MicroRNAs (miRNAs) are small, non-coding RNAs that play essential roles in plant growth, development, and stress response. We conducted a genome-wide survey of maize miRNA genes, characterizing their structure, expression, and evolution. Computational approaches based on homology and secondary structure modeling identified 150 high-confidence genes within 26 miRNA families. For 25 families, expression was verified by deep-sequencing of small RNA libraries that were prepared from an assortment of maize tissues. PCR–RACE amplification of 68 miRNA transcript precursors, representing 18 families conserved across several plant species, showed that splice variation and the use of alternative transcriptional start and stop sites is common within this class of genes. Comparison of sequence variation data from diverse maize inbred lines versus teosinte accessions suggest that the mature miRNAs are under strong purifying selection while the flanking sequences evolve equivalently to other genes. Since maize is derived from an ancient tetraploid, the effect of whole-genome duplication on miRNA evolution was examined. We found that, like protein-coding genes, duplicated miRNA genes underwent extensive gene-loss, with ∼35% of ancestral sites retained as duplicate homoeologous miRNA genes. This number is higher than that observed with protein-coding genes. A search for putative miRNA targets indicated bias towards genes in regulatory and metabolic pathways. As maize is one of the principal models for plant growth and development, this study will serve as a foundation for future research into the functional roles of miRNA genes.
MicroRNAs are non-coding RNAs that regulate gene expression post-transcriptionally and play roles in diverse pathways including those acting on development and responses to stress. Here, we describe a genome-wide computational prediction of maize miRNA genes and their characterization with respect to expression, putative targets, evolution following whole genome duplication, and allelic diversity. The structures of unprocessed primary miRNA transcripts were determined by 5′ RACE and 3′ RACE. Expression profiles were surveyed in five tissue types by deep-sequencing of small RNA libraries. We predicted miRNA targets computationally based on the most recent maize protein annotations. Analysis of the predicted functions of target genes, on the basis of gene ontology, supported their roles in regulatory processes. We identified putative orthologs in Sorghum based on an analysis of synteny and found that maize-homoeologous miRNA genes were retained more frequently than expected. We also explored miRNA nucleotide diversity among many maize inbred lines and partially inbred teosinte lines. The results indicated that mature miRNA genes were highly conserved during their evolution. This preliminary characterization based on our findings provides a framework for future analysis of miRNA genes and their roles in key traits of maize as feed, fodder, and biofuel.
Citation: Zhang L, Chia J-M, Kumari S, Stein JC, Liu Z, Narechania A, et al. (2009) A Genome-Wide Characterization of MicroRNA Genes in Maize. PLoS Genet5(11): e1000716. https://doi.org/10.1371/journal.pgen.1000716
Editor: Joseph R. Ecker, The Salk Institute for Biological Studies, United States of America
Received: July 14, 2009; Accepted: October 12, 2009; Published: November 20, 2009
This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.
Funding: This work was funded by USDA-ARS CRIS Project #1907-21000-014-00D and 3622-21000-027D and NSF DBI-0527192. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
The last decade has witnessed remarkable progress in our knowledge of the biogenesis and activity of diverse classes of small non-coding RNAs (sRNA). These include microRNAs (miRNA) , small interfering RNAs (siRNA) , trans-acting siRNAs (ta-siRNA) , and others . While the majority of plant sRNAs are transcribed from siRNA genes residing in repetitive and transposon-rich regions, and regulate chromatin silencing , a great deal of interest has been placed on miRNAs due to their ability to post-transcriptionally regulate gene expression . This is exemplified by the critical regulatory behavior of miRNAs at key positions in a variety of pathways, such as root ,, shoot , leaf – and flower , development and cell fate ,. Additionally, they also include responses to phytohormones , nutrient – and other environmental stresses –.
As a genetic model system, maize has contributed significantly to our understanding of plant development and evolution, and more recently this knowledge has been employed to elucidate the regulatory functions of miRNA genes. For instance, teosinte glume architecture 1 (tga1) is one of the major genes responsible for the evolution of maize from its ancestor teosinte and has also been identified as a target for miR156 ,. Intriguingly, mutations in Corngrass1 (Cg1) result in the over-expression of miR156 and decreased miR172 levels, resulting in alterations of the juvenile to adult phase transition ,. Taken together, it is apparent that miRNA regulation is intertwined with key plant development processes. Thus, there is considerable interest in taking advantage of the complete genome sequence of maize B73 reference genome version 1 (B73 RefGen_v1)  to systematically identify miRNA genes, their corresponding targets, and to decipher their regulatory roles.
The mature biologically active products of miRNA genes define miRNA gene families. This, along with the characteristic ‘hairpin’ structure of its precursor (pre-miRNA), allows computational detection and annotation of miRNA genes ,. Post-transcriptional regulation is accomplished by the RNA-induced silencing complex (RISC complex) which directs complementary binding of the mature miRNA product to mRNA transcripts, usually resulting in target cleavage or inhibition of translation . In plants, the near perfect complementarity between the miRNA and its substrate mRNA has permitted computational methods for the specific identification of target genes .
In this study, we systematically annotated miRNA genes based on the first nearly complete assembly of the maize B73 RefGen_v1 . This analysis includes experimental validations for many of these genes, as well as expression studies using deep small RNA transcriptome sequencing across a broad panel of maize tissues. We also conducted one of the first comprehensive characterizations of maize pri-miRNA transcripts, providing a deeper understanding of their transcription and regulation. Prediction of protein-coding targets confirmed many known regulatory substrates and provided a window into many more potentially novel pathways. We also describe the first analysis of allelic diversity of miRNAs in maize using a large panel of highly diverse inbred lines as well as its wild relative teosinte. A comparative genomic analysis with sorghum provided insight into the evolutionary dynamics of miRNA family expansions and will serve as basis for future comparative functional genomic analyses using syntenic orthologs. We believe the multi-faceted nature of this study will help accelerate our understanding of miRNAs, their regulatory roles in critical biological processes, as well as offer the community detailed annotations applicable to their own research.
Genome-wide survey of known pre–miRNAs and their paralogous pre–miRNA structures in maize
Starting with a set of 1822 plant mature miRNA sequences available in miRBase , we predicted putative pre-miRNA structures at more than 4,000 loci in the maize B73 RefGen_v1 . Of these, more than 300 passed initial filters for positional overlaps, secondary structure and orientation of mature miRNA sequences within the respective stem-loop structures. After additional screening for overlaps with transposable elements (Table S1) and removal of families not well characterized in current literature, we annotated 150 miRNAs from 26 families with high confidence. Since our search space for pre-miRNA structure included only 250nt surrounding the aligned mature miRNA, our method may have missed families that have introns in their precursors. One example is miR444, which has been reported as conserved in rice, maize, sorghum, and sugarcane –, but was missed by our pipeline due to a large intron in its pre-miRNA structure.
There are a total of 98 maize miRNA genes deposited in miRBase –, which were predicted based on the MAGI (Maize Assembled Genomic Islands: maizegdb.org) and TIGR EST collections. Of these, 89 were detected in our set and are now annotated on the B73 reference genome assembly. We failed to detect nine miRNA genes in our pipeline: miR160e, miR166e, miR166f, miR169e, miR169g, miR169h, miR172a, miR172e and miR408. It is possible that these are located in regions that are not captured in the current genome assembly. In addition to those previously characterized, we found 61 putative miRNA genes representing new members of conserved families. A summary of known and previously identified miRNA gene families is provided in Table 1. Detailed information about the genomic location of each miRNA, as well as the mature miRNA and miRNA* for each miRNA gene, is listed in Table S2.
Annotated miRNA genes were found on all maize chromosomes, having a distribution pattern similar to those of protein-coding genes (Table S2) . No miRNA genes were found in unanchored portions of the assembly. Like protein coding genes, a proportion of the miRNA genes are organized as tandem paralog clusters (see section “Genome Organization and Conservation with Sorghum”). However, a subset of these was found as unusually compact clusters, with less than 2000nt separating adjacent genes, as shown in Table 2. Two members of the miRNA156 family (miR156b and miR156c) on chromosome 3 are separated by less than 200nt, as are two members of the miRNA166 family on chromosome 5. The close distance of miR156b and miR156c is observed in several monocots and they are transcribed as one transcript (polycistronic) in maize  and rice . Clusters of two members of miR166 are also observed in several plant species, including moss –. Compact clustering was particularly prevalent in the miR395 family. Four such clusters were found on chromosomes 2 and 10, comprising 16 genes in total. In rice, compact clusters of miR395 genes have also been observed with each forming a single polycistronic transcription unit .
Characterization of the pri–miRNA
Primary transcripts of miRNA genes are known to have features typical of transcription by RNA polymerase II, including 5′ capping, 3′ polyadenylation, and intron splicing . To characterize the pri-miRNA transcripts in maize, we designed gene-specific primers for each of the 89 previously identified miRNA genes and conducted 5′ RACE and 3′ RACE using a template containing mixed tissues of seedling, immature tassel and immature ear. Overall, we were able to capture the upstream transcribed regions (5′ region) of 55 miRNA genes and the downstream transcribed regions (3′ region) of 51 miRNA genes (Table S2). Among these, we obtained both 5′ and 3′ RACE products for 40 miRNA genes, producing full-length transcript sequences. Failure to amplify some miRNA gene products might have been a limitation of the tissues sampled, as some miRNA genes might be expressed in highly specific tissue/cell types, developmental stages, or environmental conditions. From our RACE data, we were able to confirm the clusters of miR156b/c and miR166k/m, but failed to detect miR395a/b clusters. Genes of miR395 family are known to be up-regulated in response to low-sulfate conditions  and it is possible that transcript abundance was below the detection threshold given the normal nutrient status in which our plants were grown. While our work was underway, the data for maize full-length complementary DNA (FLcDNA) became available ,. To identify pri-miRNAs in this FLcDNA set, we mapped 150 miRNA precursors against 63,000 FLcDNAs and found 33 transcripts that harbor 27 pre-miRNA sequences. After excluding those pri-miRNAs that were mapped with RACE, we identified 10 additional pri-miRNAs using FLcDNA analysis (Table S3).
The overall lengths for pri-miRNA transcripts ranged from 250nt to nearly 2000nt, with an average size of 810nt, far less than the 1,433nt average for maize protein-coding transcripts . Figure 1 shows that the 3′ region of pri-miRNAs (measured from the stem-loop to the transcriptional stop site) is generally longer than the 5′ region (measured from the transcriptional start site (TSS) to the stem-loop), having mean lengths of 953nt and 523nt respectively. We observed that 83% of maize pri-miRNAs have TATA-box like motifs in the −23 to −28 positions relative to TSS (data not shown). Previous studies using rice and Arabidopsis FLcDNAs and Arabidopsis pri-miRNAs showed a high prevalence of an adenine at the TSS and a cytosine at the −1 position . Of the available 76 miRNA gene transcripts with TSS sites, most have A at the TSS and C at the −1 position (Figure 2). We also observed evidence for alternative TSS. Among the 55 miRNA transcripts with 5′ region information, 10 had two TSSs that are 3nt to 9nt apart. This is similar to FLcDNAs studies of Arabidopsis and rice showing two TSSs per locus, with an average distance of 4.2nt and 8.7nt apart, respectively ; however, the distance between various stop sites can be up to several hundred nucleotides. Examples of polycistronic transcripts and multiple TSS and stop sites are shown in Figure 3.
