Summary of sRNA sequencing data.

<p>Summary of sRNA sequencing data.</p

The conservation of miRNA families in Arthropoda.

<p>The Arthropoda phylum was divided into eight orders with different colors and 12 families; and one representative species was shown in each family. The phylogenetic topology tree was adapted from Wheat et al. (2013)<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103041#pone.0103041-Wheat1" target="_blank">[54]</a>.</p

Identification and Characterization of MicroRNAs in Small Brown Planthopper (<i>Laodephax striatellus</i>) by Next-Generation Sequencing

<div><p>MicroRNAs (miRNAs) are endogenous non-coding small RNAs that regulate gene expression at the post-transcriptional level and are thought to play critical roles in many metabolic activities in eukaryotes. The small brown planthopper (<i>Laodephax striatellus</i> Fallén), one of the most destructive agricultural pests, causes great damage to crops including rice, wheat, and maize. However, information about the genome of <i>L. striatellus</i> is limited. In this study, a small RNA library was constructed from a mixed <i>L. striatellus</i> population and sequenced by Solexa sequencing technology. A total of 501 mature miRNAs were identified, including 227 conserved and 274 novel miRNAs belonging to 125 and 250 families, respectively. Sixty-nine conserved miRNAs that are included in 38 families are predicted to have an RNA secondary structure typically found in miRNAs. Many miRNAs were validated by stem-loop RT-PCR. Comparison with the miRNAs in 84 animal species from miRBase showed that the conserved miRNA families we identified are highly conserved in the Arthropoda phylum. Furthermore, miRanda predicted 2701 target genes for 378 miRNAs, which could be categorized into 52 functional groups annotated by gene ontology. The function of miRNA target genes was found to be very similar between conserved and novel miRNAs. This study of miRNAs in <i>L. striatellus</i> will provide new information and enhance the understanding of the role of miRNAs in the regulation of <i>L. striatellus</i> metabolism and development.</p></div

Expression validation of miRNAs by stem-loop RT-PCR.

<p>(A) 21 miRNAs, including 6 conserved and 15 novel miRNAs, were validated in mixed samples of <i>L. striatellus</i>; (B) validation of two conserved and five novel miRNAs in 1, second instar larvae; 2, fourth instar larvae; 3, female adults; 4, male adults and 5, mixed samples. β-actin and 5.8S rRNA were used as internal controls. <i>M</i>, marker.</p

The conservation of miRNA families in 84 animal species.

<p>The miRNAs of 84 animal species belonging to nine phyla were extracted from miRBase and published NGS data, and the conservation was analyzed for 38 miRNA families with RNA secondary structure in <i>L. striatellus</i>. Colored boxes indicate the presence of the conserved miRNA family; the same color indicates similar species.</p

Secondary structures of six representative miRNA precursors in <i>L. striatellus</i>.

<p>A green bar indicates the mature miRNA located in the 3' end, whereas a pink bar represents the mature miRNA located in the 5' end. Red circles indicate mismatches, and black circles show complementary base pairing.</p

Verification of miRNA-mediated mRNA cleavage by RLM-5′ RACE.

<p>(A) Gel electrophoresis of 5′ RACE PCR products of target genes for miRNAs. The red triangles indicate the fragments of 1, Cluster2118-Consensus1 and 2, Cluster1528-Consensus1 cleaved by lst-miR-n59-5p and lst-miR-981-3p, respectively. (B) The upper sequences show the target mRNA, whereas the bottom sequences indicate its corresponding miRNA. Watson-Crick pairings are indicated by short black lines; the G:U wobble pair is indicated by a colon. Arrows indicate the cleavage sites with the frequency of cleavage.</p

Length distribution of sRNAs in <i>L. striatellus</i>.

<p>(A) The total number of clean and unique reads of sRNAs 15 to 30 nt in length; (B) the clean reads that excluded miR-34.</p

The 19 novel miRNA families that have at least two mature miRNAs in <i>L. striatellus</i>.

a<p>Ten miRNAs (lst-miR-n2, lst-miR-n3, lst-miR-n13, lst-miR-n17, lst-miR-n28, lst-miR-n29, lst-miR-n37, lst-miR-n84, lst-miR-n104, lst-miR-n136) have identical mature sequences, but were derived from different precursors.</p

GO classification of the <i>L. striatellus</i> target genes.

<p>A total of 2103 putative target genes including 284 for conserved miRNAs and 1342 for novel miRNAs were assigned to the first-level of GO annotations of Biological Process, Cellular Component, and Molecular Function; and the potential target genes of conserved and novel miRNAs were classified into 50 and 52 functional groups at the second level of GO assignment, respectively.</p