Complexity of miRNA and their isomiRs During Formation of an Epigenetic Memory in Norway Spruce

Epigenetic memory marks establishment in Norway spruce occur exclusively during embryogenesis in response to environmental impact, and the epitype is fixated by the time the embryo is fully developed without a change in the DNA sequence. Epigenetic mechanisms influence phenotype through altered regulation of gene expression that is mitotically propagated. Epigenetic regulation may be realized through several interconnected molecular pathways including small non-coding RNAs. MicroRNAs (miRNAs) and their variants, called isomiRs, are commonly reported in deep-sequencing studies. isomiRs are to be biologically relevant and functionally cooperative partners of canonical miRNAs that act coordinately to target pathways of related genes.

We started large scale studies aimed on identifying and characterizing of genes and regulatory elements involved in the initiation, maintenance, and heritability of epigenetic memory using high-throughput sequencing approaches. miRNA regulation during formation of epigenetic memory are studied on the same full-sibs family zygotic embryo in vitro cultures developed in cold (18°C), norma l(23°C) and warm (30°C) environmental conditions. For this, we constructed 12 small RNA libraries during 4 stages of embryo development (proliferation and 3 stages of embryo formation) and are analyzing them using high-throughput sequencing approaches on the Ion PGM^TM (Life technologies) platform, followed by CLC genomics workbench data processing.

We are expecting to determine certain stages during embryogenesis when epigenetic memory marks are forming and when differences between small RNAomes will be the most prominent. The identification of novel and conserved miRNAs and their isomiRs are in progress and the analysis of smallRNAomic changes will be presented.