Identification and evaluation of reference genes for normalization in quantitative real-time PCR analysis in the premodel tree Betula luminifera

Betula luminifera is a commercial tree species that is emerging as a new model system for tree genomics research. A draft genomic sequence is expected to be publicly available in the near future, which means that an explosion of gene expression studies awaits. Thus, the work of selecting appropriate reference genes for qPCR normalization in different tissues or under various experimental conditions is extremely valuable. In this study, ten candidate genes were analyzed in B. luminifera subjected to different abiotic stresses and at various flowering stages. The expression stability of these genes was evaluated using three distinct algorithms implemented using geNorm, NormFinder and BestKeeper. The best-ranked reference genes varied across different sample sets, though RPL39, MDH and EF1a were determined as the most stable by the three programs among all tested samples. RPL39 and EF1a should be appropriate for normalization in N-starved roots, while the combination of RPL39 and MDH should be appropriate for N-starved stems and EF1a and MDH should be appropriate in N-starved leaves. In PEG-treated (osmotic) roots, MDH was the most suitable, whereas EF1a was suitable for PEG-treated stems and leaves. TUA was also stably expressed levels in PEG-treated plants. The combination of RPL39 and TUB should be appropriate for heat-stressed leaves and flowering stage. For reference gene validation, the expression levels of SOD and NFYA-3 were investigated. This work will be beneficial to future studies on gene expression under different abiotic stress conditions and flowering status in B. luminifera.