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Background   Eucalyptus globulus  and its hybrids are very important for the cellulose and paper industry mainly due to their low lignin content and frost resistance. However, rooting of cuttings of this species is recalcitrant and exogenous auxin application is often necessary for good root development. To date one of the most accurate methods available for gene expression analysis is quantitative reverse transcription-polymerase chain reaction (qPCR); however, reliable use of this technique requires reference genes for normalization. There is no single reference gene that can be regarded as universal for all experiments and biological materials. Thus, the identification of reliable reference genes must be done for every species and experimental approach. The present study aimed at identifying suitable control genes for normalization of gene expression associated with adventitious rooting in  E. globulus  microcuttings.    Results  By the use of two distinct algorithms,  geNorm  and  NormFinder , we have assessed gene expression stability of eleven candidate reference genes in  E. globulus :  18S, ACT2, EF2, EUC12, H2B, IDH, SAND, TIP41, TUA, UBI  and  33380 . The candidate reference genes were evaluated in microccuttings rooted  in vitro , in presence or absence of auxin, along six time-points spanning the process of adventitious rooting. Overall, the stability profiles of these genes determined with each one of the algorithms were very similar. Slight differences were observed in the most stable pair of genes indicated by each program:  IDH  and  SAND  for  geNorm , and  H2B  and  TUA  for  NormFinder . Both programs indentified  UBI  and  18S  as the most variable genes. To validate these results and select the most suitable reference genes, the expression profile of the  ARGONAUTE1  gene was evaluated in relation to the most stable candidate genes indicated by each algorithm.    Conclusion  Our study showed that expression stability varied between putative reference genes tested in  E. globulus . Based on the  AGO1  relative expression profile obtained using the genes suggested by the algorithms,  H2B  and  TUA  were considered as the most suitable reference genes for expression studies in  E. globulus  adventitious rooting.  UBI  and  18S  were unsuitable for use as controls in qPCR related to this process. These findings will enable more accurate and reliable normalization of qPCR results for gene expression studies in this economically important woody plant, particularly related to rooting and clonal propagation.

bmc molecular biology

Reference gene selection for quantitative reverse transcription-polymerase chain reaction normalization during in vitro adventitious rooting in Eucalyptus globulus Labill