRNAs), which comprise a large family of endogenous small non-coding RNA molecules, play important roles in the regulation of gene expression in various biological processes. The Chinese rare minnow (Gobiocypris rarus) is a Chinese native fish species and is used extensively as an experimental fish in China; however, relevant biological data, especially miRNA transcriptome data, have not been well documented. To discover conserved and potential novel miRNAs in Chinese rare minnows, a pool of equal amounts of RNA obtained from 6 different adult rare minnow tissues (brain, eye, gill, liver, muscle and heart) was sequenced using illumina deep sequencing technology.
In the present study, 26,930,553 raw reads, representing 2,118,439 unique high-quality reads, were obtained from the pooled small RNA library. Using bioinformatics analysis, 352 conserved and 112 novel Chinese rare minnow miRNAs were first discovered and characterized in this study. Moreover, we found extensive sequence variations (isomiRs) in rare minnow miRNAs, including internal miRNA isomiRs and terminal isomiRs at both the 5′ and 3′ ends and nucleotide variants. Six conserved and 4 novel miRNAs were selected and validated in 6 different adult rare minnow tissues using quantitative real-time PCR (qPCR). The results showed that miR-30a, miR-30b, and Novel-37 are ubiquitously expressed in a variety of tissues. miR-16a, miR-9, miR-125b, miR-34a, and Novel-69 were predominantly expressed in the brain. Novel-115 and Novel-7 were highly expressed in gills, but were relatively weakly expressed in other tissues. These results provided the expression patterns of miRNA genes in Chinese rare minnow. Finally, based on bioinformatics predictions, we mainly found that Novel-94 and Novel-1b-5p were simultaneously targeted to the 3′UTR of Dmrt1, which controls sex determination and/or sexual differentiation in a variety of metazoans at different sites. Novel-29b targeted the 3′UTR of Foxl2, which is involved in the maintenance of ovarian function and the transcriptional regulation of gonadal differentiation-related genes. Novel-62 and Novel-53 targeted the 3′UTR of ERbeta1 and ERbeta2 (which regulate the transcription of target genes), respectively.
Rare minnow is a widely used model for assessing the risk of environmental pollution in China. Identifying and characterizing rare minnow miRNA genes is necessary to discover the biological function of miRNAs and to screen for new molecule biomarkers to assess the risk of environmental pollution in the future.
Electronic supplementary material
The online version of this article (doi:10.1186/s12864-016-2606-5) contains supplementary material, which is available to authorized users.
PMCID: PMC4828758  PMID: 27066897
Gobiocypris rarus; microRNA; Deep sequencing; Target prediction; IsomiRs
23.  The Viral and Cellular MicroRNA Targetome in Lymphoblastoid Cell Lines 
PLoS Pathogens  2012;8(1):e1002484.
Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus linked to a number of B cell cancers and lymphoproliferative disorders. During latent infection, EBV expresses 25 viral pre-microRNAs (miRNAs) and induces the expression of specific host miRNAs, such as miR-155 and miR-21, which potentially play a role in viral oncogenesis. To date, only a limited number of EBV miRNA targets have been identified; thus, the role of EBV miRNAs in viral pathogenesis and/or lymphomagenesis is not well defined. Here, we used photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) combined with deep sequencing and computational analysis to comprehensively examine the viral and cellular miRNA targetome in EBV strain B95-8-infected lymphoblastoid cell lines (LCLs). We identified 7,827 miRNA-interaction sites in 3,492 cellular 3′UTRs. 531 of these sites contained seed matches to viral miRNAs. 24 PAR-CLIP-identified miRNA:3′UTR interactions were confirmed by reporter assays. Our results reveal that EBV miRNAs predominantly target cellular transcripts during latent infection, thereby manipulating the host environment. Furthermore, targets of EBV miRNAs are involved in multiple cellular processes that are directly relevant to viral infection, including innate immunity, cell survival, and cell proliferation. Finally, we present evidence that myc-regulated host miRNAs from the miR-17/92 cluster can regulate latent viral gene expression. This comprehensive survey of the miRNA targetome in EBV-infected B cells represents a key step towards defining the functions of EBV-encoded miRNAs, and potentially, identifying novel therapeutic targets for EBV-associated malignancies.
