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

Abstract

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 identified 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.

Figure 1

Stages of in vitro adventitious rooting in Eucalyptus globulus. After seeding, seedlings remained 14 weeks in germination medium. After this time, apical microcuttings were obtained and used in adventitious rooting experiments. The microcuttings were kept in induction medium (presence or absence of 10 mg l^-1 IBA) for 12, 24, 48 or 96 h, depending on the harvest time. For the harvest points in formation medium, the microcuttings remained 96 h in induction medium and after that were transferred to formation medium (devoid of auxin and with activated charcoal). In this case, the samples were collected after 24 and 48 h. The samples harvested at 12, 24, 48 and 96 h of induction step and at 24 and 48 h of formation step were submitted to RNA extraction and used for the analyses.

Figure 2

RNA transcription level of reference genes tested, presented as Cq mean value ± Standard Deviation (SD). (A) Mean Cq of each reference gene. (B) Transcription level profile of reference genes along the adventitious rooting process. ind: induction step; form: formation step. Auxin: indicates addition of 10 mg l^-1 Indol Butyric Acid (IBA) in induction step culture medium; control: indicates absence of IBA in induction step culture medium.

Figure 3

Gene expression stability (M) and ranking of the 11 reference genes as calculated by geNorm in Eucalyptus globulus microcuttings during in vitro adventitious rooting. The microcuttings were rooted in presence or absence of 10 mg l^-1 Indol Butyric Acid (IBA) in the induction step. A lower average expression stability M value indicates more stable expression.

Figure 4

Determination of the optimal reference gene number as calculated by geNorm for accurate normalization during Eucalyptus globulus in vitro adventitious rooting. geNorm pairwise variation values (V values) are calculated by an algorithm which measures pairwise variation (Vn/n + 1) between two sequential normalization factors NFn and NFn + 1, where n is the number of genes involved in the normalization factor.

Figure 5

AGO1 relative expression profile during Eucalyptus globulus in vitro adventitious rooting. The AGO1 (Argonaute 1) expression profile was investigated relative to the best combination of reference genes indicated by both geNorm (A) and NormFinder (B) programs. Aux: indicates addition of 10 mg l^-1 Indol Butyric Acid (IBA) in induction step culture medium; cont: indicates absence of IBA in induction step culture medium; Columns sharing the same letter are not different by ANOVA followed by Duncan test (P ≤ 0.05); small letters correspond to analysis of variance (ANOVA) performed for control treatment samples; capital letters correspond to analysis of variance (ANOVA) performed for samples treated with auxin. *: indicates significant difference between treatments within time points by t-test (P ≤ 0.05).