BACKGROUND
eucalyptus sp.
is one of the most planted hardwood genus in the world  <cit> , mainly because of its applications for generating wood and paper products.
brazil is the largest world producer of eucalypts for short fiber pulp  <cit> , thanks to clonal forests developed from elite material with high productivity  <cit> .
in southern brazil and temperate areas, especially mediterranean europe, portugal and chile, eucalyptus globulus and its hybrids are of interest for the cellulose industry due to their relatively high frost resistance and low lignin content, which facilitates cellulose extraction  <cit> .
on the other hand, e. globulus is generally considered recalcitrant to rooting  <cit> .
the commercial eucalypt forests are generally formed through vegetative propagation which has adventitious rooting as a key step  <cit> .
this developmental process can be divided in two main steps, each with its own requirements and characteristics:  induction step, which involves biochemical and molecular events, without visible morphological changes; and  formation step, which consists of cellular divisions involved in both root meristem organization and primordium establishment, followed by root elongation and emergence out of the cutting  <cit> .
multiple factors can interfere with adventitious rooting, such as phytohormones, phenolic compounds, nutritional conditions and genetic characteristics  <cit> .
among phytohormones, auxins play a central role in rooting capacity  <cit> , particularly its endogenous content and transport rate  <cit> .
however, concentrations that are beneficial to root induction can block its elongation  <cit> .
the shoot apex is the main source of endogenous auxin.
stems have a specific basipetal active transport through vascular parenchyma carried out by both influx  and efflux  carriers  <cit> .
eucalyptus globulus needs exogenous auxin application for an adequate root development in vitro  <cit> , but it is unknown if this occurs because of defficiencies in auxin content, transport or perception mechanisms, or due to some other non-auxin related reason that can block spontaneous rooting responses.
at gene expression level, little is known about the formation of root meristems in woody plants and even less concerning the effect of auxins on this process  <cit> .
to evaluate changes in gene expression, reverse-transcription followed by quantitative, real-time polymerase chain reaction  is one of the most widely used methods.
some of the advantages of qpcr are high sensitivity and specificity, speed, ease of use and capacity to carry out simultaneous measurements of gene expression in several different samples for a limited number of genes  <cit> .
to accurately and reproducibly quantify gene expression, some factors should be taken into account, such as quality and amount of starting material, presence of inhibitors in different samples, primer design, rna extraction and reverse-transcription efficiencies  <cit> .
therefore, selection of an appropriate normalization strategy is essential for obtaining an accurate and reliable quantification of gene expression levels  <cit> .
the most commonly applied approach for normalization of qpcr is the use of one or more endogenous reference genes  <cit> , which will undergo the same type of variation and preparation steps throughout the assay as the target genes.
an ideal reference gene, also known as housekeeping or internal control gene, is assumed to be constitutive, i.e., it should be expressed at a constant level in the majority of the study organism cells, through different developmental stages and in different organs.
besides, its expression is assumed to be unaffected by experimental parameters  <cit> .
genes involved in basic cellular processes, such as cell structure maintenance or primary metabolism, are often chosen as normalizers  <cit> .
however, the transcript levels of these putative reference genes are not always stable and their systematic use without previous validation may lead to the misinterpretation of results.
indeed, the previous assurance of at least one stable gene expression in all variable situations tested is recommended to accurately validate the expression of genes under analysis  <cit> .
in recent years, a large number of reference gene validation attempts have been reported for plants, but most of them have covered model and crop species: rice  <cit> , sugarcane  <cit> , arabidopsis thaliana  <cit> , potato  <cit> , grape  <cit> , barley  <cit> , brachypodium sp.
<cit> , soybean  <cit> , tomato  <cit> , brachiaria sp.
<cit> , coffee  <cit> , peach  <cit> , wheat  <cit> , chicory  <cit> , cotton  <cit> , cucumber  <cit> , longan tree  <cit> , petunia sp.
<cit> , ryegrass  <cit> , rapeseed  <cit>  and tobacco  <cit> .
just a few studies have focused on forest genera, such as poplar  <cit>  and eucalyptus sp.
