Browsing by discipline "Soveltava biologia"

The stilbene biosynthetic pathway yielding products pinosylvin and pinosylvin monomethyl ether has been implicated in Scots pine’s (Pinus sylvestris) heartwood constitutive resistance to decay. This trait, which is very valuable in forest economies has been suggested to be genetically controlled. The molecular basis for this control is not known. Wounding by drilling in stems of Scots pine seedlings have been suggested to induce production of pinosylvin and pinosylvin monomethyl ether in xylem of these seedlings which transforms to heartwood on maturity. The aim of this study was to elucidate the transcripts co-expressed with candidate genes in the stilbene biosynthesis pathway (Ps4CL, PsPAL, PsSTS and PsPMT) and their expression patterns, reconstruct a comprehensive transcriptome and decipher the difference between half sib families in the expression of transcripts reacting to the wounding of Scots pine seedlings using the next generation sequence (ABI SOLiD) profiling of RNA (RNA-Seq). Our bioinformatic pipeline involving de novo transcriptome assembly and scaffolding, read mapping and Digital gene expression (DGE) analysis were utilized to analyze this RNA-Seq data and its results were validated with quantitative PCR. The results show that RNA-Seq analysis generated 25,010 non redundant consensus sequences (representing expressed transcripts) and DGE revealed that the Scots pine seedling transcriptome was altered with up-regulation of 830 EST tags (genes) and down-regulation of 518 EST tags. PsSTS was induced in the wounding experiment while Ps4CL, PsPAL and PsPMT appeared to be expressed constitutively. Of the total 77,326 tags from the PGI EST list (our pseudo reference transcriptome), 29% (mean) of reads from eight RNA-Seq libraries were mapped to 13,000 EST tags. QPCR results confirmed the RNA-Seq data although the latter appeared more sensitive. Read counts from mapped RNA-Seq could be used to distinguish differentially expressed genes between half sib families comprising of high and low decay resistance lines. We conclude that wounding Scots pine seedlings induced a large number of genes including PsSTS. With ABI SOLiD RNA-Seq and DGE technologies, the complex transcriptome of wounded Scots pine seedlings can be reconstructed, constituent expressed transcripts elucidated and half sib family lines with divergent decay resistant trait can be distinguished.