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Open Access Highly Accessed Research article

Identification and characterization of small non-coding RNAs from Chinese fir by high throughput sequencing

Li-Chuan Wan1, Feng Wang12, Xiangqian Guo3, Shanfa Lu4, Zongbo Qiu1, Yuanyuan Zhao12, Haiyan Zhang1* and Jinxing Lin1*

Author Affiliations

1 Key Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing, 100093, China

2 Graduate School of the Chinese Academy of Sciences, Beijing, 100049, China

3 Bioinformatics Laboratory and National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China

4 Medicinal Plant Cultivation Research Center, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, Haidian District, Beijing, 100193, China

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BMC Plant Biology 2012, 12:146  doi:10.1186/1471-2229-12-146

Published: 15 August 2012

Abstract

Background

Small non-coding RNAs (sRNAs) play key roles in plant development, growth and responses to biotic and abiotic stresses. At least four classes of sRNAs have been well characterized in plants, including repeat-associated siRNAs (rasiRNAs), microRNAs (miRNAs), trans-acting siRNAs (tasiRNAs) and natural antisense transcript-derived siRNAs. Chinese fir (Cunninghamia lanceolata) is one of the most important coniferous evergreen tree species in China. No sRNA from Chinese fir has been described to date.

Results

To obtain sRNAs in Chinese fir, we sequenced a sRNA library generated from seeds, seedlings, leaves, stems and calli, using Illumina high throughput sequencing technology. A comprehensive set of sRNAs were acquired, including conserved and novel miRNAs, rasiRNAs and tasiRNAs. With BLASTN and MIREAP we identified a total of 115 conserved miRNAs comprising 40 miRNA families and one novel miRNA with precursor sequence. The expressions of 16 conserved and one novel miRNAs and one tasiRNA were detected by RT-PCR. Utilizing real time RT-PCR, we revealed that four conserved and one novel miRNAs displayed developmental stage-specific expression patterns in Chinese fir. In addition, 209 unigenes were predicted to be targets of 30 Chinese fir miRNA families, of which five target genes were experimentally verified by 5' RACE, including a squamosa promoter-binding protein gene, a pentatricopeptide (PPR) repeat-containing protein gene, a BolA-like family protein gene, AGO1 and a gene of unknown function. We also demonstrated that the DCL3-dependent rasiRNA biogenesis pathway, which had been considered absent in conifers, existed in Chinese fir. Furthermore, the miR390-TAS3-ARF regulatory pathway was elucidated.

Conclusions

We unveiled a complex population of sRNAs in Chinese fir through high throughput sequencing. This provides an insight into the composition and function of sRNAs in Chinese fir and sheds new light on land plant sRNA evolution.

Keywords:
Chinese fir; miRNA; rasiRNA; tasiRNA; Cunninghamia lanceolata