<cit> ; however, there is a lack of investigations covering in vitro growth or adventitious rooting.
the aim of this study was to evaluate the stability of  <dig> putative reference genes for the purpose of normalization in studying e. globulus gene expression during in vitro adventitious rooting .
statistical methods implemented in genorm  <cit>  and normfinder  <cit>  were used and compared in this evaluation.
in addition, to further validate the reference genes, an expression analysis of a putative e. globulus orthologue of the a. thaliana argonaute <dig>  gene during rooting of microcuttings was carried out.
RESULTS
to identify the most stable genes in e. globulus microcuttings rooted in vitro, we selected  <dig> candidate reference genes to validate by qpcr.
the gene expression stability of the candidates was evaluated in a set of  <dig> samples covering six different stages along adventitious rooting time in the presence or absence of the phytohormone auxin.
the genes included: 18s ribosomal rna   <cit> , actin 2/ <dig>   <cit> , translation elongation factor  <dig>   <cit> , histone h2b   <cit> , nadp isocitrate dehydrogenase   <cit> , polyubiquitin   <cit> , sand protein   <cit> , tip41-like protein   <cit> , alpha-tubulin   <cit> , an eucalyptus ortholog of arabidopsis thaliana expressed protein without determined function   <cit> , and a putative rna binding protein  previously shown to be constitutive in different eucalyptus organs .
the characteristics of each of the listed genes, such as gene name, a. thaliana ortholog locus, a. thaliana locus description, genbank accession number, function and e-value are provided in table  <dig>  all arabidopsis genes were used simply to identify the corresponding orthologs in eucalyptus using the genolyptus project data base.
the specificity of the amplifications was confirmed by the presence of a single band of expected size for each primer pair in agarose gel electrophoresis after pcrs employing either cdna or chromosomal dna as templates, and by the observation of single-peak melting curves of the qpcr products .
no primer dimers or other products resulted from non-specific amplification.
amplification efficiencies of pcrs ranged from  <dig>  for ef <dig> to  <dig>  for h2b .
* gene assumed as constitutive in microarray studies employing leaf and vascular tissues of fully grown e. globulus and e. grandis trees .
** gene obtained from carvalho et al.
<cit> .
gene
temperatures
* primer obtained from carvalho et al.
<cit> .
expression levels of reference gene candidates
not surprisingly, some variations on cq values, i.e.
number of cycles needed for the amplification-related fluorescence to reach a specific threshold level of detection, occurred among the reference genes tested.
transcripts of 18s were the most abundant , followed by h2b,  <dig>  act <dig>  idh, ubi, tua, tip <dig>  ef <dig> and sand, whereas euc <dig> was the least abundant  .
the expression profiles of all candidate reference genes along the adventitious rooting process are depicted in figure 2b.
expression stability of reference gene candidates
two programs were used to evaluate the stability of the  <dig> candidate reference genes: genorm  <cit>  and normfinder  <cit> .
cq data were collected for all samples and transformed to relative quantities using the delta-cq method developed by livak and schmittgen  <cit>  .
genorm analysis
the average expression stability  of all genes was calculated by genorm .
the m value is defined as the average pairwise variation of a particular gene with all other potential reference genes.
the average m values of the candidate reference genes tested are shown in figure  <dig> and summarized in table  <dig>  the genorm program recommends using an m value below the threshold of  <dig>  to identify reference genes with stable expression, but some authors propose the maximum value of  <dig>  to obtain more accurate results  <cit> .
our results indicate idh and sand as the most stably expressed genes  and 18s as the least .
to evaluate the optimal number of reference genes for reliable normalization, genorm calculates the pairwise variation vn/vn+ <dig> between the sequential ranked normalization factors nfn and nfn+ <dig> to determine the effect of adding the next reference gene in normalization.
the normalization factor is calculated based on the geometric average among the three most stable genes relative quantities and stepwise inclusion of the other genes in the order of their expression stability.
a large pairwise variation implies that the added reference gene has a significant effect on normalization and should be included for calculation of a reliable normalization factor  <cit> .
considering the cut-off value of  <dig>  proposed by vandesompele et al.