Length distribution of 5′ (blue bars) and 3′ (red bars) regions of pri-miRNA. The 5′ region is defined as the segment from the transcription start site to the hairpin structure (HP), while the 3′ region is the segment between the hairpin structure and the transcription stop site, 3′ regions are longer, averaging 953nt versus 523nt.
Nucleotide frequencies surrounding transcriptional start sites (as determined from 5′ RACE products) are shown using sequence logos ,. TSSs are predominately marked by an adenine at the +1 position and a cytosine at the −1 position.
Examples of introns, alternative transcriptional start sites (TSSs), polyadenylation sites, and polycistronic features of pri-miRNAs seen in miRNA RACE amplicons mapped to the maize genome. Mature miRNAs are represented by red boxes, miRNA* by green boxes. miRNA precursors are represented by hairpin structures while TATA box-like motifs are also indicated in the figure. Numbers in parentheses indicate physical locations on the reference chromosome. (A) RACE products show that miRNAs pri-miR156b and pri-miR156c are transcribed as a single transcript, with a 94nt intron spliced out from the locus. (B) pri-miR172c is an example of a miRNA having multiple TSSs and polyadenylation sites. 5′ RACE products map to 4 distinct locations and 3′ RACE products carry 4 distinct polyadenylation signatures. Two of the TSSs are associated with TATA box-like motifs.
In addition we found that introns were more abundant in the 3′ region than in the 5′ region. From RACE reaction products, we found only one gene (miR164c) with an intron in the 5′ region, whereas 15 genes showed one or two introns in the 3′ region (Table S4). Intron length varied from 71nt to 2196nt, and the canonical splice motif GU…AG was found for all but one intron (Table S4). Alternative non-spliced transcripts were found for three genes: miR159a, miR166g, and miR169i.
Expression profiles of miRNAs
Using Illumina's sequencing-by-synthesis technology , we profiled the genome-wide transcript profiles of miRNA abundance across five different maize tissue types (root, seedling, tassel, ear, and pollen). Small RNA libraries were generated for each tissue and sequenced using the Illumina 1G Sequencer. The small RNAs (18–22nt) were mapped to the predicted pre-miRNAs. The number of reads mapping to each pre-miRNA were enumerated and normalized against the total count of 18 to 22 nucleotide reads, reported as reads per million (RPM), for each respective library. A summary of the quantitative expression profiles for each of the 26 miRNA families is shown in Table 3. Of the five tissues surveyed, the seedling samples showed the highest expression levels while the pollen samples showed the lowest expression levels, with RPM counts an order of magnitude larger in the seedling. We noted that miR482 lacks an expression signature in any of the five tissues, while miR162 miR394, miR395, miR398, miR399, miR408, miR528 and miR1432 had low expression counts (less than 200 RPM). In contrast, miR156, miR159, miR167, miR168, miR169, miR171, miR319, and miR529 had high expression counts (slightly over 3,000 RPM, on average). In our samples, miR529 was over-expressed in the tassel (almost 16,000 RPM) but under-expressed in the other four tissues (slightly over 300RPM, on average). Several families showed higher expression levels in juvenile tissues. The miR156, miR164, miR168, miR393, miR395, miR396, miR398, and miR399 families had higher signatures in juvenile root and seedling tissues while miR172 demonstrated a higher expression level in reproductive tissues (tassel and ear).
Within miRNA families, there are examples of tissue-specific expression differences among individual miRNAs. For example, the miR396 family is overall highly expressed in the juvenile root and seedling samples but only certain individual family members (miR396a, miR396b, and miR396g) are highly expressed in pollen (Figure 4).
Solexa reads mapping to the miR396 family were counted and summed for each family member, showing tissue specificity of expression amongst members of the same family. Read counts are plotted as Reads Per Million (RPM) on a log scale.
Prediction of miRNA protein-coding targets
We computationally predicted potential maize targets using the Filtered Gene Set transcripts (Release 4a.53) of the B73 maize genome sequence . The filtered set of transcripts contains about 32,500 entries, and is comprised of the longest representative cDNA transcripts. These are presumed free of pseudogenes and low-complexity repetitive elements such as short-tandem repeats and transposons. Our computational pipeline predicted 247 unique putative genes targeted by 150 miRNA sequences belonging to 26 miRNA families. Around 85% of the predicted targets have functional InterPro annotations . We observed that 12 out of 26 miRNA families are predicted to target transcription factors, as shown in Table S6, suggesting roles of these miRNA families in post-transcriptional regulation and transcription networks. In addition to transcription factors, other predicted target genes are involved in diverse physiological and metabolic processes. Such targets include protein kinases, signal transduction histidine kinases, antifreeze proteins, F-box proteins, cytochrome P450, cupredoxin, peroxidases, multicopper oxidases, transporters, ATP sulfurylases, and cell division proteins (Table S6).
Classification of predicted miRNA target functions using Gene Ontology (GO)
To gain a better understanding of the functional roles of the predicted miRNA target genes in maize, we looked for target enrichment in Gene Ontology (GO) molecular function and biological process categories . The targets were annotated by using the GO annotations available from the B73 RefGen_v1. Of the predicted targets, 76% had GO assignments whereas only 53% of the genes in the entire refined set were associated with GO terms. BiNGO (Biological Networks Gene Ontology)  was used to study targets enrichment and to construct a hierarchical ontology tree in Cytoscape , as shown in Figure 5. We found that miRNA families preferentially target genes involved in a wide spectrum of regulatory functions and selected biological processes including gene expression/transcription, metabolism, catalysis, transport, and response to stimuli (Table S7).
Significantly overrepresented GO terms based on GO molecular functions and biological processes were visualized in Cytoscape. The size of a node is proportional to the number of targets in the GO category. The color represents enrichment significance— the deeper the color on a color scale, the higher the enrichment significance. White color nodes are not enriched but show the hierarchical relationship among the enriched ontology branches.
The genes targeted by miRNA families showed a strong affinity for binding activity (82.3%), transcription factor activity (10.6%) and transcription regulator activity (11.2%). In the GO biological process enrichment analysis, targeted genes were found to be involved in biological regulation of cellular processes (35.2%), biosynthetic processes (32%), and metabolic processes (32%). Approximately 32% of the target genes were involved in transcription/gene expression while about 19% of targets were classified in the response to stimulus category. The latter were found to be involved in the response to abiotic stimulus, endogenous stimulus, and hormone stimulus (Table S7). Since one gene can be related to multiple GO terms, the sum of the percentages of GO terms is not a relevant parameter.
We also computationally evaluated whether specific miRNA families were preferentially enriched in certain GO categories. Target genes of the following 8 miRNA families were overrepresented in both of the biological processes i.e. the regulation of transcription and the response to stimulus. These families are: miR156, miR160, miR164, miR166, miR167, miR172, miR396, and miR528. In addition to these families, target genes of miR169, miR319, miR408 and miR529 were involved in transcription regulation, whereas miR159, miR397, and miR399 target genes were involved in response to stimulus. Targets of only miR395 family showed involvement in sulfate assimilation pathway whereas targets of both miR395 and miR399 families showed enrichment in transmembrane transport activities. Target genes of miRNA families, miR164, miR397, miR408, and miR528 showed enrichment in laccase and oxidoreductase activities and were found to be involved in secondary metabolic processes such as phenylpropanoid, amino acids, aromatic compounds and lignin catabolic processes (Table S7).
Genome organization and conservation with sorghum
Previous studies in Arabidopsis , and rice  have shown that miRNA gene families evolved from a combination of tandem, segmental, and whole-genome duplication events . Maize was derived from an ancient allotetraploid, and it is estimated that the two maize progenitor genomes diverged from the ancestor of sorghum ∼12 MYA . Comparative mapping between maize and sorghum can therefore reveal the fate of miRNA genes after whole genome duplication. In addition, synteny can be used to help infer orthologous relationships between these species.
Based on the maize high confidence set, we filtered previously annotated Sorghum bicolor miRNA genes . The distribution of these genes by family is shown in Table S8, along with corresponding information for maize. Synteny was examined in the context of orthologous protein coding genes which numbered 25,216 in maize and 20,408 in sorghum  (See Materials and Methods). In total, we found 136 maize and 106 sorghum miRNA genes within syntenic regions, corresponding to 91% and 79% of their respective totals. These values are similar to the percentages of syntenic protein-coding orthologs, 85% in maize and 89% in sorghum . The lower percentage of syntenic sorghum miRNA genes may be indicative of false positives within this set, as these did not undergo the same rigorous screening process as for maize. Synteny was found amongst all families except miR827 and miR482 (Table S8). The former has a single representative in each genome, located in non-syntenic regions; the latter has one member in maize but none annotated in sorghum. As shown in Figure 6, conserved synteny among miRNA genes was detected on all chromosomes of maize and sorghum. This figure also shows that many miRNA genes in sorghum map to both sister sites created after the genome-wide duplication event in maize.
Positions of annotated MIR genes are shown using circles that are color-coded according to the family. Links show synteny between MIR genes. Maize and Sorghum chromosomes are color-coded such that syntenic regions, based on protein coding genes, have matching colors.
Many miRNA genes are organized within paralog clusters, defined as family members having no more than two intervening genes. Some of these are comprised of compact clusters, as described above. In maize, we found 13 paralog clusters containing 40 genes in total, while sorghum has 15 clusters containing 47 genes. All of the 13 paralog clusters in maize are at syntenic positions. In sorghum, 14 of the clusters (43 genes) are in syntenic regions. Since the relationship between paralog clusters is not known between species (some may be orthologs and some paralogs depending on the timing of tandem duplications relative to speciation) we collapsed these into single sites. This led to 104 sites in sorghum and 123 sites in maize. Of these, 81 sites are syntenic in sorghum, therefore representing ancestral locations of these genes in the common ancestor of maize and sorghum. In maize, 28 of these sites are retained in sister duplicate positions. Thus, almost 35% of ancestral miRNA positions have been retained at sister homoeologous positions in maize, as detailed in Table S9. This is higher than the retention rate of protein-coding genes, measured at ∼21%.
Maize had a net gain of 30 syntenic genes compared to sorghum. All of these gains occurred at retained duplicate sites. Amongst singleton sites (those harboring miRNA genes on just one sister region), there was a deficit of five maize genes due to the reduced sizes of four paralog clusters relative to sorghum. Whether this was caused by the expansion of clusters in sorghum or the contraction of clusters in maize is not known. In contrast, the combined gene counts at the 28 retained duplicate sites in maize exceed gene counts at the corresponding sorghum sites in all but two cases. This resulted in a net gain in maize of 35 genes at retained duplicate sites. There were twenty-two sites harboring single genes in sorghum with corresponding duplicate sites in maize also harboring single genes. An additional site contains exactly two genes at each corresponding region. Assuming these gene counts were stable through evolution, then a gain of 24 genes can be directly attributed to the whole genome duplication in maize. The remaining five retained sites have clusters with varying numbers of genes in sorghum and corresponding maize sister regions. These sites contributed a net gain of eleven maize genes, the combined result of whole-genome duplication and differential expansion/contraction of paralog clusters.
The evolutionary dynamics influencing miRNA family size is illustrated by closer examination of the miR159 family. This family has the largest difference in membership, 11 genes in maize compared to 3 genes in sorghum (Table S8). This excess of eight genes includes three (miR159d/e/g) that have no syntenic counterparts in sorghum, suggesting long-distance movement in maize or loss of ancestral sites in the sorghum lineage. Another four exist within a paralog cluster that is expanded in maize relative to sorghum. In maize this cluster contains six genes (miR159a/b/h/i/j/k) on chromosome 8 while the corresponding cluster on sorghum chromosome 3 has only two genes. Finally there is a single gene (miR159f) at the homoeologous site on maize chromosome 3 which resulted from the whole genome duplication.
