Temperature Driven Changes in smallRNAomes during Formation of an Epigenetic Memory in Somatic Embryos of Norway Spruce

Epigenetic memory in Norway spruce permanently affects the timing of vitally important adaptive traits, such as dormancy, bud burst and bud set. Epigenetic memory marks are established in response to the temperature conditions prevailing during early seed formation.

The main task of the current study is to continue identification and characterization of the small non-coding part of Norway spruce transcriptomes during formation of epigenetic memory in somatic embryos, which closely mimics the natural processes of epigenetic memory formation in seeds. We prepared nine small RNA libraries from embryogenic tissues at three stages of maturation grown up in vitro at three culturing temperatures (18, 23 and 28°C). Obtained libraries were sequenced on the PGM™ (Ion Torrent™) system and analyzed using CLC genomic workbench software.

Extensive characterization of small RNAs in Norway spruce embryos during different stages of embryogenesis under different temperature treatments allowed identifying at least three different types of small RNAs: micro RNAs(miRNAs), trans-acting small interfering RNAs(ta-siRs) and 31-nt length sRNAs, similar to animal’s PIWI-interacting RNAs(piRNAs). In embryonic tissues, the most prevailing were 24-nt siRs, followed by 21-nt sRNAs with separate large fraction of sRNAs with length around 31-nt. In total, we defined around 3000 ta-siRs, originated from 50 TAS loci. We report the identification of 438 novel putative miRNAs in Norway spruce transcribed from 158 miR genes and confirming 413 genes as targets for the 99 spruce novel and conserved miRNAs and 120 conserved miRNAs from other tree species and Arabidopsis. Additionally we determined around 16000 of 31-32nt sRNAs, which could be produced from more than 1300 loci (piRNA clusters).

Based on identified sRNAs we studied their expression patterns during different stages of SE in dependence on the epitype inducing temperature prevailing during SE growth and leading to establishing of epigenetic marks. We showed that Norway spruce poses a variety of sRNAs with distinct temperature dependent expression patterns, and these sRNAs target large amount of spruce genes with a wide range of functions, including also genes involved in epigenetic regulation. Unknown mechanisms provide fine-tuning of sRNAs pool content participating in the epigenetic memory formation.