Abstract
Betula luminifera is a commercial tree species that is emerging as a new model system for tree genomics research. A draft genomic sequence is expected to be publicly available in the near future, which means that an explosion of gene expression studies awaits. Thus, the work of selecting appropriate reference genes for qPCR normalization in different tissues or under various experimental conditions is extremely valuable. In this study, ten candidate genes were analyzed in B. luminifera subjected to different abiotic stresses and at various flowering stages. The expression stability of these genes was evaluated using three distinct algorithms implemented using geNorm, NormFinder and BestKeeper. The best-ranked reference genes varied across different sample sets, though RPL39, MDH and EF1a were determined as the most stable by the three programs among all tested samples. RPL39 and EF1a should be appropriate for normalization in N-starved roots, while the combination of RPL39 and MDH should be appropriate for N-starved stems and EF1a and MDH should be appropriate in N-starved leaves. In PEG-treated (osmotic) roots, MDH was the most suitable, whereas EF1a was suitable for PEG-treated stems and leaves. TUA was also stably expressed levels in PEG-treated plants. The combination of RPL39 and TUB should be appropriate for heat-stressed leaves and flowering stage. For reference gene validation, the expression levels of SOD and NFYA-3 were investigated. This work will be beneficial to future studies on gene expression under different abiotic stress conditions and flowering status in B. luminifera.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Alscher RG, Erturk N, Heath LS (2002) Role of superoxide dismutases (SODs) in controlling oxidative stress in plants. J Exp Bot 53:1331–1341
Amil-Ruiz F, Garrido-Gala J, Blanco-Portales R, Folta KM, Munoz-Blanco J, Caballero JL (2013) Identification and validation of reference genes for transcript normalization in strawberry (Fragaria × ananassa) defense responses. PLoS One 8(8):e70603
Andersen CL, Jensen JL, Orntoft TF (2004) Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res 64:5245–5250
Bigelow SW, Canham CD (2007) Nutrient limitation of juvenile trees in a northern hardwood forest: calcium and nitrate are preeminent. For Ecol Manag 243:310–319
Brunner AM, Yakovlev IA, Strauss SH (2004) Validating internal controls for quantitative plant gene expression studies. BMC Plant Biol 4:14
Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55:611–622
Chandna R, Augustine R, Bisht NC (2012) Evaluation of Candidate Reference Genes for Gene Expression Normalization in Brassica juncea using real time quantitative RT-PCR. PLoS One 7(5):e36918
Chen L, Zhong HY, Kuang JF, Li JG, Lu WJ, Chen JY (2011) Validation of reference genes for RT-qPCR studies of gene expression in banana fruit under different experimental conditions. Planta 234:377–390
Cruz F, Kalaoun S, Nobile P, Colombo C, Almeida J, Barros LMG, Romano E, Grossi-de-Sa MF, Vaslin M, Alves-Ferreira M (2009) Evaluation of coffee reference genes for relative expression studies by quantitative real-time RT-PCR. Mol Breed 23:607–616
Czechowski T, Stitt M, Altmann T, Udvardi MK, Scheible WR (2005) Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis. Plant Physiol 139:5–17
de Carvalho K, Bespalhok JC, dos Santos TB, de Souza SGH, Vieira LGE, Pereira LFP, Domingues DS (2013) Nitrogen starvation, salt and heat stress in coffee (Coffea arabica L.): identification and validation of new genes for qPCR normalization. Mol Biotechnol 53:315–325
Ding JY, Jia JW, Yang LT, Wen HB, Zhang CM, Liu WX, Zhang DB (2004) Validation of a rice specific gene, sucrose phosphate synthase, used as the endogenous reference gene for qualitative and real-time quantitative PCR detection of transgenes. J Agric Food Chem 52:3372–3377
Guenin S, Mauriat M, Pelloux J, Van Wuytswinkel O, Bellini C, Gutierrez L (2009) Normalization of qRT-PCR data: the necessity of adopting a systematic, experimental conditions-specific, validation of references. J Exp Bot 60:487–493
Gutierrez L, Mauriat M, Guenin S, Pelloux J, Lefebvre JF, Louvet R, Rusterucci C, Moritz T, Guerineau F, Bellini C, Van Wuytswinkel O (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
