High-throughput sequencing as an effective approach in profiling small RNAs derived from a hairpin RNA expression vector in woody plants.

Hairpin RNA (hpRNA)-mediated gene silencing has proved to be an efficient approach to develop virus-resistant transgenic plants. To characterize small RNA molecules (sRNAs) derived from an hpRNA expression vector in transgenic cherry rootstock plants, we conducted small RNA sequencing of (1) a transgenic rootstock containing an inverted repeat of the partial coat protein of Prunus necrotic ring spot virus (PNRSV-hpRNA); (2) a nontransgenic rootstock; and (3) a PNRSV-infected sweet cherry plant. Analysis of the PNRSV sRNA pools indicated that 24-nt (nucleotide) small interfering RNAs (siRNAs) were the most prevalent sRNAs in the transgenic rootstock whereas the most abundant sRNAs in the PNRSV-infected nontransgenic rootstock were 21-nt siRNAs. In addition, the 24-nt siRNAs of the PNRSV-hpRNA were more abundant on the sense strand than those on the antisense strand in the transgenic rootstock. In contrast, preference in generating PNRSV sRNAs, ranging from 19-nt to 30-nt for sense and antisense strands, was not distinct in the PNRSV-infected nontransgenic sweet cherry. Taken together, this is the first report on profiling hpRNA-derived sRNAs in woody plants using high-throughput sequencing technology, which is an efficient way to verify the presence/absence, the abundance, and the sequence features of certain sRNAs.