<cit> , below which the inclusion of an additional reference gene is not necessary, the use of the two most stably expressed genes  is sufficient for accurate normalization  in e. globulus in vitro rooting .
if needed, the euc <dig> gene can be added to the analysis, resulting in a pairwise variation value of  <dig>   .
* eb = error bars
normfinder analysis
in addition to the analysis by the genorm software, the expression stability of the candidate reference genes on the experimental samples was also evaluated by normfinder.
this program takes into account the intra- and inter-group variations for normalization factor calculation and the results are not affected by occasional co-regulated genes.
the best candidate will be the one with the inter-group variation as close to zero as possible, and, at the same time, having the smallest errors bars possible  <cit> .
a change in the indication of most stable genes was observed after normfinder analysis when compared to genorm, with h2b showing the most stable expression , followed by tua.
these two genes were also among the most stably expressed genes based on genorm analysis, displaying m values below  <dig>  .
ubi and 18s were the least stable genes  .
when defining the best combination of two genes using normfinder, once again both h2b and tua were the genes of choice, with a stability value of  <dig>  when used together .
relative expression profile of ago1
to further validate the control genes and for determining the best choice of genes to use, the relative expression of a putative ortholog of a. thaliana argonaute <dig>  gene in e. globulus was investigated during rooting.
ago <dig> encodes a micro rna binding protein  <cit>  involved in regulating the expression of arf <dig> , an auxin response transcription factor with inhibitory action, possibly controling genes related to auxin homeostasis and adventitious root development in a. thalina  <cit> .
the two gene pairs indicated by genorm and normfinder were used as references to evaluate ago <dig> relative expression in eight samples, covering time points of adventitious root induction and formation, both under presence and absence of auxin during the induction step.
using idh and sand as references  ago <dig> relative expression profile was not significantly different among the adventitious rooting time points analysed both in presence or absence of auxin .
when h2b and tua were used as references , ago <dig> relative expression at  <dig> h after transfer to formation step  was significantly higher in absence of auxin , whereas no differences were detected for the auxin exposure treatment .
moreover, when comparing presence and absence of auxin within each time point, ago <dig> relative expression after  <dig> h of culture on root induction medium  and at  <dig> h after transfer to root formation medium  was higher in absence of auxin  than in presence of auxin .
thus, the control gene combinations suggested by each program yielded different relative expression profiles of a gene of interest.
discussion
in gene expression studies, qpcr is often the method of choice for a target gene expression profile investigation because it is currently one of the most sensitive techniques available  <cit> .
in this context, a correct normalization is a pre-requisite for the reliability of the final results  <cit> , especially considering possible biological relevance of small differences in gene expression or in the study of different tissues and organ samples  <cit> .
in the present work, candidate reference genes for expression studies on adventitious rooting in e. globulus microcuttings were selected based on previous reports of normalization in plants, mainly with a. thaliana, populus sp.
and eucalyptus sp., taking into account the model plant status of the first species and the woody habit of the last two genera.
additional genes frequently used for normalization in qpcr studies in other plant species were also evaluated.
the use of a single reference gene has been avoided to minimize the chances of erroneous expression estimates  <cit> .
in fact, the use of multiple reference genes is becoming the golden standard in expression studies  <cit> .
in order to select the most suitable reference genes for use in adventitious rooting, we investigated the relative expression profile of a gene of interest  during this process, using the pairs of reference genes indicated as most stable by each one of the programs .
an increase in ago <dig> expression would be expected during microcutting root development considering its function in regulating auxin metabolism-related genes during adventitious rooting in arabidopsis  <cit> .
the higher ago <dig> expression in the absence of auxin may be due to a lower requirement for the regulation of auxin-metabolism related genes in presence of exogenous auxin.
therefore, normfinder seemed to be better suited for the selection of the most stable reference genes for monitoring gene expression during adventitious rooting, detecting subtle differences that can be regarded as important for the adventitious rooting process.
recent studies have reported some discrepancies between different approaches for selection of reference genes and have chosen normfinder  <cit>  as data analysis tool.
this is mainly due to the wide applicability of this tool in any kind of experimental design, especially when involving different cell types and experimental stages associated with high variation of gene expression  <cit> .
hence, we suggest h2b and tua as the most adequate reference genes for expression studies during the rooting of e. globulus microcuttings.
h2b  is one of the four core histones that form the mononucleosome, the fundamental repeating unit of chromatin in eukaryotic cells  <cit> , being a key component for genetic material structure  <cit> .
two works have motivated us to include h2b in our analysis.
hays et al.
