GSE24226
Identification and characterization of small non-coding RNAs from Chinese fir by high throughput sequencing
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. 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.  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. Overall design: The aim of this study is to investigate the small RNA transcriptome in Cunninghamia lanceolata. Total RNAs of seeds and calli were extracted using RNAiso-mate for plant tissue and RNAiso plus (Takara, Dalian, Liaoning, China), whereas total RNAs of seedlings, adult leaves and stems were isolated with the Concert Plant RNA Reagent (Invitrogen, Carlsbad, CA, USA), and were then treated with RNase-free DNase I (Promega, Madison, WI, USA). Equal amount of total RNAs from the 5 different samples were mixed to form a single RNA pool. Twenty micrograms of total RNAs from the pool were used and 16 to 30-nt sRNAs were purified using Novex 15% TBE-¬Urea gel (Invitrogen). Two adaptors were sequentially ligated to the 5' and 3' ends of purified sRNAs. The ligation products were further purified from Novex 10% TBE-Urea gel. Reverse transcriptase SuperScript II (Invitrogen) and high-fidelity DNA polymerase Phusion (New England Biolabs, Ipswich, MA, USA) were used in the following RT-PCR reaction. The amplification products were cut from Novex 6% TBE-Urea gel. The purified DNA fragments were used for sequencing on an Illumina 1G Genome Analyzer at the Beijing Genomics Institute, Shenzhen, China.