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

Abstract

BackgroundSmall 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.ResultsTo 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.ConclusionsWe 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.

A Significant Fraction of 21-Nucleotide Small RNA Originates from Phased Degradation of Resistance Genes in Several Perennial Species

“…The apparent lack of DCL3 and the low frequency of 24-nt sRNAs are not features common to all conifers, as it was recently shown that Cunninghamia lanceolata has an sRNA distribution more similar to angiosperm species, including a large fraction of 24-nt sRNAs (Wan et al, 2012). They also reported the presence of sequences predicted to code for proteins necessary for the biogenesis of 24-nt sRNAs, including DCL3.…”

The pivotal role of small non-coding RNAs in the regulation of seed development

“…Reports on sRNA studies in gymnosperm seeds have been lacking so far, but previous work on conifer sRNAs have reached different and contradicting conclusions regarding the presence of 24-nt sRNAs, typically associated with hetsiRNAs, in the sRNA transcriptome of the conifers clade Morin et al 2008;Lee et al 2011;Chávez-Montes et al 2014;Wan et al 2012;Zhang et al 2012bZhang et al , 2013bNystedt et al 2013;Niu et al 2015). In somatic embryo-derived material from the conifer Japanese larch (Larix leptolepis) the presence of significant amounts of 24-nt sRNAs has been reported (Zhang et al 2012b(Zhang et al , 2013b, but it would be interesting to check for the presence of these sRNAs in conifer seeds.…”

The Norway spruce genome sequence and conifer genome evolution

“…There has been conflicting evidence about the presence of 24-nucleotide short RNAs (sRNAs) in gymnosperms [26][27][28][29] , a class of sRNA that silence transposable elements by the establishment of DNA methylation 30 . Across 22 samples, we identified numerous 24-nucleotide sRNAs, but these were highly specific to reproductive tissues, largely associated with repeats but present at substantially lower levels than in angiosperms ( Fig.…”