<cit>  employed it as reference in northern blot analysis to assess differential gene expression between alfalfa  varieties exhibiting long- or short-stalked glandular hairs.
later, sterky et al.
<cit> , through a transcriptomics approach, showed that transcripts for histones  were among the most ubiquitous gene products present in different tissues and species of populus.
more recently, a histone h <dig> gene was included when defining reference genes for the analysis of gene expression in longan tree somatic embryogenesis by qpcr  <cit> .
nevertheless, histone genes have been employed as references mostly for expression studies in animal systems  <cit> .
the high expression stability showed in our experiments for tua corroborates data of brunner et al.
<cit>  in a study with poplar.
on the other hand, tong and collaborators found tua as one of the most variable genes for studies with peach, except when comparing genotypes or different storage time of fruit  <cit> .
data to date point to idh as the most used control gene for normalization in eucalyptus sp.
<cit> , but without indication of a previous reference-gene detailed analysis.
although elected as the most stably expressed gene in eucalyptus clones under biotic and abiotic stress  <cit> , results from the present study did not support ranking idh as one of the best reference genes because, together with sand, it appears less sensitive to detect slight differences in the expression profile of an adventitious rooting-related gene of interest.
this fact reinforces the need of detailed reference gene analysis for specific experimental conditions and processes.
adventitious rooting is a very complex process, regulated by both environmental and endogenous factors, but the molecular mechanisms by which adventitious root formation is regulated are still poorly understood.
important advances are upcoming with genetic and microarray studies in populus, especially after the availability of the poplar genome  <cit> .
studying gene expression during rooting in pinus contorta, brinker et al.
<cit>  reported an increased expression of genes involved in protein synthesis and a decreased expression of genes related to protein degradation for the first  <dig> days after auxin treatment, whereas an opposite trend was observed during root formation and elongation.
sorin et al.
also identified proteins with altered expression during adventitious rooting in arabidopsis mutants, and, among these, there were some related to protein degradation  <cit> .
these observations may help explain the low expression stability of the gene for polyubiquitin  found in our experiments , since the product of this gene is known to function in protein degradation  <cit> .
the least stably expressed gene was 18s, both by genorm and normfinder analysis .
the use of rrna as reference gene was previously considered a good strategy in gene expression studies  <cit>  but recent works suggest that this is often not the case  <cit> .
this is possibly due to the high abundance of 18s rrna compared with target mrna transcripts, making it difficult to accurately subtract the baseline value in qpcr data analysis  <cit> .
besides, 18s rrna can not be used for this purpose when reverse transcription is carried out using oligo-dt primers or only mrna is used as template  <cit> .
the most stably expressed genes identified in this study, h2b and tua, were effective references in both phases of the rooting process, induction and formation, and also in presence or absence of auxin, i.e.
with conditions leading to profuse and scarce root development, respectively .
this is a rather important feature because it allows the use of these genes as internal controls in evaluating the expression of putative key genes in the rooting process that are likely to vary between phases and in conditions leading or not to root development  <cit> .
these key genes may include those encoding auxin transporters, auxin metabolism enzymes, auxin receptors and selective proteolysis-related proteins  <cit> .