Nucleotide diversity of miRNA genes
Because of the characteristic structure and important regulatory function of miRNAs, it would be expected that the mature miRNAs be highly conserved across maize accessions. Sequence diversity in the flanking regions of the mature miRNA can be used to imply natural or artificial selection on the miRNA loci. To explore the genetic diversity of miRNAs in maize, we selected 28 maize miRNA genes (Table S10) based on their availability in the initial reduced representation libraries of maize ,. We designed sequencing primers for ∼400nt surrounding each mature miRNA sequence. The 28 maize miRNA genes and flanking regions were sequenced in 28 maize inbred lines chosen to maximize genetic diversity . Additionally, 16 partially inbred teosinte lines  (Zea mays ssp. parviglumis) were selected to represent the diversity in the progenitor of cultivated maize. As expected, there was no polymorphism detected within the mature miRNA sequences in either the maize inbred or teosinte lines. However, the level of nucleotide diversity flanking the mature miRNA sequence is similar to the genic level of diversity: the average proportion of pairwise nucleotide differences per nucleotide site (π) is 0.0065 for miRNA genes in the maize inbred lines versus 0.0067 for a random collection of 1,095 maize genes. By comparing genetic diversity in inbred versus teosinte accessions, we tested if any of the miRNAs had a reduction in polymorphisms consistent with the selective sweep during maize domestication or improvement. We found π = 0.0105 for the miRNA genes among the teosinte lines compared to π = 0.0095 for a large collection of teosinte protein coding genes . We found no evidence of such a selective sweep (Table S11) suggesting that mature miRNA sequences are under strong purifying selection due to their functional importance, making them highly conserved over long evolutionary periods, while flanking regions evolve equivalent to other genes.
Evaluation of high-confidence miRNA prediction for maize
We have systematically annotated miRNA genes on the first complete assembly of the maize genome. Our computational methods for detection were stringent by design, distinguishing 150 genes in 26 miRNA families from many potentially spurious predictions. This high confidence set represents genuine miRNA genes is reinforced by our characterization of pre-miRNA structures, promoter features, expression profiles, genetic diversity, and identification of orthologous genes in sorghum (see further discussion below). Plant miRNA families can be historically divided into two classes: the highly conserved families, and the emerging class of lineage-specific families. Recent work suggests that a subset of the predicted lineage-specific families have characteristics of siRNA genes, associated with transposon-related repeats and showing little or no expression at the pre-miRNA level . One such family is miR414, which is currently under consideration for removal from miRBase ,. Another example is the miR437 family, which has expanded in the sorghum and maize lineages relative to rice. In maize, more than 90% of miR437 pre-miRNA structures show the presence of the Stowaway miniature inverted repeat transposonable element (MITE). Interestingly, these MITEs account for more than 87% of the repeats found in miRNA genes. In our analysis, maize transposon-related repeats were found in 11 miRNA families (miR437, miR854, miR1128, miR1132, miR1133, miR1320, miR1435, miR1436, miR1439, miR1884, and miR2102), thus making them suspect and supporting their removal from the high-confidence set (Table S1). In , Voinnet also noted the association of recently evolved families with MITEs and raised similar questions as to whether they represent true miRNAs versus siRNAs.
In animals, approximately 80% of miRNAs are found within introns of either protein-coding or non-coding genes . In contrast, most of the annotated miRNAs in plants are located in intergenic regions, with some exceptions . In the current work, 87% of the miRNAs were found in intergenic regions. Exceptions were 19 genes whose mature miRNA coordinates lie within exons of predicted protein coding genes. In six of these cases the host genes were also the predicted target of the embedded miRNA gene, oriented either on the same strand (miR159c, miR319b, miR396a and miR397a) or opposite strand (miR169j and miR399d). Of the remaining 13 miRNA genes, all except miR159e were located on the same strand as the protein-coding genes they are embedded in. miR159e was not expressed in any of the tissues sampled and has no syntenic relationship with any of the sorghum miR159 genes (see sections below), suggesting that miR159e might not be functional. The protein-coding genes with embedded miRNAs are small (encoding proteins of less than 120aa compared to the average of 358aa ) and have no identifiable InterPro domains. We speculate that a proportion of these may not be bona fide protein-coding genes but likely the result of misannotation based on populations of non-coding transcripts used in the evidence-based prediction method .
Expression of miRNA families and genes based on small RNA library profiling
MicroRNA tissue-specificity is known to play a role in plant development, an example of which is the regulation of juvenile-to-adult vegetative phase transition . To assess miRNA tissue specificity, we surveyed miRNA expression levels in five maize tissue types (root, seedling, tassel, ear, and pollen). In our data, we found that the expression of miR156 and miR172 families are anti-correlated (Table S4); miR156 is expressed higher in young roots and seedlings but lower in adult tissues (tassel, ear, and pollen), while miR172 expression has an opposite trend, albeit with a lower overall expression level. These results fit well with the current models of phase transition, whereby opposing gradients of miR156 and miR172 are responsible for the transition from juvenile to adult. This converse regulatory relationship between miR156 and miR172 has also been reported recently .
The miR529 family is expressed conspicuously higher in the tassel as compared with the other surveyed tissues. We note that the mature miRNA products of families miR529 and miR156 are identical from positions 8–14; which are key residues in miRNA-target recognition. Consequently both miRNA families have similar predicted targets consisting mainly of SQUAMOSA-promoter binding proteins (SBP). It is likely that miR529 is either related to, or is a sub-grouping of, the miR156 family, but with a distinct tissue expression profile.
As seen in the miR396 gene family, tissue expression profiles are not necessarily conserved amongst miRNA genes of the same family. This family consists of 7 members that exhibit distinct expression profiles (Figure 4, Table S2) – miR396a/b/g shows elevated expression in the pollen, miR396c/d is highly expressed in the juvenile tissues but not adult tissues, while miR396e/f expression profile shows a peak in seedling tissues. miR396c/d mature miRNA sequences carry an additional guanine residue between positions 8 and 9 as compared to the rest of the miR396 family. Other members of the miR396 family are conserved in both monocots and dicots, but miR396c/d appear to be monocot specific (equivalent to osa-miR396d/e found in rice) . The latter are also the only members of the miR396 family that target QLQ (glutamine-leucine-glutamine) and WRC (tryptophan-arginine-cysteine) domains that define the Growth-Regulating Factor (GRF) family of transcription factors . The GRF transcription factors are involved in leaf and cotyledon growth and expressed most abundantly in active developing tissues . QLQ has been found to be involved in mediating protein interactions whereas WRC plays a role in DNA binding . Therefore, miR396c/d could be acting as regulatorsr of GRF genes in juvenile tissues independently from the rest of the miR396 family.
There are also miRNA gene families that appear to be constitutively expressed in all 5 tissues. For example, miR168 family has a read count of at least 2400 RPM in all tissues surveyed, reflecting its role in maintaining a steady-state balance of the RNA silencing machinery by targeting the slicer AGO1 (ARGONAUTE1) of the RISC complex –.
Recent transcriptome analysis of Arabidopsis indicates that 15 genes involved in the miRNA pathway (including AGO1, AGO2, AGO4 AGO7, and DCL1–3) are absent in pollen . In the current study, we observed that expression of most families are lower in pollen as compared with other tissues, suggesting that the miRNA biogenesis machinery is similarly down-regulated in maize pollen tissues.
Of the 26 miRNA families on our refined list, only miR482 (consisting of a single member) failed to register any evidence of expression in the 5 tissues sampled. While miR482 had previously been annotated in poplar, pine, soybean and grape, it has not been identified in any monocots.
Characterization of pri–miRNA transcripts
Our analysis of the pri-miRNA transcripts shows that these genes exhibit many of the conserved signatures of RNA polymerase II transcripts. These include TATA boxes in conserved locations, alternative TSS and polyadenylation sites, and introns . Using the stems of the pre-miRNA for orientation, we found that, on average, the 5′ region is shorter than the 3′ region. The average length of the 3′ region is likely an underestimation due to ascertainment biases inherent in the RACE protocol, where smaller transcripts are more likely to be amplified than longer ones. This may also explain the shorter average length of pri-miRNAs amplified by RACE as compared with the average length of FL-cDNA that harbor pre-miRNA sequences (Table S3). The frequency of TATA-box appearance (83%) is similar to that of Arabidopsis miRNA transcripts (83%)  and much higher than average for protein promoters (50%) . As in , we also found a high occurrence of cytosine at the −1 position and adenine at the +1 position of TSSs (Figure 2). Together, such characteristics as the TATA box, the nucleotide frequency surrounding the TSSs and expected size of 5′ transcript regions, will help in the development of new methods to predict proximal promoters of miRNA genes.
Our target prediction method is stringent, but still allows us to capture most miRNA targets that are conserved across several plant species, including Arabidopsis , –, poplar , rice ,,, wheat , soybean , mustard , and grape . For example, miR156 targets SBP transcription factors ,, while miR159 targets the MYB family . Both miR160 and miR167 target Auxin Response Factor (ARF) transcription factors in Arabidopsis and maize ,; and are also captured by our predictions. The same trend is observed in many other miRNA families including miR164, miR166, miR169, miR171, miR172, miR319 and miR396 as they target various families of transcription factors such as NAM (No Apical Meristem) proteins, bZIP (basic-leucine Zipper) genes, CBF (CCAAT binding factor), GRAS transcription factor, AP2 (APETALA2)-EREBP (Ethylene-Responsive Element Binding Proteins), CCCH type zinc finger protein and TCP (Teosinite branched, Cycloidea, and PCF), GRF transcription factor families respectively , , –. These transcription factors are known to regulate plant development. MiRNA families miR393 and miR394 target F-box protein and are known to play a role in the expression control of genes involved in regulation of metabolic processes . It has been reported that in Arabidopsis, rice, and wheat, miR168 targets AGO1 (ARGONAUTE1) –. The prediction pipeline captured three AGO1 orthologs in maize . Our target predictions are further consistent with the literature in the case of miR395, which regulates sulfate metabolism by targeting sulphate transporter genes and ATP sulfurylase (APS) proteins, and whose expression increases in response to low sulfate growing conditions .
We also predicted potential targets of miRNA families not previously identified in maize (miR482, miR528, miR529, miR827 and miR1432). Trehalose phosphatase, cytochrome P450, pentatricopeptide, and CCHC type zinc finger protein were predicted as targets of miR482, suggesting the involvement of miR482 in a wide range of biosynthetic reactions. Similar to miR397 and miR408, miR528 also targets copper proteins cupredoxin, multicopper oxidase and laccase genes and thus might play a critical role in regulating physiological processes (photosynthetic and respiratory electron transport) and stress responses. Both miR156 and miR529 were predicted to target genes encoding the SBP box. miR827 was predicted to target NAD(P)-binding and SPX (SYG1/Pho81/XPR1) proteins, whereas miR1432 was predicted to target Poly(ADP-ribose) polymerase, catalytic region functions and calcium binding EF hand domains. Both SPX and calcium binding EF hand domains are associated with proteins that are active in signal transduction pathways.
