Selection and validation of reference genes for normalization of qRT-PCR gene expression in wheat (Triticum durum L.) under drought and salt stresses
- 79 Downloads
Eight candidate housekeeping genes were examined as internal controls for normalizing expression analysis of durum wheat (Triticum durum L.) under drought and salinity stress conditions. Quantitative real-time PCR was used to analyse gene expression of multiple stress levels, plant ages (24 and 50 days old), and plant tissues (leaf and root). The algorithms BestKeeper, NormFinder, GeNorm, the delta Ct method and the RefFinder were applied to determine the stability of candidate genes. Under drought stress, the most stable reference genes were glyceraldehyde-3 phosphate, ubiquitin and \(\beta \)-tubulin2, whereas under salinity stress conditions, eukaryotic elongation factor 1-\(\alpha \), glyceraldehyde-3 phosphate and actin were identified as the most stable reference genes. Validation with stress-responsive genes NAC29 and NAC6 demonstrated that the expression level of target genes could be determined reliably with combinations of up to three of the reference genes. This is the first report on reference genes appropriate for quantification of target gene expression in T. durum under drought and salt stresses. Results of this investigation may be applicable to other Triticum species.
Keywordsdrought stress housekeeping genes quantitative real-time PCR salt stress transcription factors Triticum durum
We are grateful to Dr Ali Hadipour, Dr Arman Salehi and Dr Negar Salehi for editing this paper and especially Mrs Sheryl Nikpoor and Navid Jamshidi for their valuable comments. Also, we are thankful to anonymous referees who helped us to improve our paper.
- Brennan J. P., Aw-Hassan A., Quade K. J. and Nordblom T. L. 2002 Impact of ICARDA research on Australian agriculture. Econ. Res. Rep. 11, NSW Agriculture, Wagga Wagga.Google Scholar
- Galli V., Borowski J. M., Perin E. C., da Silva Messias R., Labonde J., dos Santos Pereira I. et al. 2015 Validation of reference genes for accurate normalization of gene expression for real time-quantitative PCR in strawberry fruits using different cultivars and osmotic stresses. Gene 554, 205–214.CrossRefGoogle Scholar
- Gutierrez L., Mauriat M., Guénin S., Pelloux J., Lefebvre J. F., Louvet R. et al. 2008 The lack of a systematic validation of reference genes: a serious pitfall undervalued in reverse transcription-polymerase chain reaction (RT-PCR) analysis in plants. Plant Biotechnol. J. 6, 609–618.CrossRefGoogle Scholar
- Vandesompele J., De Preter K., Pattyn F., Poppe B., Van Roy N., De Paepe A. et al. 2002 Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 3 ( https://doi.org/10.1186/gb-2002-3-7-research0034).