CONCLUSIONS
this work constitutes the first in-depth study to validate optimal control genes for the quantification of transcript levels in e. globulus during in vitro adventitious rooting.
considering that both programs used are based on distinct statistical algorithms, potential discrepancies could be expected.
the comparative ago <dig> expression profile, using the combinations of reference genes indicated by each program, allowed us to suggest the most adequate combination of control genes, based on the functions described in the literature for this gene of interest.
hence, the normfinder program was considered more appropriate for our experiments, indicating h2b and tua as suitable reference genes for normalization in e. globulus microcuttings rooted in vitro.
besides, ubi and 18s are not indicated for use as internal controls in this species under the conditions tested.
in summary, these findings represent useful tools for normalization of qpcr results and will enable more accurate and reliable gene expression studies related to the vital process of clonal propagation by rooting in this important woody species.

 eucalyptus sp. is one of the most planted hardwood genus in the world, mainly because of its applications for generating wood and paper products. brazil is the largest world producer of eucalypts for short fiber pulp, thanks to clonal forests developed from elite material with high productivity. in southern brazil and temperate areas, especially mediterranean europe, portugal and chile, eucalyptus globulus and its hybrids are of interest for the cellulose industry due to their relatively high frost resistance and low lignin content, which facilitates cellulose extraction. on the other hand, e. globulus is generally considered recalcitrant to rooting. the commercial eucalypt forests are generally formed through vegetative propagation which has adventitious rooting as a key step. this developmental process can be divided in two main steps, each with its own requirements and characteristics: induction step, which involves biochemical and molecular events, without visible morphological changes; and formation step, which consists of cellular divisions involved in both root meristem organization and primordium establishment, followed by root elongation and emergence out of the cutting. multiple factors can interfere with adventitious rooting, such as phytohormones, phenolic compounds, nutritional conditions and genetic characteristics. among phytohormones, auxins play a central role in rooting capacity, particularly its endogenous content and transport rate. however, concentrations that are beneficial to root induction can block its elongation. the shoot apex is the main source of endogenous auxin. stems have a specific basipetal active transport through vascular parenchyma carried out by both influx and efflux carriers. eucalyptus globulus needs exogenous auxin application for an adequate root development in vitro, but it is unknown if this occurs because of defficiencies in auxin content, transport or perception mechanisms, or due to some other non-auxin related reason that can block spontaneous rooting responses. at gene expression level, little is known about the formation of root meristems in woody plants and even less concerning the effect of auxins on this process. to evaluate changes in gene expression, reverse-transcription followed by quantitative, real-time polymerase chain reaction is one of the most widely used methods. some of the advantages of qpcr are high sensitivity and specificity, speed, ease of use and capacity to carry out simultaneous measurements of gene expression in several different samples for a limited number of genes. to accurately and reproducibly quantify gene expression, some factors should be taken into account, such as quality and amount of starting material, presence of inhibitors in different samples, primer design, rna extraction and reverse-transcription efficiencies. therefore, selection of an appropriate normalization strategy is essential for obtaining an accurate and reliable quantification of gene expression levels. the most commonly applied approach for normalization of qpcr is the use of one or more endogenous reference genes, which will undergo the same type of variation and preparation steps throughout the assay as the target genes. an ideal reference gene, also known as housekeeping or internal control gene, is assumed to be constitutive, i.e., it should be expressed at a constant level in the majority of the study organism cells, through different developmental stages and in different organs. besides, its expression is assumed to be unaffected by experimental parameters. genes involved in basic cellular processes, such as cell structure maintenance or primary metabolism, are often chosen as normalizers. however, the transcript levels of these putative reference genes are not always stable and their systematic use without previous validation may lead to the misinterpretation of results. indeed, the previous assurance of at least one stable gene expression in all variable situations tested is recommended to accurately validate the expression of genes under analysis. in recent years, a large number of reference gene validation attempts have been reported for plants, but most of them have covered model and crop species: rice, sugarcane, arabidopsis thaliana, potato, grape, barley, brachypodium sp., soybean, tomato, brachiaria sp., coffee, peach, wheat, chicory, cotton, cucumber, longan tree, petunia sp., ryegrass, rapeseed and tobacco. just a few studies have focused on forest genera, such as poplar and eucalyptus sp.; however, there is a lack of investigations