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Abstract Real-time quantitative PCR(qPCR) has been widely used in gene expression analysis to date, and selection of suitable reference genes for qPCR according to specific experimental materials or conditions is very important for accurate normalization of target gene expression. However, only a few studies on reference genes have been done in plants. The present research here was conducted to identify appropriate reference gene(s) for normalization in future expression studies on tree peony (Paeonia suffruticosa Andr.). In this study, expression of five commonly used housekeeping genes such as β-tubulin, ubiquitin, glyceraldehyde- 3-phosphate dehydrogenase(GAPDH), Actin and 18S ribsomal RNA(18S rRNA) were assessed by qPCR in various tissues(roots, stems, leaves and petals) of tree peony and the petals of this cut flower during different opening phases or under different treatments(with ethylene or glucose). All the candidate reference genes could amplify specifically with high efficiency in qPCR reaction. To determine the expression stability of these genes, qPCR data were determined by two commonly used applets(geNorm and NormFinder), which produced highly comparable results depending on the experimental parameters. According to the analysis of geNorm program, the widely used reference genes ubiquitin and GAPDH were top ranked under all above conditions, which demonstrated they were both in the highest stability, whereas others including β-tubulin, actin and 18S rRNA were poorly ranked. Similarly, the results from NormFinder showed that ubiquitin was the most stable gene in the first two experimental conditions, while GAPDH was the most stable one in the petals of cut flower. As a result, for various tissues, as well as petals at different opening stages of tree peony cut flower, ubiquitin could be chosen as the proper reference gene, while compared to differences of gene expression in petals of this cut flower among diverse treatments, GAPDH was considered as the most appropriate reference gene. Besides, given using a normalization factor that was the combination of multiple housekeeping genes, the analysis recommended two most suitable reference genes (ubiquitin and GAPDH) were enough to achieve more accurate and reliable results among all investigated experimental conditions. The present study has provided an important reference for analysis the expression of critical genes in tree peony.
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Received: 13 December 2011
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