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

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.Identification and characterization of diverse classes of small non-coding RNAs (sRNAs) in eukaryotes has been a major research focus in recent years [1,2]. At least four classes of sRNAs have been well characterized in plants, including heterochromatic and repeat-associated small interfering RNAs (rasiRNAs) [3], microRNAs (miRNAs) [4,5], trans-acting siRNAs (tasiRNAs) [6] and natural antisense transcript-derived siRNAs (nat-siRNAs) [7]. In plants, the majority of endogenous sRNAs are 24-nt rasiRNAs [8]. They repress transposable elements and maintain