title: Reference gene selection for quantitative real-time PCR in Solanum lycopersicum L. inoculated with the mycorrhizal fungus Rhizophagus irregularis.
abstract: The gene expression stability of candidate reference genes in the roots and leaves of Solanum lycopersicum inoculated with arbuscular mycorrhizal fungi was investigated. Eight candidate reference genes including elongation factor 1 α (EF1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (PGK), protein phosphatase 2A (PP2Acs), ribosomal protein L2 (RPL2), β-tubulin (TUB), ubiquitin (UBI) and actin (ACT) were selected, and their expression stability was assessed to determine the most stable internal reference for quantitative PCR normalization in S. lycopersicum inoculated with the arbuscular mycorrhizal fungus Rhizophagus irregularis. The stability of each gene was analysed in leaves and roots together and separated using the geNorm and NormFinder algorithms. Differences were detected between leaves and roots, varying among the best-ranked genes depending on the algorithm used and the tissue analysed. PGK, TUB and EF1 genes showed higher stability in roots, while EF1 and UBI had higher stability in leaves. Statistical algorithms indicated that the GAPDH gene was the least stable under the experimental conditions assayed. Then, we analysed the expression levels of the LePT4 gene, a phosphate transporter whose expression is induced by fungal colonization in host plant roots. No differences were observed when the most stable genes were used as reference genes. However, when GAPDH was used as the reference gene, we observed an overestimation of LePT4 expression. In summary, our results revealed that candidate reference genes present variable stability in S. lycopersicum arbuscular mycorrhizal symbiosis depending on the algorithm and tissue analysed. Thus, reference gene selection is an important issue for obtaining reliable results in gene expression quantification.
authors.affiliation: Laboratorio Biorremediacion, Departamento de Ciencias Forestales, Facultad de Ciencias Agropecuarias y Forestales, Universidad de La Frontera, Temuco 4811230, Chile; Programa de Doctorado en Ciencias Mención Biología Celular y Molecular Aplicada, Universidad de la Frontera, Temuco 4811230, Chile.
authors.forename: Alejandra
authors.initials: A
authors.lastname: Fuentes
authors.affiliation: Laboratorio Biorremediacion, Departamento de Ciencias Forestales, Facultad de Ciencias Agropecuarias y Forestales, Universidad de La Frontera, Temuco 4811230, Chile.
authors.forename: Javier
authors.initials: J
authors.lastname: Ortiz
authors.affiliation: Center of Molecular Biology and Pharmacogenetics, Scientific and Technological Bioresource Nucleus (BIOREN), Universidad de La Frontera, Temuco 4811230, Chile.
authors.forename: Nicolás
authors.initials: N
authors.lastname: Saavedra
authors.affiliation: Center of Molecular Biology and Pharmacogenetics, Scientific and Technological Bioresource Nucleus (BIOREN), Universidad de La Frontera, Temuco 4811230, Chile.
authors.forename: Luis A
authors.initials: LA
authors.lastname: Salazar
authors.affiliation: Centro de Biotecnología Vegetal. Universidad Andres Bello, Republica 217, Santiago, Chile.
authors.forename: Claudio
authors.initials: C
authors.lastname: Meneses
authors.affiliation: Laboratorio Biorremediacion, Departamento de Ciencias Forestales, Facultad de Ciencias Agropecuarias y Forestales, Universidad de La Frontera, Temuco 4811230, Chile. Electronic address: cesar.arriagada@ufrontera.cl.
authors.forename: Cesar
authors.initials: C
authors.lastname: Arriagada
mesh_terms: D020508:Fungi, Unclassified / Q000378:metabolism*; D018506:Gene Expression Regulation, Plant / Q000502:physiology*; D018551:Solanum lycopersicum / Q000378:metabolism*; D038821:Mycorrhizae / Q000378:metabolism*; D010940:Plant Proteins / Q000096:biosynthesis*; D060888:Real-Time Polymerase Chain Reaction; D013559:Symbiosis / Q000502:physiology*
chemical_list: D010940:Plant Proteins
keywords: Arbuscular mycorrhizal fungi; Reference genes; qPCR