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. 2012 Nov 19;12:218.
doi: 10.1186/1471-2229-12-218.

Identification and Profiling of Novel microRNAs in the Brassica Rapa Genome Based on Small RNA Deep Sequencing

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Free PMC article

Identification and Profiling of Novel microRNAs in the Brassica Rapa Genome Based on Small RNA Deep Sequencing

Bumjin Kim et al. BMC Plant Biol. .
Free PMC article

Abstract

Background: MicroRNAs (miRNAs) are one of the functional non-coding small RNAs involved in the epigenetic control of the plant genome. Although plants contain both evolutionary conserved miRNAs and species-specific miRNAs within their genomes, computational methods often only identify evolutionary conserved miRNAs. The recent sequencing of the Brassica rapa genome enables us to identify miRNAs and their putative target genes. In this study, we sought to provide a more comprehensive prediction of B. rapa miRNAs based on high throughput small RNA deep sequencing.

Results: We sequenced small RNAs from five types of tissue: seedlings, roots, petioles, leaves, and flowers. By analyzing 2.75 million unique reads that mapped to the B. rapa genome, we identified 216 novel and 196 conserved miRNAs that were predicted to target approximately 20% of the genome's protein coding genes. Quantitative analysis of miRNAs from the five types of tissue revealed that novel miRNAs were expressed in diverse tissues but their expression levels were lower than those of the conserved miRNAs. Comparative analysis of the miRNAs between the B. rapa and Arabidopsis thaliana genomes demonstrated that redundant copies of conserved miRNAs in the B. rapa genome may have been deleted after whole genome triplication. Novel miRNA members seemed to have spontaneously arisen from the B. rapa and A. thaliana genomes, suggesting the species-specific expansion of miRNAs. We have made this data publicly available in a miRNA database of B. rapa called BraMRs. The database allows the user to retrieve miRNA sequences, their expression profiles, and a description of their target genes from the five tissue types investigated here.

Conclusions: This is the first report to identify novel miRNAs from Brassica crops using genome-wide high throughput techniques. The combination of computational methods and small RNA deep sequencing provides robust predictions of miRNAs in the genome. The finding of numerous novel miRNAs, many with few target genes and low expression levels, suggests the rapid evolution of miRNA genes. The development of a miRNA database, BraMRs, enables us to integrate miRNA identification, target prediction, and functional annotation of target genes. BraMRs will represent a valuable public resource with which to study the epigenetic control of B. rapa and other closely related Brassica species. The database is available at the following link: http://bramrs.rna.kr [1].

Figures

Figure 1
Figure 1
Workflow to identify B. rapa miRNA genes. Candidate miRNA genes were predicted based on a combination of small RNA deep sequencing (blue arrows) and similarity searches using known A. thaliana miRNA sequences (green arrows). Sequence reads generated from small RNA sequencing were aligned to the draft genome sequences of B. rapa followed by secondary structure modeling. The resulting reads were further analyzed for structural and compositional features, genomic position, and expression level validation. Similarity searches using known A. thaliana miRNAs followed by secondary structure modeling and structural and compositional feature analysis identified matches of conserved miRNA candidates in the B. rapa genome. Integration and clustering of both data sets resulted in the final miRNA candidate genes (brown arrows).
Figure 2
Figure 2
Functional classification of miRNA target genes in B. rapa and A. thaliana based on gene ontology mapping using GO molecular function and GO biological process term databases, and pathway mapping using the KEGG pathway database.
Figure 3
Figure 3
Expression profiles of miRNA genes in B. rapa . (A) Venn diagram summarizing conserved and novel miRNAs identified in B. rapa. (B) Boxplots of expression levels for 37 conserved miRNAs and 216 novel miRNAs in the five tissue types. Expression levels are presented as the log10 ratio of normalized RPM. (C) Venn diagram showing the number of miRNAs expressed in specific tissues. The numbers in parentheses refer to the conserved miRNAs.
Figure 4
Figure 4
Circos diagram of miRNA gene pairs between B. rapa and A. thaliana. Conserved miRNAs genes in B. rapa are plotted against their syntenic counterparts in A. thaliana. The individual chromosomes of B. rapa (Br) and A. thaliana (At) have 24 ancestral karyotype genome building blocks demonstrating the shared ancestral origin of their genomes. The black and gray dots represent novel and conserved miRNA genes, respectively, of each genome within a 500 kb interval. The syntenic counterparts of conserved miRNAs between the genomes are interconnected by colored lines.
Figure 5
Figure 5
Screenshots of sample output pictures from the BraMRs database. (A) Snapshot of the “miRNA Prediction” unit providing search options for different tissues, chromosomes, and target genes. (B) An example of search results from the “miRNA Prediction” function presenting a list of predicted miRNAs with relevant sequence information and links to the target gene and expression characteristics. (C) Snapshot of the “miRNA Target Prediction” unit for searching for the putative target genes of the miRNA. (D) An example of a search result from the “miRNA Target Prediction” function presenting a list of predicted miRNA target genes with information on the binding site, alignment score, number/type of mismatches, and links to putative function and EST matches.

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References

    1. The BraMRs database. http://bramrs.rna.kr.
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