The stringent criteria used to predict targets could potentially reduce false positive rates at the cost of missing several authentic targets. For example, miR162 targets DCL1 , but this target (GRMZM2G040762 in maize) was excluded by our pipeline due to the presence of a 1nt bulge in the alignment with miR162. In addition, for about 15% of the predicted targets, functional information is not available at this time. We hypothesize that some of these targets may be novel but this needs to be further verified based on experimental evidence.
Retention of miRNA genes following whole-genome duplication
The maize lineage tetraploidy event occurred an estimated 5 to 15 MYA –, and precipitated large-scale chromosomal rearrangements as well as massive losses of duplicate genes in the process of returning to a genetically diploid state , –. Our analysis showed that greater than 1/3 of ancestral miRNA positions were retained at both homoeologous sites and these accounted for the majority of gene family expansions in maize relative to sorghum. Interestingly this proportion of retained sites was greater than that seen for protein-coding genes. Gene duplication, especially by polyploidization, has long been thought to provide raw material for the evolution of functional novelty . The initial redundancy of duplicate genes is followed by a period of relaxed selection with gene loss being the ultimate fate for most duplicates ,. Classical models for retention of duplicate homoeologs include neofunctionialization, subfunctionalization, and maintenance of stochiometry among interacting components of pathways . Evidence for subfunctionalization has been reported in tandem duplicate paralogs of miR168 in the Brassicaceae. However, more recent hypotheses on the causes of retention following genome-wide duplication events are based on common characteristics of retained genes. For example, studies of the after effects of polyploidy in Arabidopsis , rice  and most recently maize  show bias for retention of transcription and other regulatory factors. Such classes may have indispensable functions such that their retention provides buffering against gene loss . As shown here and elsewhere, miRNA genes are largely associated with regulatory functions and thus mutations in miRNAs can be expected to have profound deleterious effects on growth and development . Thus the observed preferential retention of miRNA genes compared to protein coding genes is consistent with both the genetic buffering hypothesis and with previous observations of biased retention of regulatory genes.
The majority of maize miRNA genes (91%) lie within ancestral locations that existed prior to the speciation of maize and sorghum. Those no longer at syntenic positions could represent lineage-specific movements, duplications, or losses. Identification of ortholog genes has important practical application, allowing inferences to be drawn between functional studies in each species. Conservation of gene position has proven useful for determining orthologous relationships, particularly in cases where multiple highly related paralogs exist at different genome locations ,. Such is the case for miRNA genes, which tend to occur in large yet highly conserved families. Although synteny provides a strong indication of orthology, potentially complex relationships are suggested in the case of paralog clusters, in which closer phylogenetic analysis may be required to sort out duplication histories . Nevertheless, we found simple synteny relationships for 69 sorghum genes and 81 maize genes, allowing presumptive assignment of orthology among these genes.
Conservation of mature miRNA in diverse lines
To investigate the evolution of miRNA loci we sequenced 28 loci and flanking regions in panels of inbred and teosinte lines. This germplasm set was selected as it provides an excellent representation of the allelic diversity in maize and wild relatives. As expected, there was no polymorphism detected within the mature miRNA sequences. Their conservation within maize throughout its evolution is expected given the importance of miRNA genes in suppressing target gene expression during development and stress. The flanking regions displayed diversity levels similar to protein coding genes indicating that purifying selection is limited to mature miRNAs. None of the 28 loci tested exhibited the extreme reductions in diversity in inbreds relative to teosinte accessions that would be indicative of artificial selection during domestication or crop improvement. MicroRNA loci may control such fundamental processes in development that alterations of sequence or expression are not tolerated. Our results are similar to studies in Arabidopsis , and rice  where strong purifying selection has acted on the mature miRNA, whereas flanking regions generally reflect species wide diversity levels.
In summary, we have investigated genome-wide maize miRNA genes from several aspects: their pre- and pri- structure, their expression level, their targets, their conservation, and their evolution, providing evidence of the important function of miRNAs in regulating metabolic and developmental process and adaptation to stress. Identification and characterization of this important class of regulatory genes in maize may enable breeders to engineer crops with improved architecture and stress responses necessary to increase yields for food and fuel, while addressing environmental concerns in our changing climate and ever-increasing human population.
Materials and Methods
Maize (Zea mays) inbred line B73 was used in this study. The seedlings were collected from plants grown in soil in a green house at 22°C with a 16 hr light cycle and harvested 7 days after germination. Roots were collected from B73 seeds grown in sterile water without light at 30°C for three days. Immature ear (size 0.5–2cm) and immature tassel (size 0.5–2.5cm) were harvested from plants grown in the field. All the tissues were harvested and immediately frozen in liquid nitrogen and stored at −80°C. Tissues used in the RACE analysis included seedlings and immature inflorescence (tassel and ear).
A list of plant mature miRNAs were obtained from miRBase (version 13.0) and aligned using Vmatch, an alignment algorithm that employs suffix arrays (www.vmatch.de), against a suffix array index of the maize genome sequence built using mkvtree from the vmatch package. Up to 2 mismatches were allowed in the alignment. A 250-nt contextual sequence surrounding each aligned mature miRNA was extracted and secondary structure modeling of the DNA fragment was performed using Mfold . Segments that could potentially form the canonical stem-loop signature of a precursor miRNA were tagged as putative miRNAs. This putative list was further trimmed based on the following criteria: (1) overlapping loci were reduced to non-redundant representations, (2) loci that overlapped transposable and other repetitive elements were removed, (3) loci in which the matched mature miRNA was in the wrong orientation were removed, (4) each predicted secondary structure was curated manually and those with spurious stem-loop structures were removed , and (5) miRNA families that had no evidence of expression in the small RNA libraries or RACE were removed (for more details see Figure S2). The final refined list of predicted miRNAs consisted of 150 individual miRNAs from 26 miRNA families. Based on the hairpin structure of the pre-miRNA, the mature miRNA and corresponding miRNA* sequence were identified for each.
Small RNA libraries for sequence-by-synthesis (Illumina):
We used a modified method based on Llave et al to prepare small RNA libraries . Total RNA from different tissues was extracted with TRIzol solution (Invitrogen). 100µg of total RNA were separated on 15% Acrylamide/8M Urea gel (Sequagel, National Diagnostic) along with 19 and 24nt RNA samples as marker. Small RNAs were extracted from the gel slices corresponding to 19 and 24nt RNA using high salt. Small RNAs were ligated with Modban (Linker 1 from IDT). Ligated samples were separated again on 15% Acrylamide/8M Urea gel. The gel fragments corresponding to 37–42nt were excised. Small RNA was purified from the gel fragments, and 5′ sequencing adaptors were added using T4 RNA ligase. This RNA was amplified using Superscript III Reverse transcriptase (Invitrogen) and PCR amplification (Phusion HF polymerase, NEB). Amplified cDNA were separated on 3% MetaPhor Agarose (VWR) and the bands corresponding to correct size (108–115nt) were cut and purified with QIAGENE gel purification kit and sent for Illumina sequencing.
Small RNA sequencing and sequence processing
Small RNA libraries were sequenced on an Illumina Genome Analyzer using the 36-cycle Solexa Sequencing Kit (Illumina). The Illumina Gerald pipeline was used to process and extract the first 36 bases of the runs. Adaptor sequences were identified and trimmed from each read using a customized Perl script. Reads in which the adaptor could not be identified were discarded. Novoalign (version 2.03, http://www.novocraft.com/) was used to align the trimmed reads to the high-confidence set of 150 pre-miRNAs. For each library, we counted the number of trimmed reads within the 18–22nt range that were mapping to each pre-miRNA and normalized by the total number of 18–22nt trimmed reads in the library. Trimmed reads that were <18nt or > = 23nt were not considered in this analysis.
RACE analysis of miRNA precursor
Total RNA was extracted from maize seedlings and immature inflorescence using Spectrum Plant Total RNA kit (Sigma) and treated with RNase Free DNase (QIAGEN). cDNA templates were prepared by following the instruction of FirstChoice RLM-RACE Kit (Ambion). For each miRNA precursor, two sets of gene-specific primers, mostly immediate upstream and downstream of predicted hairpin structures (for 5′ RACE, for 3′ RACE, respectively), were designed using Primer 3 (Table S5). These primers were used for two rounds of PCR amplification. Nested PCR products were analyzed on agarose gel. Positive PCR products were cloned into pCR2.1-TOPO vector using TOPO TA cloning kit (Invitrogen). Each transcript sequence was confirmed by at least 8 unique clones. Sequences corresponding to the transcripts were mapped to the genome and listed in Table S3.
Computational prediction of miRNA protein-coding target transcripts
The miRNA target transcript prediction pipeline was developed using Vmatch. For the maize protein coding transcripts, the predicted cDNA of the longest consensus maize transcript from the filtered gene sets were used in this analysis and indexed by using mkvtree (Vmatch). The mature miRNA sequences from the refined miRNA set were reverse complemented and matched against the indexed maize transcript database, with the parameters relaxed to allow up to six mismatches. The matched miRNA target transcripts were further filtered by applying empirical rules defined by Schwab et al . Briefly, we scored each miRNA complementary site. Perfect matches were given a score of 0, and all other mismatches were scored 1. Only 1 mismatch score was allowed between positions 2 to 12 inclusive. However, no mismatches were allowed at position 10 and 11 and no more than 2 consecutive mismatches were allowed after position 12. A maximum of three mismatches (excluding mismatch at position 1) was allowed across the length of the mature miRNA.
Maize miRNA targets functional enrichment analysis
We subjected potential miRNA targets for functional enrichment analysis against GO molecular function and GO biological process terms database by using BiNGO  which is a Cytoscape  plugin that maps over-represented functional themes present in a given gene-set onto the GO hierarchy. For enrichment P-value calculation (at a significance level of 0.05 or better), a hypergeometric distribution statistical testing method was selected to ensure that target genes are not hitting their corresponding biological function/process classes purely by random chance. For multiple hypotheses testing, the Benjamini and Hochberg false discovery rate (FDR) correction  was applied to reduce false negatives at the cost of a few more false positives.
In order to fully understand the function of each target, we also extracted information based on phylogenetic trees of putative genes from maize, sorghum, Arabidopsis, and rice genomes. The trees were computed using the Ensembl Compara GeneTree method . The input sequences used for the GeneTree analysis were the longest translation at a given gene locus, filtered for transposons and other low-confidence genes, from the following genome annotation resources: the maize genome (www.maizesequence.org) release 4a from June 2009 , the Sorghum genome  JGI release Sbi 1.4 from March 2008, The Arabidopsis Information Resource  release 8 from April 2008, and the MSU/TIGR Rice Genome Annotation Resource  release 5 from January 2007.
Synteny analysis of maize and sorghum
Shared synteny among homologous maize and sorghum miRNA genes was evaluated in the context of orthologous protein-coding genes. Orthologous protein-coding genes were identified using the Ensembl Compara pipeline, which is based on a phylogenetic analysis . In all, the orthologue sets included 25,216 maize genes and 20,408 sorghum genes, which participated in 27,275 relationships. DAGchainer  was used to identify colinear chains amongst these orthologs, together with all pairwise combinations of within-family miRNA genes. Such chains were required to have at least five colinear genes with no more than ten intervening genes between neighbors. Additional syntenies among non-colinear genes were searched based on distance to colinear anchors that flank the orthologue of the gene in question in the other genome. The gene in question was considered syntenic if positioned within five genes of its nearest anchor. Paralogous clusters of miRNA genes were identified as family members separated by no more than two intervening genes.
