Skip to main content
SpringerLink
Log in

Identification of suitable reference genes in Taxodium ‘Zhongshanshan’ under abiotic stresses

  • Original Article
  • Published:
Trees Aims and scope Submit manuscript

Abstract

Key message

This work provides a guideline for application of Taxodium ‘Zhongshanshan’ reference gene usage in the qRT-PCR system, and also for reference gene selection for other Taxodiaceae species.

Abstract

Quantitative real-time PCR (qRT-PCR) is a practical method which is frequently used for analysis of gene expression. However, many factors can affect the qRT-PCR analysis by influencing the quantitative measurement of the expression of genes. To avoid erroneous experimental analyses, it is necessary to select suitable reference genes for normalization of the variation. Taxodium ‘Zhongshanshan’ (T. ‘Zhongshanshan’) is an economically and ecologically important tree species in China for its extreme tolerance to several stresses. Although some responses of T. ‘Zhongshanshan’ to abiotic stresses have been studied, reference genes suitable for the normalization of qRT-PCR data under stresses have not previously been identified. Ten candidate reference genes were used to validate expression stability in this study, including actin (ACT), adenine phosphoribosyl transferase (APRT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), protein phosphatase 2A (PP2A), tubulin (TUB), 18S ribosomal RNA (18S rRNA), 25S ribosomal RNA (25S rRNA), ubiquitin extension protein (UBI), ubiquitin (UBQ), and SKP1/ASK-interacting protein (SKIP). To select appropriate reference genes, we selected a diverse set of samples representing different stress conditions. GeNorm and NormFinder were made to evaluate the result systematically. The results indicated that no single reference gene had an optimal performance across all the experimental systems, while, PP2A and APRT can be proposed as good starting points for gene-expression studies. The qRT-PCR analysis of pyruvate decarboxylase (PDC) expression patterns in response to half-waterlogging stress confirmed the reliability of selected stable reference genes. Our results have provided a guideline for application of T. ‘Zhongshanshan’ reference gene usage in the qRT-PCR system.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Barsalobres-Cavallari CF, Severino FE, Maluf MP, Maia IG (2009) Identification of suitable internal control genes for expression studies in Coffea arabica under different experimental conditions. BMC Mol Biol 10:1

    Article  PubMed  PubMed Central  Google Scholar 

  • Bertolde FZ, Almeida A-AF, Pirovani CP (2014) Analysis of gene expression and proteomic profiles of clonal genotypes from Theobroma cacao subjected to soil flooding. PLoS One 9:e108705

    Article  PubMed  PubMed Central  Google Scholar 

  • Borowski JM, Galli V, da Silva Messias R, Perin EC, Buss JH, e Silva SD, Rombaldi CV (2014) Selection of candidate reference genes for real-time PCR studies in lettuce under abiotic stresses. Planta 239:1187–1200

    CAS  PubMed  Google Scholar 

  • Campos MD, Frederico AM, Nothnagel T, Arnholdt-Schmitt B, Cardoso H (2015) Selection of suitable reference genes for reverse transcription quantitative real-time PCR studies on different experimental systems from carrot (Daucus carota L.). Sci Hortic 186:115–123

    Article  CAS  Google Scholar 

  • Chang E, Shi S, Liu J, Cheng T, Xue L, Yang X, Yang W, Lan Q, Jiang Z (2012) Selection of reference genes for quantitative gene expression studies in Platycladus orientalis (Cupressaceae) using real-time PCR. PLoS One 7:e33278

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Chen J, Li X, Wang D, Li L, Zhou H, Liu Z, Wu J, Wang P, Jiang X, Fabrice MR (2015a) Identification and testing of reference genes for gene expression analysis in pollen of Pyrus bretschneideri. Sci Hortic 190:43–56

    Article  CAS  Google Scholar 

  • Chen Y, Hu B, Tan Z, Liu J, Yang Z, Li Z, Huang B (2015b) Selection of reference genes for quantitative real-time PCR normalization in creeping bentgrass involved in four abiotic stresses. Plant Cell Rep 34:1825–1834

    Article  CAS  PubMed  Google Scholar 

  • Chen H, Yang Z, Hu Y, Tan J, Jia J, Xu H, Chen X (2016) Reference genes selection for quantitative gene expression studies in Pinus massoniana L. Trees 30:685–696

    Article  CAS  Google Scholar 

  • Costa JH, Saraiva KD, Morais VD, Oliveira JT, Sousa DO, de Melo DF, Morais JK, Vasconcelos IM (2016) Reference gene identification for real-time PCR analyses in soybean leaves under fungus (Cercospora kikuchii) infection and treatments with salicylic and jasmonic acids Australasian. Plant Pathol 45:191–199

    CAS  Google Scholar 

  • Czechowski T, Stitt M, Altmann T, Udvardi MK, Scheible W-R (2005) Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis. Plant Physiol 139:5–17

