Techniques for Small Non-Coding RNA Analysis in Seeds of Forest Tree Species

In recent years, the scientific community has become aware that epigenetic mechanisms play a more important role in gene regulatory networks (GRNs) than was hitherto thought, as accumulating evidence has shown that changes in epigenetics without genetic variation can affect complex traits over multiple generations. Within the epigenetic machinery, small non-coding RNAs (sRNAs, 18-24 nucleotides in length) are evolutionarily conserved RNA molecules that target mRNAs for deregulation or translational repression. They commonly have high-level regulatory functions in GRNs by mediating DNA and/or histone methylation and gene silencing essential for plant developmental programs and adaptability. Local adaptation enables plants to acquire a high fitness by, for example, properly timing developmental transitions to match plant growth stages with organism's favorable seasons. In particular, the seed represents a key evolutionary adaptation of seed plants that facilitates dispersal and reinitiates the development coupled in time with suitable environmental conditions. With the advent of high-throughput sequencing for sRNAs and computational approaches for sRNA detection and categorization, it is now feasible to unravel how sRNAs contribute to the fitness of tree species that can survive hundreds of years (e.g., conifers). Of particular interest is to disentangle the roles of sRNAs from complex genomic information in tree species with intimidating genomic sizes (commonly 20-30 Gb in conifers) and abundant nongenic components (e.g., >60% transposable elements). In this chapter, we use seeds of the conifer Picea glauca as a study system to describe the methods and protocols we used or have recently updated, from high-quality RNA isolation to sRNA identification, sequence conservation, abundance comparison, and functional analysis.