title: Establishing references for gene  expression  analyses by RT-qPCR in Theobroma cacao tissues.
abstract: Lack of  continuous progress in Theobroma cacao (Malvaceae) breeding, especially  associated with seed quality traits, requires more efficient selection methods  based on genomic information. Reverse transcript quantitative PCR (RT-qPCR) has  become the method of choice for gene expression analysis, but relative  expression analysis requires various reference genes, which must be stable  across various biological conditions. We sought suitable reference genes for  various tissues of cacao, especially developing seeds. Ten potential reference  genes were analyzed for stability at various stages of embryo development,  leaves, stems, roots, flowers, and pod epicarp; seven of them were also  evaluated in shoot tips treated either with hormones (salicylate; ethefon;  methyl-jasmonate) or after inoculation with the fungus Moniliophthora  perniciosa (Marasmiaceae sensu lato). For developing embryos, the  three most stable genes were actin (ACT), polyubiquitin (PUB), and ribosomal  protein L35 (Rpl35). In the analyses of various tissues, the most stable genes  were malate dehydrogenase (MDH), glyceraldehyde 3-phosphate dehydrogenase  (GAPDH), and acyl-carrier protein B (ACP B). GAPDH, MDH and tubulin (TUB) were  the most appropriate for normalization when shoot apexes were treated with  hormones, while ACT, TUB and Rpl35 were the most appropriate after inoculation  with M. perniciosa. We conclude that for each plant system and  biological or ontogenetical condition, there is a need to define suitable  reference genes. This is the first report to define reference genes for  expression studies in cacao.
authors.affiliation: Centro de Energia Nuclear na Agricultura, Universidade de São Paulo, Piracicaba, SP, Brasil.
authors.forename: T T
authors.initials: TT
authors.lastname: Pinheiro
authors.forename: C G
authors.initials: CG
authors.lastname: Litholdo
authors.forename: M L
authors.initials: ML
authors.lastname: Sereno
authors.forename: G A
authors.initials: GA
authors.lastname: Leal
authors.forename: P S B
authors.initials: PS
authors.lastname: Albuquerque
authors.forename: A
authors.initials: A
authors.lastname: Figueira
mesh_terms: D002099:Cacao / Q000235:genetics*; D020869:Gene Expression Profiling; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant / Q000235:genetics; D056726:Genetic Association Studies; D009928:Organ Specificity / Q000235:genetics*; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000592:standards*; D012984:Software