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • da Costa M, Duro N, Batista-Santos P, Ramalho JC, Ribeiro-Barros AI (2015) Validation of candidate reference genes for qRT-PCR studies in symbiotic and non-symbiotic Casuarina glauca Sieb. ex Spreng. under salinity conditions. Symbiosis 66:21–35

    Article  Google Scholar 

  • de Lima JC, de Costa F, Füller TN, da Silva Rodrigues-Corrêa KC, Kerber MR, Lima MS, Fett JP, Fett-Neto AG (2016) Reference genes for qPCR analysis in Resin-Tapped Adult Slash Pine as a tool to address the molecular basis of commercial resinosis. Front Plant Sci 7:849

    PubMed  PubMed Central  Google Scholar 

  • De Santis C, Smith-Keune C, Jerry DR (2011) Normalizing RT-qPCR data: are we getting the right answers? An appraisal of normalization approaches and internal reference genes from a case study in the finfish Lates calcarifer. Mar Biotechnol 13:170–180

    Article  CAS  PubMed  Google Scholar 

  • Die JV, Román B, Nadal S, González-Verdejo CI (2010) Evaluation of candidate reference genes for expression studies in Pisum sativum under different experimental conditions. Planta 232:145–153

    Article  CAS  PubMed  Google Scholar 

  • Gadkar VJ, Filion M (2015) Validation of endogenous reference genes in Buglossoides arvensis for normalizing RT-qPCR-based gene expression data. SpringerPlus 4:1

    Article  CAS  Google Scholar 

  • Galli V, Borowski JM, Perin EC, da Silva Messias R, Labonde J, dos Santos Pereira I, dos Anjos Silva SD, Rombaldi CV (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

    Article  CAS  PubMed  Google Scholar 

  • Gu C, Chen S, Liu Z, Shan H, Luo H, Guan Z, Chen F (2011) Reference gene selection for quantitative real-time PCR in Chrysanthemum subjected to biotic and abiotic stress. Mol Biotechnol 49:192–197

    Article  CAS  PubMed  Google Scholar 

  • Gu C, Song A, Zhang X, Wang H, Li T, Chen Y, Jiang J, Chen F, Chen S (2016) Cloning of chrysanthemum high-affinity nitrate transporter family (CmNRT2) and characterization of CmNRT2.1. Sci Rep 6:23462

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Guo F, Song Z, Sullivan L, Wang H, Liu X, Wang X, Li Z, Zhao Y (2015) Enhancing phytolith carbon sequestration in rice ecosystems through basalt powder amendment. Sci Bull 60:591–597

    Article  CAS  Google Scholar 

  • Han L, Han J, Yin Y, Yu C, Shi Q (2015) Purification effect of four Taxodium ‘Zhongshanshan’ varieties in entrophic water. Chin J Environ Eng 7:3311–3318 (in Chinese)

    Google Scholar 

  • Heid CA, Stevens J, Livak KJ, Williams PM (1996) Real time quantitative PCR. Genome Res 6:986–994

    Article  CAS  PubMed  Google Scholar 

  • Hua J, Yin Y, Zhou D, Yu C, Xu J (2011) Effects of soil water conditions on growth and physiology of Taxodium ‘Zhongshanshan 406’. EMG 20(8/9):1221–1225 (in Chinese)

    Google Scholar 

  • Huang L, Yan H, Jiang X, Yin G, Zhang X, Qi X, Zhang Y, Yan Y, Ma X, Peng Y (2014a) Identification of candidate reference genes in perennial ryegrass for quantitative RT-PCR under various abiotic stress conditions PloS One 9:e93724

    PubMed  Google Scholar 

  • Huang L, Yan H, Jiang X, Zhang X, Zhang Y, Huang X, Zhang Y, Miao J, Xu B, Frazier T (2014b) Evaluation of candidate reference genes for normalization of quantitative RT-PCR in switchgrass under various abiotic stress conditions. Bioenergy Res 7:1201–1211

    Article  CAS  Google Scholar 

  • Klie M, Debener T (2011) Identification of superior reference genes for data normalisation of expression studies via quantitative PCR in hybrid roses (Rosa hybrida). BMC Res Notes 4:1

    Article  Google Scholar 

  • Kong F, Cao M, Sun P, Liu W, Mao Y (2015) Selection of reference genes for gene expression normalization in Pyropia yezoensis using quantitative real-time PCR. J Appl Phycol 27:1003–1010

    Article  CAS  Google Scholar 

  • Kong Q, Gao L, Cao L, Liu Y, Saba H, Huang Y, Bie Z (2016) Assessment of suitable reference genes for quantitative gene expression studies in melon fruits. Front Plant Sci 7:1178

