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Identification of superior reference genes for data normalisation of expression studies via quantitative PCR in hybrid roses (Rosa hybrida)

DOI: 10.1186/1756-0500-4-518

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We used three different algorithms (BestKeeper, geNorm and NormFinder) to validate the expression stability of nine candidate reference genes in different rose tissues from three different genotypes of Rosa hybrida and in leaves treated with various stress factors. The candidate genes comprised the classical "housekeeping genes" (Actin, EF-1α, GAPDH, Tubulin and Ubiquitin), and genes showing stable expression in studies in Arabidopsis (PP2A, SAND, TIP and UBC). The programs identified no single gene that showed stable expression under all of the conditions tested, and the individual rankings of the genes differed between the algorithms. Nevertheless the new candidate genes, specifically, PP2A and UBC, were ranked higher as compared to the other traditional reference genes. In general, Tubulin showed the most variable expression and should be avoided as a reference gene.Reference genes evaluated as suitable in experiments with Arabidopsis thaliana were stably expressed in roses under various experimental conditions. In most cases, these genes outperformed conventional reference genes, such as EF1-α and Tubulin. We identified PP2A, SAND and UBC as suitable reference genes, which in different combinations may be used for normalisation in expression analyses via qPCR for different rose tissues and stress treatments. However, the vast genetic variation found within the genus Rosa, including differences in ploidy levels, might also influence expression stability of reference genes, so that future research should also consider different genotypes and ploidy levels.Roses are one of the economically most important ornamentals worldwide. They are produced as cut and potted plants and garden and landscaping plants with a production value of 24 billion Euros from 1995 to 2007 [1]. Other, less prominent uses include medicinal applications or the consumption in teas and soups [2]. Apart from the beauty of their flowers, roses are also admired for their delicate scent. Their scent


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