Diversity of miRNA loci
Two diverse sets of maize materials were used for DNA sequence analysis: maize inbred lines, and partially inbred teosinte accessions (Table S11). The 28 maize inbred lines (B73, B97, CML103, CML228, CML247, CML277, CML322, CML333, CML52, CML69, Hp301, IL14H, Ki11, Ki3, Ky21, M162W, M37W, Mo17, Mo18W, MS71, NC350, NC358, Oh43, Oh7B, P39, Tx303, Tzi8 and W22) were chosen to maximize allelic diversity. The 16 partial inbred teosinte (Zea mays ssp. parviglumis) accessions are identical to those used previously ,. We conducted extended DNA sequencing of the known 28 maize miRNAs. Primer 3 was used to design the sets of PCR primers for analysis of the selected candidate miRNA genes. The genomic PCRs were performed using PCR master Mix I (Promega) or Takara LA Taq polymerase. Unincorporated primers and dNTPs were removed by exonuclease I (NEB) and shrimp alkaline phosphatase (USB). The PCR products were ethanol precipitated and sequenced with forward, reverse, and internal primers using Illumina machines. Base calling, quality assessment, and trimming of trace files were conducted with PHRED and sequence assembly was performed by PHRAP. The multiple sequences for each gene were aligned with ClustalW. We used DNAAlign Editor  for aligning the amplicon sequences across the germplasm set to accurately identify nucleotide variations, insertions, and deletions. The alignments are available to the community from the www.panzea.org .
The data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus  and are accessible through GEO Series accession number GSE17943 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE17943).
miRNA families removed based on TE annotation.
(0.04 MB DOC)
High-confidence maize miRNA genes.
(0.06 MB XLS)
Maize miRNA genes mapped to maize genome.
(0.04 MB XLS)
Maize miRNA genes with intron.
(0.04 MB DOC)
Primers used in 5′ and 3′ RACE.
(0.03 MB XLS)
Complete list of predicted miRNA targets of maize miRNAs. For each miRNA family, total number of targets, their function, Arabidopsis orthologs, and Interpro IDs are given.
(0.10 MB XLS)
Functional classification of targets with miRNA families based on Gene Ontology (GO). Classification of miRNA families of significantly overrepresented predicted miRNA targets based on GO full annotations are given. Adjusted P value, based on Benjamini and Hochberg correction applied on hypergeometric test is given. Cluster frequency represents total number of genes annotated to that GO term divided by total no of genes in the test set. Total frequency represents total number of genes annotated to that GO term divided by total no of genes in the reference set. Since one gene can be related to multiple GO terms, the sum of the percentages in each column is not a relevant parameter.
(0.05 MB XLS)
miRNA genes in maize and sorghum.
(0.02 MB XLS)
Syntenic sites harboring MIR genes in maize and sorghum. The 82 ancestral sites are numbered according to their order in sorghum. “Homoeologue status” indicates whether the site is retained on only one or both homoeologous positions in maize. If “Single” then information is provided under “Maize Homoeologue A.” If “Both” then information is additionally given under “Maize Homoeologue B.” “Size” indicates the number of clustered paralog genes at a site. Only one representative gene is listed for each site; for paralog clusters this is the first in the series.
(0.04 MB XLS)
Panzea accession of maize miRNA genes.
(0.04 MB XLS)
The authors thank Bonnie Hurwitz for helping review this manuscript, Michael Levy for his contribution to the data analysis, and Shiran Pasternak for his help in editing this manuscript. We are also grateful to Peter Van Buren for his technical support of our computational resources. We would also like to thank David Jackson at Cold Spring Harbor Lab for sharing the B73 seeds.
Conceived and designed the experiments: LZ JMC SK JCS DW. Performed the experiments: LZ JMC SK JCS ZL KG MDM. Analyzed the data: LZ JMC SK JCS AN CAM. Wrote the paper: LZ JMC SK JCS CAM MDM DW.
- 1. Reinhart BJ, Weinstein EG, Rhoades MW, Bartel B, Bartel DP (2002) MicroRNAs in plants. Genes Dev 16: 1616–1626.BJ ReinhartEG WeinsteinMW RhoadesB. BartelDP Bartel2002MicroRNAs in plants.Genes Dev1616161626
- 2. Llave C, Kasschau KD, Rector MA, Carrington JC (2002) Endogenous and silencing-associated small RNAs in plants. Plant Cell 14: 1605–1619.C. LlaveKD KasschauMA RectorJC Carrington2002Endogenous and silencing-associated small RNAs in plants.Plant Cell1416051619
- 3. Vazquez F, Vaucheret H, Rajagopalan R, Lepers C, Gasciolli V, et al. (2004) Endogenous trans-acting siRNAs regulate the accumulation of Arabidopsis mRNAs. Mol Cell 16: 69–79.F. VazquezH. VaucheretR. RajagopalanC. LepersV. Gasciolli2004Endogenous trans-acting siRNAs regulate the accumulation of Arabidopsis mRNAs.Mol Cell166979
- 4. Adai A, Johnson C, Mlotshwa S, Archer-Evans S, Manocha V, et al. (2005) Computational prediction of miRNAs in Arabidopsis thaliana. Genome Res 15: 78–91.A. AdaiC. JohnsonS. MlotshwaS. Archer-EvansV. Manocha2005Computational prediction of miRNAs in Arabidopsis thaliana.Genome Res157891
- 5. Jones-Rhoades MW, Bartel DP, Bartel B (2006) MicroRNAS and their regulatory roles in plants. Annu Rev Plant Biol 57: 19–53.MW Jones-RhoadesDP BartelB. Bartel2006MicroRNAS and their regulatory roles in plants.Annu Rev Plant Biol571953
- 6. Wang JW, Wang LJ, Mao YB, Cai WJ, Xue HW, et al. (2005) Control of root cap formation by MicroRNA-targeted auxin response factors in Arabidopsis. Plant Cell 17: 2204–2216.JW WangLJ WangYB MaoWJ CaiHW Xue2005Control of root cap formation by MicroRNA-targeted auxin response factors in Arabidopsis.Plant Cell1722042216
- 7. Boualem A, Laporte P, Jovanovic M, Laffont C, Plet J, et al. (2008) MicroRNA166 controls root and nodule development in Medicago truncatula. Plant J 54: 876–887.A. BoualemP. LaporteM. JovanovicC. LaffontJ. Plet2008MicroRNA166 controls root and nodule development in Medicago truncatula.Plant J54876887
- 8. Golz JF (2006) Signalling between the shoot apical meristem and developing lateral organs. Plant Mol Biol 60: 889–903.JF Golz2006Signalling between the shoot apical meristem and developing lateral organs.Plant Mol Biol60889903
- 9. Kidner CA, Martienssen RA (2004) Spatially restricted microRNA directs leaf polarity through ARGONAUTE1. Nature 428: 81–84.CA KidnerRA Martienssen2004Spatially restricted microRNA directs leaf polarity through ARGONAUTE1.Nature4288184
- 10. Xu L, Yang L, Huang H (2007) Transcriptional, post-transcriptional and post-translational regulations of gene expression during leaf polarity formation. Cell Res 17: 512–519.L. XuL. YangH. Huang2007Transcriptional, post-transcriptional and post-translational regulations of gene expression during leaf polarity formation.Cell Res17512519
- 11. Chitwood DH, Guo M, Nogueira FT, Timmermans MC (2007) Establishing leaf polarity: the role of small RNAs and positional signals in the shoot apex. Development 134: 813–823.DH ChitwoodM. GuoFT NogueiraMC Timmermans2007Establishing leaf polarity: the role of small RNAs and positional signals in the shoot apex.Development134813823
- 12. Chen X (2004) A microRNA as a translational repressor of APETALA2 in Arabidopsis flower development. Science 303: 2022–2025.X. Chen2004A microRNA as a translational repressor of APETALA2 in Arabidopsis flower development.Science30320222025
- 13. Mallory AC, Dugas DV, Bartel DP, Bartel B (2004) MicroRNA regulation of NAC-domain targets is required for proper formation and separation of adjacent embryonic, vegetative, and floral organs. Curr Biol 14: 1035–1046.AC MalloryDV DugasDP BartelB. Bartel2004MicroRNA regulation of NAC-domain targets is required for proper formation and separation of adjacent embryonic, vegetative, and floral organs.Curr Biol1410351046
- 14. Carraro N, Peaucelle A, Laufs P, Traas J (2006) Cell differentiation and organ initiation at the shoot apical meristem. Plant Mol Biol 60: 811–826.N. CarraroA. PeaucelleP. LaufsJ. Traas2006Cell differentiation and organ initiation at the shoot apical meristem.Plant Mol Biol60811826
- 15. Chuck G, Meeley R, Irish E, Sakai H, Hake S (2007) The maize tasselseed4 microRNA controls sex determination and meristem cell fate by targeting Tasselseed6/indeterminate spikelet1. Nat Genet 39: 1517–1521.G. ChuckR. MeeleyE. IrishH. SakaiS. Hake2007The maize tasselseed4 microRNA controls sex determination and meristem cell fate by targeting Tasselseed6/indeterminate spikelet1.Nat Genet3915171521
- 16. Liu Q, Zhang YC, Wang CY, Luo YC, Huang QJ, et al. (2009) Expression analysis of phytohormone-regulated microRNAs in rice, implying their regulation roles in plant hormone signaling. FEBS Lett 583: 723–728.Q. LiuYC ZhangCY WangYC LuoQJ Huang2009Expression analysis of phytohormone-regulated microRNAs in rice, implying their regulation roles in plant hormone signaling.FEBS Lett583723728
- 17. Fujii H, Chiou TJ, Lin SI, Aung K, Zhu JK (2005) A miRNA involved in phosphate-starvation response in Arabidopsis. Curr Biol 15: 2038–2043.H. FujiiTJ ChiouSI LinK. AungJK Zhu2005A miRNA involved in phosphate-starvation response in Arabidopsis.Curr Biol1520382043
- 18. Ding YF, Zhu C (2009) The role of microRNAs in copper and cadmium homeostasis. Biochem Biophys Res Commun 386: 6–10.YF DingC. Zhu2009The role of microRNAs in copper and cadmium homeostasis.Biochem Biophys Res Commun386610
- 19. Huang SQ, Peng J, Qiu CX, Yang ZM (2009) Heavy metal-regulated new microRNAs from rice. J Inorg Biochem 103: 282–287.SQ HuangJ. PengCX QiuZM Yang2009Heavy metal-regulated new microRNAs from rice.J Inorg Biochem103282287
- 20. Zhao B, Liang R, Ge L, Li W, Xiao H, et al. (2007) Identification of drought-induced microRNAs in rice. Biochem Biophys Res Commun 354: 585–590.B. ZhaoR. LiangL. GeW. LiH. Xiao2007Identification of drought-induced microRNAs in rice.Biochem Biophys Res Commun354585590
