Expression of juvenility related microRNAs and target genes during micropropagation of silver birch (Betula pendula Roth).

Abstract Silver birch (Betula pendula Roth.) is an ecologically and economically important deciduous tree species in Northern Europe. In vitro culture initiation and shoot rejuvenation from birches in their reproductive phase is difficult and often unsuccessful. Currently, markers to evaluate the degree of juvenility of birch in vitro shoots, which could be used to track the rejuvenation process and to determine factors affecting it, have not been developed. The aims of this study were to assess expression of juvenility related microRNAs and target genes during micropropagation of silver birch, and to investigate factors affecting juvenility of birch genotypes with different in vitro morphogenic ability. Potential precursor sequences and target genes of the microRNAs miR156 and miR172 were determined by alignment to the birch unigene set. Ten potential miR156 and miR172 precursor sequences were identified, and were tested for the ability to form the required stem-loop structure. Based on precursor sequences, primers were designed for real time PCR analysis of precursor miRNA expression. Expression patterns of two miR156 family precursors (miR156_511 and miR156_789) and one miR172 precursor (miR172_1931) and two target genes (BpSPL1 and BpAP2) had the best correlation with juvenility/maturity in in vitro propagated silver birch. Expression patterns of these miRNA precursors and target genes were also investigated in samples cultured under different in vitro conditions. This study provides an initial survey of molecular markers for assessment of phase change in birch in vitro micropropagation.