covering in vitro growth or adventitious rooting. the aim of this study was to evaluate the stability of putative reference genes for the purpose of normalization in studying e. globulus gene expression during in vitro adventitious rooting. statistical methods implemented in genorm and normfinder were used and compared in this evaluation. in addition, to further validate the reference genes, an expression analysis of a putative e. globulus orthologue of the a. thaliana argonaute gene during rooting of microcuttings was carried out.  to identify the most stable genes in e. globulus microcuttings rooted in vitro, we selected candidate reference genes to validate by qpcr. the gene expression stability of the candidates was evaluated in a set of samples covering six different stages along adventitious rooting time in the presence or absence of the phytohormone auxin. the genes included: 18s ribosomal rna, actin 2/, translation elongation factor, histone h2b, nadp isocitrate dehydrogenase, polyubiquitin, sand protein, tip41-like protein, alpha-tubulin, an eucalyptus ortholog of arabidopsis thaliana expressed protein without determined function, and a putative rna binding protein previously shown to be constitutive in different eucalyptus organs. the characteristics of each of the listed genes, such as gene name, a. thaliana ortholog locus, a. thaliana locus description, genbank accession number, function and e-value are provided in table all arabidopsis genes were used simply to identify the corresponding orthologs in eucalyptus using the genolyptus project data base. the specificity of the amplifications was confirmed by the presence of a single band of expected size for each primer pair in agarose gel electrophoresis after pcrs employing either cdna or chromosomal dna as templates, and by the observation of single-peak melting curves of the qpcr products. no primer dimers or other products resulted from non-specific amplification. amplification efficiencies of pcrs ranged from for ef to for h2b. * gene assumed as constitutive in microarray studies employing leaf and vascular tissues of fully grown e. globulus and e. grandis trees. ** gene obtained from carvalho et al.. gene temperatures * primer obtained from carvalho et al.. expression levels of reference gene candidates not surprisingly, some variations on cq values, i.e. number of cycles needed for the amplification-related fluorescence to reach a specific threshold level of detection, occurred among the reference genes tested. transcripts of 18s were the most abundant, followed by h2b, act idh, ubi, tua, tip ef and sand, whereas euc was the least abundant. the expression profiles of all candidate reference genes along the adventitious rooting process are depicted in figure 2b. expression stability of reference gene candidates two programs were used to evaluate the stability of the candidate reference genes: genorm and normfinder. cq data were collected for all samples and transformed to relative quantities using the delta-cq method developed by livak and schmittgen. genorm analysis the average expression stability of all genes was calculated by genorm. the m value is defined as the average pairwise variation of a particular gene with all other potential reference genes. the average m values of the candidate reference genes tested are shown in figure and summarized in table the genorm program recommends using an m value below the threshold of to identify reference genes with stable expression, but some authors propose the maximum value of to obtain more accurate results. our results indicate idh and sand as the most stably expressed genes and 18s as the least. to evaluate the optimal number of reference genes for reliable normalization, genorm calculates the pairwise variation vn/vn+ between the sequential ranked normalization factors nfn and nfn+ to determine the effect of adding the next reference gene in normalization. the normalization factor is calculated based on the geometric average among the three most stable genes relative quantities and stepwise inclusion of the other genes in the order of their expression stability. a large pairwise variation implies that the added reference gene has a significant effect on normalization and should be included for calculation of a reliable normalization factor. considering the cut-off value of proposed by vandesompele et al., below which the inclusion of an additional reference gene is not necessary, the use of the two most stably expressed genes is sufficient for accurate normalization in e. globulus in vitro rooting. if needed, the euc gene can be added to the analysis, resulting in a pairwise variation value of. * eb = error bars normfinder analysis in addition to the analysis by the genorm software, the expression stability of the candidate reference genes on the experimental samples was also evaluated by normfinder. this program takes into account the intra- and inter-group variations for normalization factor calculation and the results are not affected by occasional co-regulated genes. the best candidate will be the one with the inter-group variation as close to zero as possible, and, at the same time, having the smallest errors bars possible. a change in the indication of most stable genes was observed after normfinder analysis when compared to genorm, with h2b showing the most stable expression, followed by tua. these two genes were also among the most stably expressed genes based on genorm analysis, displaying m values below. ubi and 18s were the least stable genes. when defining the best combination of two genes using normfinder, once again both h2b and tua were the genes of choice, with a stability value