Identification of novel and candidate miRNAs in rice by high throughput sequencing

Abstract

Background: Small RNA-guided gene silencing at the transcriptional and post-transcriptional levels has emerged as an important mode of gene regulation in plants and animals. Thus far, conventional sequencing of small RNA libraries from rice led to the identification of most of the conserved miRNAs. Deep sequencing of small RNA libraries is an effective approach to uncover rare and lineage- and/or species-specific microRNAs (miRNAs) in any organism.

Results: In order to identify new miRNAs and possibly abiotic-stress regulated small RNAs in rice, three small RNA libraries were constructed from control rice seedlings and seedlings exposed to drought or salt stress, and then subjected to pyrosequencing. A total of 58,781, 43,003 and 80,990 unique genome-matching small RNAs were obtained from the control, drought and salt stress libraries, respectively. Sequence analysis confirmed the expression of most of the conserved miRNAs in rice. Importantly, 23 new miRNAs mostly each derived from a unique locus in rice genome were identified. Six of the new miRNAs are conserved in other monocots. Additionally, we identified 40 candidate miRNAs. Allowing not more than 3 mis-matches between a miRNA and its target mRNA, we predicted 20 targets for 9 of the new miRNAs.

Conclusion: Deep sequencing proved to be an effective strategy that allowed the discovery of 23 low-abundance new miRNAs and 40 candidate miRNAs in rice.

Figure 1

miRNA sequence alignments of the new miRNAs conserved in monocots. Figure 1a). Osa-miR1436 sequence conserved between rice and Aegilops. Figure b-f). miRNA sequence alignment of the newly identified rice miRNAs with the Osa-miR444. The Newly found miRNAs are conserved in related monocots and are included in the alignment. Osa-miR444 is highlighted with the yellow background. Red colored nucleotides in the newly identified miRNA in rice and other monocots indicate a nucleotide that is different from Osa-miR444.

Figure 2

Some of the newly identified miRNAs are conserved in monocots. Predicted fold-back structure using the miRNA precursor sequences from rice and other monocots. Used EST sequences for prediction of fold-back structures are indicated in parentheses. Cloned miRNA sequence in the fold-back structures is shown in red letters.

Figure 3

Expression patterns of the new miRNAs in rice seedlings. (a-c), Small RNA blots of low molecular weight RNA isolated from rice seedlings which were untreated (control) or treated with salt or drought stress. The blots were probed with ³²P-end-labelled oligonucleotides; (d-h) Blots of low molecular weight RNA isolated from untreated rice seedlings in duplicates. The blots were stripped and re-probed with U6 as loading controls.

Figure 4

Expression patterns of the newly identified putative miRNAs in rice seedlings. (a-b), Small RNA blots of low molecular weight RNA isolated from rice seedlings which were untreated (control) or treated with salt or drought stress. The blots were probed with ³²P-end-labelled oligonucleotides; (c-g) Blots of low molecular weight RNA isolated from untreated rice seedlings in duplicates. The blots were stripped and re-probed with U6 as loading controls.