title: Validation of reference genes from Eucalyptus spp. under different stress conditions.
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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: 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.
authors.forename: Jullyana Cristina Magalhães Silva
authors.initials: JC
authors.lastname: Moura
authors.forename: Pedro
authors.initials: P
authors.lastname: Araújo
authors.forename: Michael dos Santos
authors.initials: Mdos S
authors.lastname: Brito
authors.forename: Uiara Romero
authors.initials: UR
authors.lastname: Souza
authors.forename: Juliana de Oliveira Fernandes
authors.initials: Jde O
authors.lastname: Viana
authors.forename: Paulo
authors.initials: P
authors.lastname: Mazzafera
mesh_terms: D000199:Actins / Q000235:genetics; D002138:Calibration; D003080:Cold Temperature; D019295:Computational Biology; D018744:DNA, Plant / Q000378:metabolism*; D003624:Darkness; D055864:Droughts; D005052:Eucalyptus / Q000145:classification / Q000235:genetics* / Q000528:radiation effects; D018506:Gene Expression Regulation, Plant / Q000528:radiation effects; D007521:Isocitrate Dehydrogenase / Q000235:genetics; D008027:Light; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism; D018547:Plant Stems; D060888:Real-Time Polymerase Chain Reaction / Q000592:standards*; D012015:Reference Standards; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000592:standards*; D013312:Stress, Physiological / Q000235:genetics*; D014404:Tubulin / Q000235:genetics; D025801:Ubiquitin / Q000235:genetics; D014867:Water
chemical_list: D000199:Actins; D018744:DNA, Plant; D010940:Plant Proteins; D014404:Tubulin; D025801:Ubiquitin; D014867:Water; D007521:Isocitrate Dehydrogenase