Author Summary
Over 90% of adults worldwide are infected with Epstein-Barr virus (EBV). While EBV infection is normally controlled by a healthy immune system, in immuno-compromised individuals, EBV can cause serious disease and/or cancer. During infection, EBV expresses viral microRNAs (miRNAs) and induces the expression of specific cellular miRNAs. In general, miRNAs inhibit target gene expression by binding to complementary regions on target messenger RNAs (mRNA). While cellular miRNAs regulate important biological processes such as cell growth and differentiation, and many miRNAs have been linked to cancer progression, the functions of EBV miRNAs are largely unknown. To identify targets of EBV miRNAs and cellular miRNAs in EBV-infected cells, we used a high-throughput method based on next-generation sequencing technology to give a global picture of miRNA-regulated gene expression. Our analysis showed that over 500 mRNAs can be regulated by viral miRNAs, many of which are directly relevant to EBV infection. This study provides a comprehensive survey of viral and cellular miRNA targets in B cells, which is a positive step towards identifying novel therapeutic targets for EBV-associated cancers.
PMCID: PMC3266933  PMID: 22291592
24.  Genome-Wide Identification of Schistosoma japonicum MicroRNAs Using a Deep-Sequencing Approach 
PLoS ONE  2009;4(12):e8206.
Human schistosomiasis is one of the most prevalent and serious parasitic diseases worldwide. Schistosoma japonicum is one of important pathogens of this disease. MicroRNAs (miRNAs) are a large group of non-coding RNAs that play important roles in regulating gene expression and protein translation in animals. Genome-wide identification of miRNAs in a given organism is a critical step to facilitating our understanding of genome organization, genome biology, evolution, and posttranscriptional regulation.
Methodology/Principal Findings
We sequenced two small RNA libraries prepared from different stages of the life cycle of S. japonicum, immature schistosomula and mature pairing adults, through a deep DNA sequencing approach, which yielded ∼12 million high-quality short sequence reads containing a total of ∼2 million non-redundant tags. Based on a bioinformatics pipeline, we identified 176 new S. japonicum miRNAs, of which some exhibited a differential pattern of expression between the two stages. Although 21 S. japonicum miRNAs are orthologs of known miRNAs within the metazoans, some nucleotides at many positions of Schistosoma miRNAs, such as miR-8, let-7, miR-10, miR-31, miR-92, miR-124, and miR-125, are indeed significantly distinct from other bilaterian orthologs. In addition, both miR-71 and some miR-2 family members in tandem are found to be clustered in a reversal direction model on two genomic loci, and two pairs of novel S. japonicum miRNAs were derived from sense and antisense DNA strands at the same genomic loci.
The collection of S. japonicum miRNAs could be used as a new platform to study the genomic structure, gene regulation and networks, evolutionary processes, development, and host-parasite interactions. Some S. japonicum miRNAs and their clusters could represent the ancestral forms of the conserved orthologues and a model for the genesis of novel miRNAs.
PMCID: PMC2785426  PMID: 19997615
25.  Characterization of bovine miRNAs by sequencing and bioinformatics analysis 
BMC Molecular Biology  2009;10:90.
MicroRNAs (miRNAs) are a family of ~22 nucleotide small RNA molecules which regulate gene expression by fully or partially binding to their complementary sequences in mRNAs or promoters. A large number of miRNAs and their expression patterns have been reported in human, mouse and rat. However, miRNAs and their expression patterns in live stock species such as beef cattle are not well studied.
We constructed and sequenced small-RNA libraries to yield a total of 13,541 small-RNA sequences from 11 bovine tissues including brain, subcutaneous fat, muscle, liver, kidney, spleen and thymus. In total, 228 miRNAs including 29 novel miRNA candidates were identified. Of the 199 miRNAs, 101 have been previously reported as bovine miRNAs and the other 98 are bovine orthologs of known miRNAs that have been identified in at least one other mammalian species. Of the 29 novel miRNA candidates, 17 appeared at this point in time to be bovine specific, while the remaining 12 had evidence of evolutionary conservation in other mammalian species. Five miRNAs (miR-23a, -23b, -99a, -125b and -126-5p) were very abundant across the 11 tissues, accounting for 44.3% of all small RNA sequences. The expression analysis of selected miRNAs using qRT-PCR also showed that miR-26a and -99a were highly expressed in all tissues, while miR-122 and miR-133a were predominantly expressed in liver and muscle, respectively.
The miRNA expression patterns among 11 tissues from beef cattle revealed that most miRNAs were ubiquitously expressed in all tissues, while only a few miRNAs were tissue specific. Only 60% miRNAs in this study were found to display strand bias, suggesting that there are some key factors for mature miRNA selection other than internal stability. Most bovine miRNAs are highly conserved in other three mammalian species, indicating that these miRNAs may have a role in different species that are potential molecular markers for evolution.
PMCID: PMC2761914  PMID: 19758457

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