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Open Access Research article

Validation of reference genes from Eucalyptus spp. under different stress conditions

Jullyana Cristina Magalhães Silva Moura12, Pedro Araújo2, Michael dos Santos Brito23, Uiara Romero Souza2, Julianade Oliveira Fernandes Viana4 and Paulo Mazzafera2*

Author Affiliations

1 Departamento de Ciências Biológicas, Instituto de Ciências Exatas, Naturais e Educação, Universidade Federal do Triângulo Mineiro, Av. Getúlio Guaritá 159, Bairro Abadia, Uberaba, MG, 38025-440, Brazil

2 Departamento de Biologia Vegetal, Instituto de Biologia, CP 6109, Universidade Estadual de Campinas, Campinas, SP, 13083-970, Brazil

3 Centro Avançado da Pesquisa Tecnológica do Agronegócio de Cana, Ribeirão Preto, SP, CP 206 14001-970, Brazil

4 Fibria Celulose S.A., Rodovia Euryale de Jesus Zerbine, Km 84, São Silvestre, Jacareí, SP, 12340-010, Brazil

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BMC Research Notes 2012, 5:634  doi:10.1186/1756-0500-5-634

Published: 14 November 2012

Abstract

Background

The genus Eucalyptus consists of approximately 600 species and subspecies and has a physiological plasticity that allows some species to propagate in different regions of the world. Eucalyptus is a major source of cellulose for paper manufacturing, and its cultivation is limited by weather conditions, particularly water stress and low temperatures. Gene expression studies using quantitative reverse transcription polymerase chain reaction (qPCR) require reference genes, which must have stable expression to facilitate the comparison of the results from analyses using different species, tissues, and treatments. Such studies have been limited in eucalyptus.

Results

Eucalyptus globulus Labill, Eucalyptus urograndis (hybrid from Eucalyptus urophylla S.T. Blake X Eucalyptus grandis Hill ex-Maiden) and E. uroglobulus (hybrid from E. urograndis X E. globulus) were subjected to different treatments, including water deficiency and stress recovery, low temperatures, presence or absence of light, and their respective controls. Except for treatment with light, which examined the seedling hypocotyl or apical portion of the stem, the expression analyses were conducted in the apical and basal parts of the stem. To select the best pair of genes, the bioinformatics tools GeNorm and NormFinder were compared. Comprehensive analyses that did not differentiate between species, treatments, or tissue types, showed that IDH (isocitrate dehydrogenase), SAND (SAND protein), ACT (actin), and A-Tub (α-tubulin) genes were the most stable. IDH was the most stable gene in all of the treatments.

Conclusion

Comparing these results with those of other studies on eucalyptus, we concluded that five genes are stable in different species and experimental conditions: IDH, SAND, ACT, A-Tub, and UBQ (ubiquitin). It is usually recommended a minimum of two reference genes is expression analysis; therefore, we propose that IDH and two others genes among the five identified genes in this study should be used as reference genes for a wide range of conditions in eucalyptus.

Keywords:
Eucalyptus; Drought; Cold; Light; Reference genes