- 21. Lu S, Sun YH, Chiang VL (2008) Stress-responsive microRNAs in Populus. Plant J 55: 131–151.S. LuYH SunVL Chiang2008Stress-responsive microRNAs in Populus.Plant J55131151
- 22. Ding D, Zhang L, Wang H, Liu Z, Zhang Z, et al. (2009) Differential expression of miRNAs in response to salt stress in maize roots. Ann Bot (Lond) 103: 29–38.D. DingL. ZhangH. WangZ. LiuZ. Zhang2009Differential expression of miRNAs in response to salt stress in maize roots.Ann Bot (Lond)1032938
- 23. Zhou X, Wang G, Sutoh K, Zhu JK, Zhang W (2008) Identification of cold-inducible microRNAs in plants by transcriptome analysis. Biochim Biophys Acta 1779: 780–788.X. ZhouG. WangK. SutohJK ZhuW. Zhang2008Identification of cold-inducible microRNAs in plants by transcriptome analysis.Biochim Biophys Acta1779780788
- 24. Chuck G, Cigan AM, Saeteurn K, Hake S (2007) The heterochronic maize mutant Corngrass1 results from overexpression of a tandem microRNA. Nature Genetics 39: 544–549.G. ChuckAM CiganK. SaeteurnS. Hake2007The heterochronic maize mutant Corngrass1 results from overexpression of a tandem microRNA.Nature Genetics39544549
- 25. Lauter N, Kampani A, Carlson S, Goebel M, Moose SP (2005) microRNA172 down-regulates glossy15 to promote vegetative phase change in maize. Proc Natl Acad Sci U S A 102: 9412–9417.N. LauterA. KampaniS. CarlsonM. GoebelSP Moose2005microRNA172 down-regulates glossy15 to promote vegetative phase change in maize.Proc Natl Acad Sci U S A10294129417
- 26. Schnable PS, Ware D, Fulton RS, Stein JC, Wei F, et al. (2009) The B73 maize genome: complexity, diversity and dynamics. Science 326: PS SchnableD. WareRS FultonJC SteinF. Wei2009The B73 maize genome: complexity, diversity and dynamics.Science326
- 27. Voinnet O (2009) Origin, biogenesis, and activity of plant microRNAs. Cell 136: 669–687.O. Voinnet2009Origin, biogenesis, and activity of plant microRNAs.Cell136669687
- 28. Griffiths-Jones S, Saini HK, van Dongen S, Enright AJ (2008) miRBase: tools for microRNA genomics. Nucleic Acids Res 36: D154–158.S. Griffiths-JonesHK SainiS. van DongenAJ Enright2008miRBase: tools for microRNA genomics.Nucleic Acids Res36D154158
- 29. Paterson AH, Bowers JE, Bruggmann R, Dubchak I, Grimwood J, et al. (2009) The Sorghum bicolor genome and the diversification of grasses. Nature 457: 551–556.AH PatersonJE BowersR. BruggmannI. DubchakJ. Grimwood2009The Sorghum bicolor genome and the diversification of grasses.Nature457551556
- 30. Sunkar R, Zhou X, Zheng Y, Zhang W, Zhu JK (2008) Identification of novel and candidate miRNAs in rice by high throughput sequencing. BMC Plant Biol 8: 25.R. SunkarX. ZhouY. ZhengW. ZhangJK Zhu2008Identification of novel and candidate miRNAs in rice by high throughput sequencing.BMC Plant Biol825
- 31. Lu C, Jeong DH, Kulkarni K, Pillay M, Nobuta K, et al. (2008) Genome-wide analysis for discovery of rice microRNAs reveals natural antisense microRNAs (nat-miRNAs). Proc Natl Acad Sci U S A 105: 4951–4956.C. LuDH JeongK. KulkarniM. PillayK. Nobuta2008Genome-wide analysis for discovery of rice microRNAs reveals natural antisense microRNAs (nat-miRNAs).Proc Natl Acad Sci U S A10549514956
- 32. Dezulian T, Remmert M, Palatnik JF, Weigel D, Huson DH (2006) Identification of plant microRNA homologs. Bioinformatics 22: 359–360.T. DezulianM. RemmertJF PalatnikD. WeigelDH Huson2006Identification of plant microRNA homologs.Bioinformatics22359360
- 33. Maher C, Timmermans MC, Stein L, Ware D (2004) Identifying microRNAs in plant genomes. Proc IEEE CSB 718–723.C. MaherMC TimmermansL. SteinD. Ware2004Identifying microRNAs in plant genomes.Proc IEEE CSB718723
- 34. Zhang BH, Pan XP, Anderson TA (2006) Identification of 188 conserved maize microRNAs and their targets. Febs Letters 580: 3753–3762.BH ZhangXP PanTA Anderson2006Identification of 188 conserved maize microRNAs and their targets.Febs Letters58037533762
- 35. Wang S, Zhu QH, Guo X, Gui Y, Bao J, et al. (2007) Molecular evolution and selection of a gene encoding two tandem microRNAs in rice. FEBS Lett 581: 4789–4793.S. WangQH ZhuX. GuoY. GuiJ. Bao2007Molecular evolution and selection of a gene encoding two tandem microRNAs in rice.FEBS Lett58147894793
- 36. Axtell MJ, Snyder JA, Bartel DP (2007) Common functions for diverse small RNAs of land plants. Plant Cell 19: 1750–1769.MJ AxtellJA SnyderDP Bartel2007Common functions for diverse small RNAs of land plants.Plant Cell1917501769
- 37. Maher C, Stein L, Ware D (2006) Evolution of Arabidopsis microRNA families through duplication events. Genome Res 16: 510–519.C. MaherL. SteinD. Ware2006Evolution of Arabidopsis microRNA families through duplication events.Genome Res16510519
- 38. Guddeti S, Zhang DC, Li AL, Leseberg CH, Kang H, et al. (2005) Molecular evolution of the rice miR395 gene family. Cell Res 15: 631–638.S. GuddetiDC ZhangAL LiCH LesebergH. Kang2005Molecular evolution of the rice miR395 gene family.Cell Res15631638
- 39. Xie Z, Allen E, Fahlgren N, Calamar A, Givan SA, et al. (2005) Expression of Arabidopsis MIRNA genes. Plant Physiol 138: 2145–2154.Z. XieE. AllenN. FahlgrenA. CalamarSA Givan2005Expression of Arabidopsis MIRNA genes.Plant Physiol13821452154
- 40. Chiou TJ, Aung K, Lin SI, Wu CC, Chiang SF, et al. (2006) Regulation of phosphate homeostasis by MicroRNA in Arabidopsis. Plant Cell 18: 412–421.TJ ChiouK. AungSI LinCC WuSF Chiang2006Regulation of phosphate homeostasis by MicroRNA in Arabidopsis.Plant Cell18412421
- 41. Alexandrov NN, Brover VV, Freidin S, Troukhan ME, Tatarinova TV, et al. (2009) Insights into corn genes derived from large-scale cDNA sequencing. Plant Mol Biol 69: 179–194.NN AlexandrovVV BroverS. FreidinME TroukhanTV Tatarinova2009Insights into corn genes derived from large-scale cDNA sequencing.Plant Mol Biol69179194
- 42. Soderlund C, Descour A, Kudrna D, Bomhoff M, Boyd L, et al. (2009) Sequencing, Mapping, and Analysis of 27,455 Maize Full-Length cDNAs. PLoS Genet 5(11): e1000740.C. SoderlundA. DescourD. KudrnaM. BomhoffL. Boyd2009Sequencing, Mapping, and Analysis of 27,455 Maize Full-Length cDNAs.PLoS Genet5(11)e1000740
- 43. Alexandrov NN, Troukhan ME, Brover VV, Tatarinova T, Flavell RB, et al. (2006) Features of Arabidopsis genes and genome discovered using full-length cDNAs. Plant Mol Biol 60: 69–85.NN AlexandrovME TroukhanVV BroverT. TatarinovaRB Flavell2006Features of Arabidopsis genes and genome discovered using full-length cDNAs.Plant Mol Biol606985
- 44. Tanaka T, Koyanagi KO, Itoh T (2009) Highly diversified molecular evolution of downstream transcription start sites in rice and Arabidopsis. Plant Physiol 149: 1316–1324.T. TanakaKO KoyanagiT. Itoh2009Highly diversified molecular evolution of downstream transcription start sites in rice and Arabidopsis.Plant Physiol14913161324
- 45. Bentley DR (2006) Whole-genome re-sequencing. Curr Opin Genet Dev 16: 545–552.DR Bentley2006Whole-genome re-sequencing.Curr Opin Genet Dev16545552
- 46. Mulder NJ, Apweiler R, Attwood TK, Bairoch A, Bateman A, et al. (2007) New developments in the InterPro database. Nucleic Acids Res 35: D224–228.NJ MulderR. ApweilerTK AttwoodA. BairochA. Bateman2007New developments in the InterPro database.Nucleic Acids Res35D224228
- 47. Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, et al. (2000) Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet 25: 25–29.M. AshburnerCA BallJA BlakeD. BotsteinH. Butler2000Gene ontology: tool for the unification of biology. The Gene Ontology Consortium.Nat Genet252529
- 48. Maere S, Heymans K, Kuiper M (2005) BiNGO: a Cytoscape plugin to assess overrepresentation of gene ontology categories in biological networks. Bioinformatics 21: 3448–3449.S. MaereK. HeymansM. Kuiper2005BiNGO: a Cytoscape plugin to assess overrepresentation of gene ontology categories in biological networks.Bioinformatics2134483449
- 49. Shannon P, Markiel A, Ozier O, Baliga NS, Wang JT, et al. (2003) Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res 13: 2498–2504.P. ShannonA. MarkielO. OzierNS BaligaJT Wang2003Cytoscape: a software environment for integrated models of biomolecular interaction networks.Genome Res1324982504
- 50. Gazzani S, Li M, Maistri S, Scarponi E, Graziola M, et al. (2009) Evolution of MIR168 paralogs in Brassicaceae. BMC Evol Biol 9: 62.S. GazzaniM. LiS. MaistriE. ScarponiM. Graziola2009Evolution of MIR168 paralogs in Brassicaceae.BMC Evol Biol962
- 51. Li A, Mao L (2007) Evolution of plant microRNA gene families. Cell Res 17: 212–218.A. LiL. Mao2007Evolution of plant microRNA gene families.Cell Res17212218
- 52. Swigonova Z, Lai J, Ma J, Ramakrishna W, Llaca V, et al. (2004) Close split of sorghum and maize genome progenitors. Genome Res 14: 1916–1923.Z. SwigonovaJ. LaiJ. MaW. RamakrishnaV. Llaca2004Close split of sorghum and maize genome progenitors.Genome Res1419161923
- 53. Palmer LE, Rabinowicz PD, O'Shaughnessy AL, Balija VS, Nascimento LU, et al. (2003) Maize genome sequencing by methylation filtration. Science 302: 2115–2117.LE PalmerPD RabinowiczAL O'ShaughnessyVS BalijaLU Nascimento2003Maize genome sequencing by methylation filtration.Science30221152117
- 54. Whitelaw CA, Barbazuk WB, Pertea G, Chan AP, Cheung F, et al. (2003) Enrichment of gene-coding sequences in maize by genome filtration. Science 302: 2118–2120.CA WhitelawWB BarbazukG. PerteaAP ChanF. Cheung2003Enrichment of gene-coding sequences in maize by genome filtration.Science30221182120
- 55. Yu J, Holland JB, McMullen MD, Buckler ES (2008) Genetic design and statistical power of nested association mapping in maize. Genetics 178: 539–551.J. YuJB HollandMD McMullenES Buckler2008Genetic design and statistical power of nested association mapping in maize.Genetics178539551