    PubMed  PubMed Central  Google Scholar 

  • Kozera B, Rapacz M (2013) Reference genes in real-time PCR. J Appl genet 54:391–406

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kumar G, Singh AK (2015) Reference gene validation for qRT-PCR based gene expression studies in different developmental stages and under biotic stress in apple. Sci Hortic 197:597–606

    Article  CAS  Google Scholar 

  • Kundu A, Patel A, Pal A (2013) Defining reference genes for qPCR normalization to study biotic and abiotic stress responses in Vigna mungo. Plant Cell Rep 32:1647–1658

    Article  CAS  PubMed  Google Scholar 

  • Kürsteiner O, Dupuis I, Kuhlemeier C (2003) The pyruvate decarboxylase1 gene of Arabidopsis is required during anoxia but not other environmental stresses. Plant Physiol 132:968–978

    Article  PubMed  PubMed Central  Google Scholar 

  • Ling H, Wu Q, Guo J, Xu L, Que Y (2014) Comprehensive selection of reference genes for gene expression normalization in sugarcane by real time quantitative RT-PCR. PLoS One 9:e97469

    Article  PubMed  PubMed Central  Google Scholar 

  • Liu M, Wu Z, Jiang F (2015a) Selection and validation of garlic reference genes for quantitative real-time PCR normalization. Plant Cell Tissue Organ Cult (PCTOC) 122:435–444

    Article  CAS  Google Scholar 

  • Liu W, Zhao T, Wang H, Zeng J, Xiang L, Zhu S, Chen M, Lan X, Liu X, Liao Z (2015b) Reference gene selection in Artemisia annua L., a plant species producing anti-malarial artemisinin. Plant Cell. Tissue Organ Cult (PCTOC) 121:141–152

    Article  Google Scholar 

  • Long X, He B, Gao X, Qin Y, Yang J, Fang Y, Qi J, Tang C (2015) Validation of reference genes for quantitative real-time PCR during latex regeneration in rubber tree. Gene 563:190–195

    Article  CAS  PubMed  Google Scholar 

  • Maroufi A, Van Bockstaele E, De Loose M (2010) Validation of reference genes for gene expression analysis in chicory (Cichorium intybus) using quantitative real-time PCR. BMC Mol Biol 11:15

    Article  PubMed  PubMed Central  Google Scholar 

  • Meng Y, Li N, Tian J, Gao J, Zhang C (2013) Identification and validation of reference genes for gene expression studies in postharvest rose flower (Rosa hybrida). Sci Hortic 158:16–21

    Article  CAS  Google Scholar 

  • Monteiro F, Sebastiana M, Pais MS, Figueiredo A (2013) Reference gene selection and validation for the early responses to downy mildew infection in susceptible and resistant Vitis vinifera cultivars. PLoS One 8:e72998

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Nicot N, Hausman J-F, Hoffmann L, Evers D (2005) Housekeeping gene selection for real-time RT-PCR normalization in potato during biotic and abiotic stress. J Exp Bot 56:2907–2914

    Article  CAS  PubMed  Google Scholar 

  • Niu L, Tao Y-B, Chen M-S, Fu Q, Li C, Dong Y, Wang X, He H, Xu Z-F (2015a) Selection of reliable reference genes for gene expression studies of a promising oilseed crop, Plukenetia volubilis, by real-time quantitative PCR. Int J Mol Sci 16:12513–12530

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Niu X, Qi J, Zhang G, Xu J, Tao A, Fang P, Su J (2015b) Selection of reliable reference genes for quantitative real-time PCR gene expression analysis in Jute (Corchorus capsularis) under stress treatments. Front Plant Sci 6:848

    Article  PubMed  PubMed Central  Google Scholar 

  • Peng Y-L, Wang Y-S, Gu J-D (2015) Identification of suitable reference genes in mangrove Aegiceras corniculatum under abiotic stresses. Ecotoxicology 24:1714–1721

    Article  CAS  PubMed  Google Scholar 

  • Qi B, Yang Y, Yin Y, Xu M, Li H (2014) De novo sequencing, assembly, and analysis of the Taxodium ‘Zhongshansa’roots and shoots transcriptome in response to short-term waterlogging. BMC Plant Biol 14:1

    Article  Google Scholar 

  • Rocha M, Licausi F, Araujo WL, Nunes-Nesi A, Sodek L, Fernie AR, van Dongen JT (2010) Glycolysis and the tricarboxylic acid cycle are linked by alanine aminotransferase during hypoxia induced by waterlogging of Lotus japonicus. Plant Physiol 152:1501–1513

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sinha P, Singh VK, Suryanarayana V, Krishnamurthy L, Saxena RK, Varshney RK (2015) Evaluation and validation of housekeeping genes as reference for gene expression studies in pigeonpea (Cajanus cajan) under drought stress conditions. PLoS One 10:e0122847

