Deep Sequencing and Microarray Hybridization Identify Conserved and Species-Specific MicroRNAs during Somatic Embryogenesis in Hybrid Yellow Poplar

Tingting Li,Jinhui Chen,Pengkai Wang,Ye Lu,Yanjuan Zhang,Liwei Yang,Shuai Qiu,Jisen Shi,Baohong Zhang

doi:10.1371/journal.pone.0043451

Abstract

BackgroundTo date, several studies have indicated a major role for microRNAs (miRNAs) in regulating plant development, but miRNA-mediated regulation of the developing somatic embryo is poorly understood, especially during early stages of somatic embryogenesis in hardwood plants. In this study, Solexa sequencing and miRNA microfluidic chips were used to discover conserved and species-specific miRNAs during somatic embryogenesis of hybrid yellow poplar (Liriodendron tulipifera×L. chinense).Methodology/Principal FindingsA total of 17,214,153 reads representing 7,421,623 distinct sequences were obtained from a short RNA library generated from small RNAs extracted from all stages of somatic embryos. Through a combination of deep sequencing and bioinformatic analyses, we discovered 83 sequences with perfect matches to known miRNAs from 33 conserved miRNA families and 273 species-specific candidate miRNAs. MicroRNA microarray results demonstrated that many conserved and species-specific miRNAs were expressed in hybrid yellow poplar embryos. In addition, the microarray also detected another 149 potential miRNAs, belonging to 29 conserved families, which were not discovered by deep sequencing analysis. The biological processes and molecular functions of the targets of these miRNAs were predicted by carrying out BLAST search against Arabidopsis thaliana GenBank sequences and then analyzing the results with Gene Ontology.ConclusionsSolexa sequencing and microarray hybridization were used to discover 232 candidate conserved miRNAs from 61 miRNA families and 273 candidate species-specific miRNAs in hybrid yellow poplar. In these predicted miRNAs, 64 conserved miRNAs and 177 species-specific miRNAs were detected by both sequencing and microarray hybridization. Our results suggest that miRNAs have wide-ranging characteristics and important roles during all stages of somatic embryogenesis in this economically important species.

Highlights

Plant microRNAs are 20- to 24-nucleotide noncoding RNAs that regulate gene expression at transcriptional and post-transcriptional levels, either by endonucleolytic cleavage or by translational inhibition [1]
Our results suggest that miRNAs have wide-ranging characteristics and important roles during all stages of somatic embryogenesis in this economically important species
The limited number of expressed sequence tags (ESTs) made it difficult to perform a comprehensive study of hybrid yellow poplar miRNAs using only a computational analysis, and Solexa sequencing technology was used to directly obtain information on small RNA (sRNA)

Summary

Introduction

Plant microRNAs (miRNAs) are 20- to 24-nucleotide (nt) noncoding RNAs that regulate gene expression at transcriptional and post-transcriptional levels, either by endonucleolytic cleavage or by translational inhibition [1]. Plant miRNAs are high-level regulators of gene expression that affect numerous aspects of plant biology, especially developmental patterning [2]. Increasing evidence indicates that miRNAs play important roles in plant embryo development. In somatic embryogenesis of Citrus, ten conserved miRNAs showed stage- and tissue-specific expression in different embryonic tissues [6]. Hard wood plants have a more complex hereditary background and regulation mechanisms, but little is known about miRNA-mediated modulation in early-stage embryonic tissues in these plants. For economically important species, understanding the mechanism of miRNA regulation in embryogenesis would have a positive impact on hardwood plant production. Several studies have indicated a major role for microRNAs (miRNAs) in regulating plant development, but miRNA-mediated regulation of the developing somatic embryo is poorly understood, especially during early stages of somatic embryogenesis in hardwood plants. Solexa sequencing and miRNA microfluidic chips were used to discover conserved and species-specific miRNAs during somatic embryogenesis of hybrid yellow poplar (Liriodendron tulipifera6L. chinense)

Methods

Results

Conclusion