- 56. Wright SI, Bi IV, Schroeder SG, Yamasaki M, Doebley JF, et al. (2005) The effects of artificial selection on the maize genome. Science 308: 1310–1314.SI WrightIV BiSG SchroederM. YamasakiJF Doebley2005The effects of artificial selection on the maize genome.Science30813101314
- 57. Yamasaki M, Tenaillon MI, Bi IV, Schroeder SG, Sanchez-Villeda H, et al. (2005) A large-scale screen for artificial selection in maize identifies candidate agronomic loci for domestication and crop improvement. Plant Cell 17: 2859–2872.M. YamasakiMI TenaillonIV BiSG SchroederH. Sanchez-Villeda2005A large-scale screen for artificial selection in maize identifies candidate agronomic loci for domestication and crop improvement.Plant Cell1728592872
- 58. Xue LJ, Zhang JJ, Xue HW (2009) Characterization and expression profiles of miRNAs in rice seeds. Nucleic Acids Res 37: 916–930.LJ XueJJ ZhangHW Xue2009Characterization and expression profiles of miRNAs in rice seeds.Nucleic Acids Res37916930
- 59. Brown JW, Marshall DF, Echeverria M (2008) Intronic noncoding RNAs and splicing. Trends Plant Sci 13: 335–342.JW BrownDF MarshallM. Echeverria2008Intronic noncoding RNAs and splicing.Trends Plant Sci13335342
- 60. Rajagopalan R, Vaucheret H, Trejo J, Bartel DP (2006) A diverse and evolutionarily fluid set of microRNAs in Arabidopsis thaliana. Genes Dev 20: 3407–3425.R. RajagopalanH. VaucheretJ. TrejoDP Bartel2006A diverse and evolutionarily fluid set of microRNAs in Arabidopsis thaliana.Genes Dev2034073425
- 61. Willmann MR, Poethig RS (2005) Time to grow up: the temporal role of smallRNAs in plants. Curr Opin Plant Biol 8: 548–552.MR WillmannRS Poethig2005Time to grow up: the temporal role of smallRNAs in plants.Curr Opin Plant Biol8548552
- 62. Chuck G, Candela H, Hake S (2009) Big impacts by small RNAs in plant development. Curr Opin Plant Biol 12: 81–86.G. ChuckH. CandelaS. Hake2009Big impacts by small RNAs in plant development.Curr Opin Plant Biol128186
- 63. Sunkar R, Jagadeeswaran G (2008) In silico identification of conserved microRNAs in large number of diverse plant species. BMC Plant Biol 8: 37.R. SunkarG. Jagadeeswaran2008In silico identification of conserved microRNAs in large number of diverse plant species.BMC Plant Biol837
- 64. van der Knaap E, Kim JH, Kende H (2000) A novel gibberellin-induced gene from rice and its potential regulatory role in stem growth. Plant Physiol 122: 695–704.E. van der KnaapJH KimH. Kende2000A novel gibberellin-induced gene from rice and its potential regulatory role in stem growth.Plant Physiol122695704
- 65. Kim JH, Choi D, Kende H (2003) The AtGRF family of putative transcription factors is involved in leaf and cotyledon growth in Arabidopsis. Plant J 36: 94–104.JH KimD. ChoiH. Kende2003The AtGRF family of putative transcription factors is involved in leaf and cotyledon growth in Arabidopsis.Plant J3694104
- 66. Baumberger N, Baulcombe DC (2005) Arabidopsis ARGONAUTE1 is an RNA Slicer that selectively recruits microRNAs and short interfering RNAs. Proc Natl Acad Sci U S A 102: 11928–11933.N. BaumbergerDC Baulcombe2005Arabidopsis ARGONAUTE1 is an RNA Slicer that selectively recruits microRNAs and short interfering RNAs.Proc Natl Acad Sci U S A1021192811933
- 67. Sunkar R, Girke T, Zhu JK (2005) Identification and characterization of endogenous small interfering RNAs from rice. Nucleic Acids Res 33: 4443–4454.R. SunkarT. GirkeJK Zhu2005Identification and characterization of endogenous small interfering RNAs from rice.Nucleic Acids Res3344434454
- 68. Vaucheret H, Vazquez F, Crete P, Bartel DP (2004) The action of ARGONAUTE1 in the miRNA pathway and its regulation by the miRNA pathway are crucial for plant development. Genes Dev 18: 1187–1197.H. VaucheretF. VazquezP. CreteDP Bartel2004The action of ARGONAUTE1 in the miRNA pathway and its regulation by the miRNA pathway are crucial for plant development.Genes Dev1811871197
- 69. Yao Y, Guo G, Ni Z, Sunkar R, Du J, et al. (2007) Cloning and characterization of microRNAs from wheat (Triticum aestivum L.). Genome Biol 8: R96.Y. YaoG. GuoZ. NiR. SunkarJ. Du2007Cloning and characterization of microRNAs from wheat (Triticum aestivum L.).Genome Biol8R96
- 70. Pina C, Pinto F, Feijo JA, Becker JD (2005) Gene family analysis of the Arabidopsis pollen transcriptome reveals biological implications for cell growth, division control, and gene expression regulation. Plant Physiol 138: 744–756.C. PinaF. PintoJA FeijoJD Becker2005Gene family analysis of the Arabidopsis pollen transcriptome reveals biological implications for cell growth, division control, and gene expression regulation.Plant Physiol138744756
- 71. Alves-Junior L, Niemeier S, Hauenschild A, Rehmsmeier M, Merkle T (2009) Comprehensive prediction of novel microRNA targets in Arabidopsis thaliana. Nucleic Acids Res 37: 4010–4021.L. Alves-JuniorS. NiemeierA. HauenschildM. RehmsmeierT. Merkle2009Comprehensive prediction of novel microRNA targets in Arabidopsis thaliana.Nucleic Acids Res3740104021
- 72. Bonnet E, Wuyts J, Rouze P, Van de Peer Y (2004) Detection of 91 potential conserved plant microRNAs in Arabidopsis thaliana and Oryza sativa identifies important target genes. Proc Natl Acad Sci U S A 101: 11511–11516.E. BonnetJ. WuytsP. RouzeY. Van de Peer2004Detection of 91 potential conserved plant microRNAs in Arabidopsis thaliana and Oryza sativa identifies important target genes.Proc Natl Acad Sci U S A1011151111516
- 73. Jones-Rhoades MW, Bartel DP (2004) Computational identification of plant microRNAs and their targets, including a stress-induced miRNA. Mol Cell 14: 787–799.MW Jones-RhoadesDP Bartel2004Computational identification of plant microRNAs and their targets, including a stress-induced miRNA.Mol Cell14787799
- 74. Luo YC, Zhou H, Li Y, Chen JY, Yang JH, et al. (2006) Rice embryogenic calli express a unique set of microRNAs, suggesting regulatory roles of microRNAs in plant post-embryogenic development. FEBS Lett 580: 5111–5116.YC LuoH. ZhouY. LiJY ChenJH Yang2006Rice embryogenic calli express a unique set of microRNAs, suggesting regulatory roles of microRNAs in plant post-embryogenic development.FEBS Lett58051115116
- 75. Wang JF, Zhou H, Chen YQ, Luo QJ, Qu LH (2004) Identification of 20 microRNAs from Oryza sativa. Nucleic Acids Res 32: 1688–1695.JF WangH. ZhouYQ ChenQJ LuoLH Qu2004Identification of 20 microRNAs from Oryza sativa.Nucleic Acids Res3216881695
- 76. Jin W, Li N, Zhang B, Wu F, Li W, et al. (2008) Identification and verification of microRNA in wheat (Triticum aestivum). J Plant Res 121: 351–355.W. JinN. LiB. ZhangF. WuW. Li2008Identification and verification of microRNA in wheat (Triticum aestivum).J Plant Res121351355
- 77. Subramanian S, Fu Y, Sunkar R, Barbazuk WB, Zhu JK, et al. (2008) Novel and nodulation-regulated microRNAs in soybean roots. BMC Genomics 9: 160.S. SubramanianY. FuR. SunkarWB BarbazukJK Zhu2008Novel and nodulation-regulated microRNAs in soybean roots.BMC Genomics9160
- 78. Xie FL, Huang SQ, Guo K, Xiang AL, Zhu YY, et al. (2007) Computational identification of novel microRNAs and targets in Brassica napus. FEBS Lett 581: 1464–1474.FL XieSQ HuangK. GuoAL XiangYY Zhu2007Computational identification of novel microRNAs and targets in Brassica napus.FEBS Lett58114641474
- 79. Carra A, Mica E, Gambino G, Pindo M, Moser C, et al. (2009) Cloning and characterization of small non-coding RNAs from grape. Plant J. A. CarraE. MicaG. GambinoM. PindoC. Moser2009Cloning and characterization of small non-coding RNAs from grape.Plant J
- 80. Schwarz S, Grande AV, Bujdoso N, Saedler H, Huijser P (2008) The microRNA regulated SBP-box genes SPL9 and SPL15 control shoot maturation in Arabidopsis. Plant Mol Biol 67: 183–195.S. SchwarzAV GrandeN. BujdosoH. SaedlerP. Huijser2008The microRNA regulated SBP-box genes SPL9 and SPL15 control shoot maturation in Arabidopsis.Plant Mol Biol67183195
- 81. Wang JW, Schwab R, Czech B, Mica E, Weigel D (2008) Dual effects of miR156-targeted SPL genes and CYP78A5/KLUH on plastochron length and organ size in Arabidopsis thaliana. Plant Cell 20: 1231–1243.JW WangR. SchwabB. CzechE. MicaD. Weigel2008Dual effects of miR156-targeted SPL genes and CYP78A5/KLUH on plastochron length and organ size in Arabidopsis thaliana.Plant Cell2012311243
- 82. Millar AA, Gubler F (2005) The Arabidopsis GAMYB-like genes, MYB33 and MYB65, are MicroRNA-regulated genes that redundantly facilitate anther development. Plant Cell 17: 705–721.AA MillarF. Gubler2005The Arabidopsis GAMYB-like genes, MYB33 and MYB65, are MicroRNA-regulated genes that redundantly facilitate anther development.Plant Cell17705721
- 83. Mica E, Gianfranceschi L, Pe ME (2006) Characterization of five microRNA families in maize. J Exp Bot 57: 2601–2612.E. MicaL. GianfranceschiME Pe2006Characterization of five microRNA families in maize.J Exp Bot5726012612
- 84. Wu MF, Tian Q, Reed JW (2006) Arabidopsis microRNA167 controls patterns of ARF6 and ARF8 expression, and regulates both female and male reproduction. Development 133: 4211–4218.MF WuQ. TianJW Reed2006Arabidopsis microRNA167 controls patterns of ARF6 and ARF8 expression, and regulates both female and male reproduction.Development13342114218
- 85. Aukerman MJ, Sakai H (2003) Regulation of flowering time and floral organ identity by a MicroRNA and its APETALA2-like target genes. Plant Cell 15: 2730–2741.MJ AukermanH. Sakai2003Regulation of flowering time and floral organ identity by a MicroRNA and its APETALA2-like target genes.Plant Cell1527302741
- 86. Juarez MT, Twigg RW, Timmermans MC (2004) Specification of adaxial cell fate during maize leaf development. Development 131: 4533–4544.MT JuarezRW TwiggMC Timmermans2004Specification of adaxial cell fate during maize leaf development.Development13145334544
- 87. Zhang BH, Pan XP, Wang QL, Cobb GP, Anderson TA (2006) Computational identification of microRNAs and their targets. Computational Biology and Chemistry 30: 395–407.BH ZhangXP PanQL WangGP CobbTA Anderson2006Computational identification of microRNAs and their targets.Computational Biology and Chemistry30395407