Hong SM, Bahn SC, Lyu A, Jung HS, Ahn JH (2010) Identification and testing of superior reference genes for a starting pool of transcript normalization in Arabidopsis. Plant Cell Physiol 51:1694–1706
Huang HH, Jiang C, Tong ZK, Cheng LJ, Zhu MY, Lin EP (2014) Eight distinct cellulose synthase catalytic subunit genes from Betula luminifera are associated with primary and secondary cell wall biosynthesis. Cellulose 21:2183–2198
Jian B, Liu B, Bi YR, Hou WS, Wu CX, Han TF (2008) Validation of internal control for gene expression study in soybean by quantitative real-time PCR. BMC Mol Biol 9:1
Kong QS, Yuan JX, Niu PH, Xie JJ, Jiang W, Huang Y, Bie ZL (2014) Screening suitable reference genes for normalization in reverse transcription quantitative real-time PCR analysis in melon. PLoS One 9(1):e87197
Lovdal T, Lillo C (2009) Reference gene selection for quantitative real-time PCR normalization in tomato subjected to nitrogen, cold, and light stress. Anal Biochem 387:238–242
Mittler R, Blumwald E (2010) Genetic engineering for modern agriculture: challenges and perspectives. Annu Rev Plant Biol 61:443–462
Obrero A, Die JV, Roman B, Gomez P, Nadal S, Gonzalez-Verdejo CI (2011) Selection of reference genes for gene expression studies in Zucchini (Cucurbita pepo) using qPCR. J Agric Food Chem 59:5402–5411
Paolacci AR, Tanzarella OA, Porceddu E, Ciaffi M (2009) Identification and validation of reference genes for quantitative RT-PCR normalization in wheat. BMC Mol Biol 10:1
Park SC, Kim YH, Ji CY, Park S, Jeong JC, Lee HS, Kwak SS (2012) Stable internal reference genes for the normalization of real-time PCR in different sweetpotato cultivars subjected to abiotic stress conditions. PLoS One 7(12):e51502
Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29:e45
Pfaffl MW, Tichopad A, Prgomet C, Neuvians TP (2004) Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: bestKeeper—excel-based tool using pair-wise correlations. Biotechnol Lett 26:509–515
Podevin N, Krauss A, Henry I, Swennen R, Remy S (2012) Selection and validation of reference genes for quantitative RT-PCR expression studies of the non-model crop Musa. Mol Breed 30:1237–1252
Silveira ED, Alves-Ferreira M, Guimaraes LA, da Silva FR, Carneiro VT (2009) Selection of reference genes for quantitative real-time PCR expression studies in the apomictic and sexual grass Brachiaria brizantha. BMC Plant Biol 9:84
Udvardi MK, Czechowski T, Scheible WR (2008) Eleven golden rules of quantitative RT-PCR. Plant Cell 20:1736–1737
Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3(7):1
Wan HJ, Zhao ZG, Qian CT, Sui YH, Malik AA, Chen JF (2010) Selection of appropriate reference genes for gene expression studies by quantitative real-time polymerase chain reaction in cucumber. Anal Biochem 399:257–261
Yin H, Chen QM, Yi MF (2008) Effects of short-term heat stress on oxidative damage and responses of antioxidant system in Lilium longiflorum. Plant Growth Regul 54:45–54
Zhao M, Ding H, Zhu JK, Zhang F, Li WX (2011) Involvement of miR169 in the nitrogen-starvation responses in Arabidopsis. New Phytol 190:906–915
Zhong HY, Chen JW, Li CQ, Chen L, Wu JY, Chen JY, Lu WJ, Li JG (2011) Selection of reliable reference genes for expression studies by reverse transcription quantitative real-time PCR in litchi under different experimental conditions. Plant Cell Rep 30:641–653
Zhu J, Zhang L, Li W, Han S, Yang W, Qi L (2013) Reference gene selection for quantitative real-time PCR normalization in Caragana intermedia under different abiotic stress conditions. PLoS One 8:e53196
Author information
Authors and Affiliations
Corresponding authors
Additional information
Project funding
This work was financially supported by the National Natural Science Foundation of China (No. 31300566) and Zhejiang Province Science and Technology Support Program (No. 2012C12908-8).
The online version is available at http://www.springerlink.com
Corresponding Editor: Chai Ruihai
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Wu, J., Zhang, J., Pan, Y. et al. Identification and evaluation of reference genes for normalization in quantitative real-time PCR analysis in the premodel tree Betula luminifera . J. For. Res. 28, 273–282 (2017). https://doi.org/10.1007/s11676-016-0314-2
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11676-016-0314-2