title: Validation of reference genes for accurate normalization of gene expression for real time-quantitative PCR in strawberry fruits using different cultivars and osmotic stresses.
abstract: The increasing demand of strawberry (Fragaria×ananassa Duch) fruits is associated mainly with their sensorial characteristics and the content of antioxidant compounds. Nevertheless, the strawberry production has been hampered due to its sensitivity to abiotic stresses. Therefore, to understand the molecular mechanisms highlighting stress response is of great importance to enable genetic engineering approaches aiming to improve strawberry tolerance. However, the study of expression of genes in strawberry requires the use of suitable reference genes. In the present study, seven traditional and novel candidate reference genes were evaluated for transcript normalization in fruits of ten strawberry cultivars and two abiotic stresses, using RefFinder, which integrates the four major currently available software programs: geNorm, NormFinder, BestKeeper and the comparative delta-Ct method. The results indicate that the expression stability is dependent on the experimental conditions. The candidate reference gene DBP (DNA binding protein) was considered the most suitable to normalize expression data in samples of strawberry cultivars and under drought stress condition, and the candidate reference gene HISTH4 (histone H4) was the most stable under osmotic stresses and salt stress. The traditional genes GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and 18S (18S ribosomal RNA) were considered the most unstable genes in all conditions. The expression of phenylalanine ammonia lyase (PAL) and 9-cis epoxycarotenoid dioxygenase (NCED1) genes were used to further confirm the validated candidate reference genes, showing that the use of an inappropriate reference gene may induce erroneous results. This study is the first survey on the stability of reference genes in strawberry cultivars and osmotic stresses and provides guidelines to obtain more accurate RT-qPCR results for future breeding efforts.
authors.affiliation: Embrapa Clima Temperado, Rodovia BR 396, Km 78 Caixa Postal 403, CEP 96001-970 Pelotas, RS, Brazil; Universidade Federal de Pelotas, Faculdade de Agronomia Eliseu Maciel, Campus Universitário s/n, Caixa Postal 354, CEP 96010-900 Pelotas, RS, Brazil. Electronic address: vane.galli@yahoo.com.br.
authors.forename: Vanessa
authors.initials: V
authors.lastname: Galli
authors.affiliation: Embrapa Clima Temperado, Rodovia BR 396, Km 78 Caixa Postal 403, CEP 96001-970 Pelotas, RS, Brazil; Universidade Federal de Pelotas, Faculdade de Agronomia Eliseu Maciel, Campus Universitário s/n, Caixa Postal 354, CEP 96010-900 Pelotas, RS, Brazil.
authors.forename: Joyce Moura
authors.initials: JM
authors.lastname: Borowski
authors.affiliation: Embrapa Clima Temperado, Rodovia BR 396, Km 78 Caixa Postal 403, CEP 96001-970 Pelotas, RS, Brazil; Universidade Federal de Pelotas, Faculdade de Agronomia Eliseu Maciel, Campus Universitário s/n, Caixa Postal 354, CEP 96010-900 Pelotas, RS, Brazil.
authors.forename: Ellen Cristina
authors.initials: EC
authors.lastname: Perin
authors.affiliation: Embrapa Clima Temperado, Rodovia BR 396, Km 78 Caixa Postal 403, CEP 96001-970 Pelotas, RS, Brazil; Universidade Federal de Pelotas, Faculdade de Agronomia Eliseu Maciel, Campus Universitário s/n, Caixa Postal 354, CEP 96010-900 Pelotas, RS, Brazil.
authors.forename: Rafael da Silva
authors.initials: Rda S
authors.lastname: Messias
authors.affiliation: Universidade Federal de Pelotas, Centro de Ciências Biológicas, Campus Universitário s/n, Caixa Postal 354, CEP 96010-900 Pelotas, RS, Brazil.
authors.forename: Julia
authors.initials: J
authors.lastname: Labonde
authors.affiliation: Embrapa Clima Temperado, Rodovia BR 396, Km 78 Caixa Postal 403, CEP 96001-970 Pelotas, RS, Brazil.
authors.forename: Ivan dos Santos
authors.initials: Idos S
authors.lastname: Pereira
authors.affiliation: Embrapa Clima Temperado, Rodovia BR 396, Km 78 Caixa Postal 403, CEP 96001-970 Pelotas, RS, Brazil.
authors.forename: Sérgio Delmar Dos Anjos
authors.initials: SD
authors.lastname: Silva
authors.affiliation: Universidade Federal de Pelotas, Faculdade de Agronomia Eliseu Maciel, Campus Universitário s/n, Caixa Postal 354, CEP 96010-900 Pelotas, RS, Brazil.
authors.forename: Cesar Valmor
authors.initials: CV
authors.lastname: Rombaldi
mesh_terms: D019295:Computational Biology; D031985:Fragaria / Q000145:classification / Q000235:genetics*; D020869:Gene Expression Profiling; D018506:Gene Expression Regulation, Plant; D009997:Osmotic Pressure; D010940:Plant Proteins / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012015:Reference Standards; D012984:Software; D013312:Stress, Physiological
chemical_list: D010940:Plant Proteins
keywords: Abiotic stresses; Fragaria×ananassa; RefFinder; Reference genes; Strawberry cultivars