- 88. Sunkar R, Chinnusamy V, Zhu J, Zhu JK (2007) Small RNAs as big players in plant abiotic stress responses and nutrient deprivation. Trends Plant Sci 12: 301–309.R. SunkarV. ChinnusamyJ. ZhuJK Zhu2007Small RNAs as big players in plant abiotic stress responses and nutrient deprivation.Trends Plant Sci12301309
- 89. Kidner CA, Martienssen RA (2005) The role of ARGONAUTE1 (AGO1) in meristem formation and identity. Dev Biol 280: 504–517.CA KidnerRA Martienssen2005The role of ARGONAUTE1 (AGO1) in meristem formation and identity.Dev Biol280504517
- 90. Xie Z, Kasschau KD, Carrington JC (2003) Negative feedback regulation of Dicer-Like1 in Arabidopsis by microRNA-guided mRNA degradation. Curr Biol 13: 784–789.Z. XieKD KasschauJC Carrington2003Negative feedback regulation of Dicer-Like1 in Arabidopsis by microRNA-guided mRNA degradation.Curr Biol13784789
- 91. Blanc G, Wolfe KH (2004) Widespread paleopolyploidy in model plant species inferred from age distributions of duplicate genes. Plant Cell 16: 1667–1678.G. BlancKH Wolfe2004Widespread paleopolyploidy in model plant species inferred from age distributions of duplicate genes.Plant Cell1616671678
- 92. Bowers JE, Chapman BA, Rong J, Paterson AH (2003) Unravelling angiosperm genome evolution by phylogenetic analysis of chromosomal duplication events. Nature 422: 433–438.JE BowersBA ChapmanJ. RongAH Paterson2003Unravelling angiosperm genome evolution by phylogenetic analysis of chromosomal duplication events.Nature422433438
- 93. Gaut BS, Doebley JF (1997) DNA sequence evidence for the segmental allotetraploid origin of maize. Proc Natl Acad Sci U S A 94: 6809–6814.BS GautJF Doebley1997DNA sequence evidence for the segmental allotetraploid origin of maize.Proc Natl Acad Sci U S A9468096814
- 94. Lai J, Ma J, Swigonova Z, Ramakrishna W, Linton E, et al. (2004) Gene loss and movement in the maize genome. Genome Res 14: 1924–1931.J. LaiJ. MaZ. SwigonovaW. RamakrishnaE. Linton2004Gene loss and movement in the maize genome.Genome Res1419241931
- 95. Bruggmann R, Bharti AK, Gundlach H, Lai J, Young S, et al. (2006) Uneven chromosome contraction and expansion in the maize genome. Genome Res 16: 1241–1251.R. BruggmannAK BhartiH. GundlachJ. LaiS. Young2006Uneven chromosome contraction and expansion in the maize genome.Genome Res1612411251
- 96. Langham RJ, Walsh J, Dunn M, Ko C, Goff SA, et al. (2004) Genomic duplication, fractionation and the origin of regulatory novelty. Genetics 166: 935–945.RJ LanghamJ. WalshM. DunnC. KoSA Goff2004Genomic duplication, fractionation and the origin of regulatory novelty.Genetics166935945
- 97. Swigonova Z, Bennetzen JL, Messing J (2005) Structure and evolution of the r/b chromosomal regions in rice, maize and sorghum. Genetics 169: 891–906.Z. SwigonovaJL BennetzenJ. Messing2005Structure and evolution of the r/b chromosomal regions in rice, maize and sorghum.Genetics169891906
- 98. Ohno S (1970) Evolution by gene duplication. Springer-Verlag. S. Ohno1970Evolution by gene duplicationSpringer-Verlag
- 99. Doyle JJ, Flagel LE, Paterson AH, Rapp RA, Soltis DE, et al. (2008) Evolutionary genetics of genome merger and doubling in plants. Annu Rev Genet 42: 443–461.JJ DoyleLE FlagelAH PatersonRA RappDE Soltis2008Evolutionary genetics of genome merger and doubling in plants.Annu Rev Genet42443461
- 100. Lynch M, Conery JS (2003) The origins of genome complexity. Science 302: 1401–1404.M. LynchJS Conery2003The origins of genome complexity.Science30214011404
- 101. Semon M, Wolfe KH (2007) Consequences of genome duplication. Curr Opin Genet Dev 17: 505–512.M. SemonKH Wolfe2007Consequences of genome duplication.Curr Opin Genet Dev17505512
- 102. Seoighe C, Gehring C (2004) Genome duplication led to highly selective expansion of the Arabidopsis thaliana proteome. Trends Genet 20: 461–464.C. SeoigheC. Gehring2004Genome duplication led to highly selective expansion of the Arabidopsis thaliana proteome.Trends Genet20461464
- 103. Tian CG, Xiong YQ, Liu TY, Sun SH, Chen LB, et al. (2005) Evidence for an ancient whole-genome duplication event in rice and other cereals. Yi Chuan Xue Bao 32: 519–527.CG TianYQ XiongTY LiuSH SunLB Chen2005Evidence for an ancient whole-genome duplication event in rice and other cereals.Yi Chuan Xue Bao32519527
- 104. Chapman BA, Bowers JE, Feltus FA, Paterson AH (2006) Buffering of crucial functions by paleologous duplicated genes may contribute cyclicality to angiosperm genome duplication. Proc Natl Acad Sci U S A 103: 2730–2735.BA ChapmanJE BowersFA FeltusAH Paterson2006Buffering of crucial functions by paleologous duplicated genes may contribute cyclicality to angiosperm genome duplication.Proc Natl Acad Sci U S A10327302735
- 105. Wright MW, Bruford EA (2006) Human and orthologous gene nomenclature. Gene 369: 1–6.MW WrightEA Bruford2006Human and orthologous gene nomenclature.Gene36916
- 106. Barbazuk WB, Korf I, Kadavi C, Heyen J, Tate S, et al. (2000) The syntenic relationship of the zebrafish and human genomes. Genome Res 10: 1351–1358.WB BarbazukI. KorfC. KadaviJ. HeyenS. Tate2000The syntenic relationship of the zebrafish and human genomes.Genome Res1013511358
- 107. de Meaux J, Hu JY, Tartler U, Goebel U (2008) Structurally different alleles of the ath-MIR824 microRNA precursor are maintained at high frequency in Arabidopsis thaliana. Proc Natl Acad Sci U S A 105: 8994–8999.J. de MeauxJY HuU. TartlerU. Goebel2008Structurally different alleles of the ath-MIR824 microRNA precursor are maintained at high frequency in Arabidopsis thaliana.Proc Natl Acad Sci U S A10589948999
- 108. Ehrenreich IM, Purugganan MD (2008) Sequence variation of MicroRNAs and their binding sites in Arabidopsis. Plant Physiol 146: 1974–1982.IM EhrenreichMD Purugganan2008Sequence variation of MicroRNAs and their binding sites in Arabidopsis.Plant Physiol14619741982
- 109. Zuker M (2003) Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31: 3406–3415.M. Zuker2003Mfold web server for nucleic acid folding and hybridization prediction.Nucleic Acids Res3134063415
- 110. Rozen S, Skaletsky H (2000) Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol 132: 365–386.S. RozenH. Skaletsky2000Primer3 on the WWW for general users and for biologist programmers.Methods Mol Biol132365386
- 111. Schwab R, Palatnik JF, Riester M, Schommer C, Schmid M, et al. (2005) Specific effects of microRNAs on the plant transcriptome. Dev Cell 8: 517–527.R. SchwabJF PalatnikM. RiesterC. SchommerM. Schmid2005Specific effects of microRNAs on the plant transcriptome.Dev Cell8517527
- 112. Benjamini Y, Hochberg Y (1995) Controlling the false discovery rate: A practical and powerful approach to multiple testing. J Roy Statist Soc Ser 289–300.Y. BenjaminiY. Hochberg1995Controlling the false discovery rate: A practical and powerful approach to multiple testing.J Roy Statist Soc Ser289300
- 113. Vilella AJ, Severin J, Ureta-Vidal A, Heng L, Durbin R, et al. (2009) EnsemblCompara GeneTrees: Complete, duplication-aware phylogenetic trees in vertebrates. Genome Res 19: 327–335.AJ VilellaJ. SeverinA. Ureta-VidalL. HengR. Durbin2009EnsemblCompara GeneTrees: Complete, duplication-aware phylogenetic trees in vertebrates.Genome Res19327335
- 114. Swarbreck D, Wilks C, Lamesch P, Berardini TZ, Garcia-Hernandez M, et al. (2008) The Arabidopsis Information Resource (TAIR): gene structure and function annotation. Nucleic Acids Res 36: D1009–1014.D. SwarbreckC. WilksP. LameschTZ BerardiniM. Garcia-Hernandez2008The Arabidopsis Information Resource (TAIR): gene structure and function annotation.Nucleic Acids Res36D10091014
- 115. Ouyang S, Zhu W, Hamilton J, Lin H, Campbell M, et al. (2007) The TIGR Rice Genome Annotation Resource: improvements and new features. Nucleic Acids Res 35: D883–887.S. OuyangW. ZhuJ. HamiltonH. LinM. Campbell2007The TIGR Rice Genome Annotation Resource: improvements and new features.Nucleic Acids Res35D883887
- 116. Hubbard TJ, Aken BL, Ayling S, Ballester B, Beal K, et al. (2009) Ensembl 2009. Nucleic Acids Res 37: D690–697.TJ HubbardBL AkenS. AylingB. BallesterK. Beal2009Ensembl 2009.Nucleic Acids Res37D690697
- 117. Haas BJ, Delcher AL, Wortman JR, Salzberg SL (2004) DAGchainer: a tool for mining segmental genome duplications and synteny. Bioinformatics 20: 3643–3646.BJ HaasAL DelcherJR WortmanSL Salzberg2004DAGchainer: a tool for mining segmental genome duplications and synteny.Bioinformatics2036433646
- 118. Sanchez-Villeda H, Schroeder S, Flint-Garcia S, Guill KE, Yamasaki M, et al. (2008) DNAAlignEditor: DNA alignment editor tool. BMC Bioinformatics 9: 154.H. Sanchez-VilledaS. SchroederS. Flint-GarciaKE GuillM. Yamasaki2008DNAAlignEditor: DNA alignment editor tool.BMC Bioinformatics9154
- 119. Jaiswal P, Ni J, Yap I, Ware D, Spooner W, et al. (2006) Gramene: a bird's eye view of cereal genomes. Nucleic Acids Res 34: D717–723.P. JaiswalJ. NiI. YapD. WareW. Spooner2006Gramene: a bird's eye view of cereal genomes.Nucleic Acids Res34D717723
- 120. Edgar R, Domrachev M, Lash AE (2002) Gene Expression Omnibus: NCBI gene expression and hybridization array data repository. Nucleic Acids Res 30: 207–210.R. EdgarM. DomrachevAE Lash2002Gene Expression Omnibus: NCBI gene expression and hybridization array data repository.Nucleic Acids Res30207210
- 121. Crooks GE, Hon G, Chandonia JM, Brenner SE (2004) WebLogo: a sequence logo generator. Genome Res 14: 1188–1190.GE CrooksG. HonJM ChandoniaSE Brenner2004WebLogo: a sequence logo generator.Genome Res1411881190
- 122. Schneider TD, Stephens RM (1990) Sequence logos: a new way to display consensus sequences. Nucleic Acids Res 18: 6097–6100.TD SchneiderRM Stephens1990Sequence logos: a new way to display consensus sequences.Nucleic Acids Res1860976100