title: Quantitative real-time PCR normalization for gene expression studies in the plant pathogenic fungi Lasiodiplodia theobromae.
abstract: Lasiodiplodia theobromae is a highly virulent plant pathogen. It has been suggested that heat stress increases its virulence. The aim of this work was to evaluate, compare, and recommend normalization strategies for gene expression analysis of the fungus growing with grapevine wood under heat stress. Using RT-qPCR-derived data, reference gene stability was evaluated through geNorm, NormFinder and Bestkeeper applications. Based on the geometric mean using the ranking position obtained for each independent analysis, genes were ranked from least to most stable as follows: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), β-tubulin (TUB) and elongation factor-1α (EF1α). Using RNAseq-derived data based on the calculated tagwise dispersion these genes were ordered by increasing stability as follows: GAPDH, ACT, TUB, and EF1α. The correlation between RNAseq and RTqPCR results was used as criteria to identify the best RT-qPCR normalization approach. The gene TUB is recommended as the best option for normalization among the commonly used reference genes, but alternative fungal reference genes are also suggested.
authors.affiliation: Departamento de Microbiología, Centro de Investigación Científica y de Educación Superior de Ensenada (CICESE), Ensenada, Baja California 22860, Mexico. Electronic address: marcos_paolinelli@yahoo.com.ar.
authors.forename: Marcos
authors.initials: M
authors.lastname: Paolinelli-Alfonso
authors.affiliation: Departamento de Biotecnología Marina, Centro de Investigación Científica y de Educación Superior de Ensenada (CICESE), Ensenada, Baja California 22860, Mexico. Electronic address: cgalindo@cicese.mx.
authors.forename: Clara Elizabeth
authors.initials: CE
authors.lastname: Galindo-Sánchez
authors.affiliation: Departamento de Microbiología, Centro de Investigación Científica y de Educación Superior de Ensenada (CICESE), Ensenada, Baja California 22860, Mexico. Electronic address: ruhernan@cicese.mx.
authors.forename: Rufina
authors.initials: R
authors.lastname: Hernandez-Martinez
mesh_terms: D001203:Ascomycota / Q000235:genetics*; D020869:Gene Expression Profiling / Q000379:methods; D005800:Genes, Fungal; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D010935:Plant Diseases / Q000382:microbiology*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D014404:Tubulin / Q000235:genetics; D027843:Vitis / Q000382:microbiology*
chemical_list: D014404:Tubulin; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases
keywords: Botryosphaeria dieback; Botryosphaeria rhodina; Botryosphaeriaceae; Fungal RNA extraction; Trunk disease fungi; Vitis vinifera