    Article  PubMed  PubMed Central  Google Scholar 

  • 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 3:1

    Article  Google Scholar 

  • Vandesompele J, Kubista M, Pfaffl MW (2009) Reference gene validation software for improved normalization. Real-time PCR: current technology and applications, vol 47. Caister Academic Press, Norfolk, p 64

    Google Scholar 

  • Wan H, Zhao Z, Qian C, Sui Y, Malik AA, Chen J (2010) Selection of appropriate reference genes for gene expression studies by quantitative real-time polymerase chain reaction in cucumber. Anal Biochem 399:257–261

    Article  CAS  PubMed  Google Scholar 

  • Wei L, Miao H, Zhao R, Han X, Zhang T, Zhang H (2013) Identification and testing of reference genes for Sesame gene expression analysis by quantitative real-time PCR. Planta 237:873–889

    Article  CAS  PubMed  Google Scholar 

  • Xu Y, Li H, Li X, Lin J, Wang Z, Yang Q, Chang Y (2015) Systematic selection and validation of appropriate reference genes for gene expression studies by quantitative real-time PCR in pear. Acta Physiol Plant 37:1–16

    Article  Google Scholar 

  • Yi S, Qian Y, Han L, Sun Z, Fan C, Liu J, Ju G (2012) Selection of reliable reference genes for gene expression studies in Rhododendron micranthum. Turcz Scientia Horticulturae 138:128–133

    Article  CAS  Google Scholar 

  • You-Xiong Q, Zhi-Xia Y, Li-Ping X, Ru-Kai C (2009) Isolation and identification of differentially expressed genes in sugarcane infected by Ustilago scitaminea. Acta Agron Sin 35:452–458

    Google Scholar 

  • Yu C, Xu S, Yin Y (2016) Transcriptome analysis of the Taxodium ‘Zhongshanshan 405’ roots in response to salinity stress. Plant Physiol Biochem 100:156–165

    Article  CAS  PubMed  Google Scholar 

  • Zhang Z, Hu J (2007) Development and validation of endogenous reference genes for expression profiling of medaka (Oryzias latipes) exposed to endocrine disrupting chemicals by quantitative real-time RT-PCR. Toxicol Sci 95:356–368

    Article  CAS  PubMed  Google Scholar 

  • Zhang Y, Zhang J, Wu X, Wang J, Duan F (2015) Flooding tolerance of Taxodium hybrid ‘Zhongshanshan’ along the hydro-fluctuation belt of the Three Gorges Reservoir. Sci Soil Water Conserv 13(2):56–62 (in Chinese)

    Google Scholar 

  • Zhang J-Y, Huang S-N, Wang G, Xuan J-P, Guo Z-R (2016) Overexpression of Actinidia deliciosa pyruvate decarboxylase 1 gene enhances waterlogging stress in transgenic Arabidopsis thaliana. Plant Physiol Biochem 106:244–252

    Article  CAS  PubMed  Google Scholar 

  • Zhuang H, Fu Y, He W, Wang L, Wei Y (2015) Selection of appropriate reference genes for quantitative real-time PCR in Oxytropis ochrocephala Bunge using transcriptome datasets under abiotic stress treatments. Front Plant Sci 6:475

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Author information

Author notes

  1. Yunlong Yin and Fangyuan Yu contributed equally to this work.

Authors and Affiliations

  1. Collaborative Innovation Center of Sustainable Forestry in Southern China, College of Forest Sciences, Nanjing Forestry University, Nanjing, Jiangsu, China

    Zhiquan Wang, Yunlong Yin & Fangyuan Yu

  2. Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, Nanjing, Jiangsu, China

    Zhiquan Wang, Chunsun Gu, Lei Xuan, Jianfeng Hua, Qin Shi, Wencai Fan & Yunlong Yin

Authors
  1. Zhiquan Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Chunsun Gu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Lei Xuan
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jianfeng Hua
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Qin Shi
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Wencai Fan
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Yunlong Yin
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Fangyuan Yu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Yunlong Yin or Fangyuan Yu.

Ethics declarations

Funding

This work was supported by the National Natural Science Foundation of China (No. 31570593), the Program of Innovation Capacity Construction of Jiangsu Province (No. BM 2015019), the Natural Science Foundation of Jiangsu Province (No. BK 20150551), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), and the Jiangsu Agriculture Science and Technology Innovation Fund (JASTIF) (No. CX(16)1005).

Conflict of interest

The research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The authors did not receive payment or services from a third party for any aspect of the submitted work.

Additional information

Communicated by F. Canovas.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 58 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Wang, Z., Gu, C., Xuan, L. et al. Identification of suitable reference genes in Taxodium ‘Zhongshanshan’ under abiotic stresses. Trees 31, 1519–1530 (2017). https://doi.org/10.1007/s00468-017-1566-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00468-017-1566-y

Keywords

Search

Navigation