of when used together. relative expression profile of ago1 to further validate the control genes and for determining the best choice of genes to use, the relative expression of a putative ortholog of a. thaliana argonaute gene in e. globulus was investigated during rooting. ago encodes a micro rna binding protein involved in regulating the expression of arf, an auxin response transcription factor with inhibitory action, possibly controling genes related to auxin homeostasis and adventitious root development in a. thalina. the two gene pairs indicated by genorm and normfinder were used as references to evaluate ago relative expression in eight samples, covering time points of adventitious root induction and formation, both under presence and absence of auxin during the induction step. using idh and sand as references ago relative expression profile was not significantly different among the adventitious rooting time points analysed both in presence or absence of auxin. when h2b and tua were used as references, ago relative expression at h after transfer to formation step was significantly higher in absence of auxin, whereas no differences were detected for the auxin exposure treatment. moreover, when comparing presence and absence of auxin within each time point, ago relative expression after h of culture on root induction medium and at h after transfer to root formation medium was higher in absence of auxin than in presence of auxin. thus, the control gene combinations suggested by each program yielded different relative expression profiles of a gene of interest. discussion in gene expression studies, qpcr is often the method of choice for a target gene expression profile investigation because it is currently one of the most sensitive techniques available. in this context, a correct normalization is a pre-requisite for the reliability of the final results, especially considering possible biological relevance of small differences in gene expression or in the study of different tissues and organ samples. in the present work, candidate reference genes for expression studies on adventitious rooting in e. globulus microcuttings were selected based on previous reports of normalization in plants, mainly with a. thaliana, populus sp. and eucalyptus sp., taking into account the model plant status of the first species and the woody habit of the last two genera. additional genes frequently used for normalization in qpcr studies in other plant species were also evaluated. the use of a single reference gene has been avoided to minimize the chances of erroneous expression estimates. in fact, the use of multiple reference genes is becoming the golden standard in expression studies. in order to select the most suitable reference genes for use in adventitious rooting, we investigated the relative expression profile of a gene of interest during this process, using the pairs of reference genes indicated as most stable by each one of the programs. an increase in ago expression would be expected during microcutting root development considering its function in regulating auxin metabolism-related genes during adventitious rooting in arabidopsis. the higher ago expression in the absence of auxin may be due to a lower requirement for the regulation of auxin-metabolism related genes in presence of exogenous auxin. therefore, normfinder seemed to be better suited for the selection of the most stable reference genes for monitoring gene expression during adventitious rooting, detecting subtle differences that can be regarded as important for the adventitious rooting process. recent studies have reported some discrepancies between different approaches for selection of reference genes and have chosen normfinder as data analysis tool. this is mainly due to the wide applicability of this tool in any kind of experimental design, especially when involving different cell types and experimental stages associated with high variation of gene expression. hence, we suggest h2b and tua as the most adequate reference genes for expression studies during the rooting of e. globulus microcuttings. h2b is one of the four core histones that form the mononucleosome, the fundamental repeating unit of chromatin in eukaryotic cells, being a key component for genetic material structure. two works have motivated us to include h2b in our analysis. hays et al. employed it as reference in northern blot analysis to assess differential gene expression between alfalfa varieties exhibiting long- or short-stalked glandular hairs. later, sterky et al., through a transcriptomics approach, showed that transcripts for histones were among the most ubiquitous gene products present in different tissues and species of populus. more recently, a histone h gene was included when defining reference genes for the analysis of gene expression in longan tree somatic embryogenesis by qpcr. nevertheless, histone genes have been employed as references mostly for expression studies in animal systems. the high expression stability showed in our experiments for tua corroborates data of brunner et al. in a study with poplar. on the other hand, tong and collaborators found tua as one of the most variable genes for studies with peach, except when comparing genotypes or different storage time of fruit. data to date point to idh as the most used control gene for normalization in eucalyptus sp., but without indication of a previous reference-gene detailed analysis. although elected as the most stably expressed gene in eucalyptus clones under biotic and abiotic stress, results from the present study did not support ranking idh as one of the best reference genes because, together with sand, it appears less