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. 2012;7(3):e33040.
doi: 10.1371/journal.pone.0033040. Epub 2012 Mar 30.

A combined approach of high-throughput sequencing and degradome analysis reveals tissue specific expression of microRNAs and their targets in cucumber

Weihua Mao  1 , Zeyun Li, Xiaojian Xia, Yadan Li, Jingquan Yu
Affiliations

A combined approach of high-throughput sequencing and degradome analysis reveals tissue specific expression of microRNAs and their targets in cucumber

Weihua Mao et al. PLoS One. 2012.

Abstract

MicroRNAs (miRNAs) are endogenous small RNAs playing an important regulatory function in plant development and stress responses. Among them, some are evolutionally conserved in plant and others are only expressed in certain species, tissue or developmental stages. Cucumber is among the most important greenhouse species in the world, but only a limited number of miRNAs from cucumber have been identified and the experimental validation of the related miRNA targets is still lacking. In this study, two independent small RNA libraries from cucumber leaves and roots were constructed, respectively, and sequenced with the high-throughput Illumina Solexa system. Based on sequence similarity and hairpin structure prediction, a total of 29 known miRNA families and 2 novel miRNA families containing a total of 64 miRNA were identified. QRT-PCR analysis revealed that some of the cucumber miRNAs were preferentially expressed in certain tissues. With the recently developed 'high throughput degradome sequencing' approach, 21 target mRNAs of known miRNAs were identified for the first time in cucumber. These targets were associated with development, reactive oxygen species scavenging, signaling transduction and transcriptional regulation. Our study provides an overview of miRNA expression profile and interaction between miRNA and target, which will help further understanding of the important roles of miRNAs in cucumber plants.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. The size distribution of the small RNAs in leaves and roots libraries of cucumber.
Figure 2
Figure 2. Predicted secondary structures of novel cucumber miRNAs.
The mature miRNA and miRNA* sequences are written with red and blue capital letters, respectively.
Figure 3
Figure 3. Expression analysis of miRNAs in cucumber leaves and roots by qRT-PCR.
The amount of expression was normalized by the level of U6 in qRT-PCR. All reactions of qRT-PCR were repeated three times for each sample. Left indicates the miRNA relative expression generated from the high-throughput sequencing; Right indicates the miRNA relative expression tested by qRT-PCR.
Figure 4
Figure 4. Target plots (t-plots) of miRNA targets in different categories confirmed by degradome sequencing.
(A) T-plot (top) and miRNA: mRNA alignments (bottom) for two category I targets, Csa020279 and Csa009014 transcripts. The arrow indicates signatures consistent with miRNA-directed cleavage. The solid lines and dot in miRNA: mRNA alignments indicate matched RNA base pairs and GU mismatch, respectively, and the red letter indicates the cleavage site. (B) As in (A) for Csa18310 and Csa008131, a category II target for csa-miR172 and csa-miR858. (C) As in (A) for Csa014411, a category III target for csa-miR169.
See this image and copyright information in PMC

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