sensitive to detect slight differences in the expression profile of an adventitious rooting-related gene of interest. this fact reinforces the need of detailed reference gene analysis for specific experimental conditions and processes. adventitious rooting is a very complex process, regulated by both environmental and endogenous factors, but the molecular mechanisms by which adventitious root formation is regulated are still poorly understood. important advances are upcoming with genetic and microarray studies in populus, especially after the availability of the poplar genome. studying gene expression during rooting in pinus contorta, brinker et al. reported an increased expression of genes involved in protein synthesis and a decreased expression of genes related to protein degradation for the first days after auxin treatment, whereas an opposite trend was observed during root formation and elongation. sorin et al. also identified proteins with altered expression during adventitious rooting in arabidopsis mutants, and, among these, there were some related to protein degradation. these observations may help explain the low expression stability of the gene for polyubiquitin found in our experiments, since the product of this gene is known to function in protein degradation. the least stably expressed gene was 18s, both by genorm and normfinder analysis. the use of rrna as reference gene was previously considered a good strategy in gene expression studies but recent works suggest that this is often not the case. this is possibly due to the high abundance of 18s rrna compared with target mrna transcripts, making it difficult to accurately subtract the baseline value in qpcr data analysis. besides, 18s rrna can not be used for this purpose when reverse transcription is carried out using oligo-dt primers or only mrna is used as template. the most stably expressed genes identified in this study, h2b and tua, were effective references in both phases of the rooting process, induction and formation, and also in presence or absence of auxin, i.e. with conditions leading to profuse and scarce root development, respectively. this is a rather important feature because it allows the use of these genes as internal controls in evaluating the expression of putative key genes in the rooting process that are likely to vary between phases and in conditions leading or not to root development. these key genes may include those encoding auxin transporters, auxin metabolism enzymes, auxin receptors and selective proteolysis-related proteins.  this work constitutes the first in-depth study to validate optimal control genes for the quantification of transcript levels in e. globulus during in vitro adventitious rooting. considering that both programs used are based on distinct statistical algorithms, potential discrepancies could be expected. the comparative ago expression profile, using the combinations of reference genes indicated by each program, allowed us to suggest the most adequate combination of control genes, based on the functions described in the literature for this gene of interest. hence, the normfinder program was considered more appropriate for our experiments, indicating h2b and tua as suitable reference genes for normalization in e. globulus microcuttings rooted in vitro. besides, ubi and 18s are not indicated for use as internal controls in this species under the conditions tested. in summary, these findings represent useful tools for normalization of qpcr results and will enable more accurate and reliable gene expression studies related to the vital process of clonal propagation by rooting in this important woody species. 
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. to validate these results and select the most suitable reference genes, the expression profile of the argonaute gene was evaluated in relation to the most stable candidate genes indicated by each algorithm. both programs indentified ubi and 18s as the most variable genes. the present study aimed at identifying suitable control genes for normalization of gene expression associated with adventitious rooting in e. globulus microcuttings. 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, act ef euc h2b, idh, sand, tip tua, ubi and 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. our study showed that expression stability varied between putative reference genes tested in e. globulus. based on the ago 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. eucalyptus globulus and its hybrids are very important for the cellulose and paper industry mainly due to their low lignin content and frost resistance. to date one of the most accurate methods available for gene expression analysis is quantitative reverse transcription-polymerase chain reaction; however, reliable use of this technique requires reference genes for normalization. thus, the identification of reliable reference genes must be done for every species and experimental approach. 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. there is no single reference gene that can be regarded as universal for all experiments and biological materials.
 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; 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.  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, act ef euc h2b, idh, sand, tip tua, ubi and 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 argonaute gene was evaluated in relation to the most stable candidate genes indicated by each algorithm.  our study showed that expression stability varied between putative reference genes tested in e. globulus. based on the ago 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. 