Skip to main content

Advertisement

SpringerLink
Log in

Validation of reference genes for gene expression analysis in Valsa mali var. mali using real-time quantitative PCR

  • Original Paper
  • Published:
World Journal of Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

Valsa mali var. mali (Vmm), is the predominant species of apple valsa canker in China. Modern analysis of genes involved in virulence or pathogenicity usually implicate gene expression analysis most often performed using real-time quantitative polymerase chain reaction (RT-qPCR). However, for relative gene expression analysis pertinent reference genes have to be validated before using them as internal reference. This has not been reported for Vmm, so far. Therefore, eight commonly used housekeeping genes (ACT, CYP, EF1-α, G6PDH, GAPDH, L13, TUB, and UBQ) were cloned and evaluated for their expression stability by geNorm and NormFinder. Overall, all of the candidate reference genes were found to be suitable for gene expression analysis. After analysis of 10 samples from different strains and abiotic stress treatments, G6PDH appeared to be the most suitable reference gene, whereas GAPDH was the least suitable. Moreover, taking G6PDH combined with L13 or CYP as reference genes, improved the reliability of RT-qPCR significantly. The influence of the reference system on expression data was demonstrated by analyzing Vmmpg-1 encoding an endo-polygalacturonase gene. Pectinases are considered key pathogenicity factors for this fungus. In order to better understand the role of pectinases in pathogenicity of Vmm, RT-qPCR was used for expression analysis. Our results may provide a guideline for future studies on gene expression of V. mali var. mali by using RT-qPCR.

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

  • Abe K, Kotoda N, Kato H, Soejima J (2007) Resistance sources to valsa canker (Valsa ceratosperma) in a germplasm collection of diverse Malus species. Plant Breeding 126(4):449–453

    Article  Google Scholar 

  • Altschul Gish SF, Miller W, Myers W, Lipman EW, David J (1990) Basic local alignment search tool. J Mol Biol 215(3):403–410

    CAS  Google Scholar 

  • Andersen CL, Jensen JL, Ørntoft 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(15):5245

    Article  CAS  Google Scholar 

  • Bohle K, Jungebloud A, Göcke Y, Dalpiaz A, Cordes C, Horn H, Hempel DC (2007) Selection of reference genes for normalisation of specific gene quantification data of Aspergillus niger. J Biotechnol 132(4):353–358

    Article  CAS  Google Scholar 

  • Bustin S (2002) Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. J Mol Endocrinol 29(1):23–39

    Article  CAS  Google Scholar 

  • Bustin S, Dorudi S (1998) Molecular assessment of tumour stage and disease recurrence using PCR-based assays. Mol Med Today 4(9):389–396

    Article  CAS  Google Scholar 

  • Expósito-Rodríguez M, Borges A, Borges-Pérez A, Pérez J (2008) Selection of internal control genes for quantitative real-time RT-PCR studies during tomato development process. BMC Plant Biol 8(1):131

    Article  Google Scholar 

  • Feng H, Huang X, Zhang Q, Wei G, Wang X, Kang Z (2012) Selection of suitable inner reference genes for relative quantification expression of microRNA in wheat. Plant Physiol Biochem 51:116–122

    Article  CAS  Google Scholar 

  • Frohman MA, Dush MK, Martin GR (1988) Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc Natl Acad Sci USA 85(23):8998–9002

    Article  CAS  Google Scholar 

  • Gachon C, Mingam A, Charrier B (2004) Real-time PCR: what relevance to plant studies? J Exp Bot 55(402):1445–1454

    Article  CAS  Google Scholar 

  • Glare E, Divjak M, Bailey M, Walters E (2002) β-Actin and GAPDH housekeeping gene expression in asthmatic airways is variable and not suitable for normalising mRNA levels. Thorax 57(9):765–770

    Article  CAS  Google Scholar 

  • Hoondal G, Tiwari R, Tewari R, Dahiya N, Beg Q (2002) Microbial alkaline pectinases and their industrial applications: a review. Appl Microbiol Biotechnol 59(4):409–418

    CAS  Google Scholar 

  • Huggett J, Dheda K, Bustin S, Zumla A (2005) Real-time RT-PCR normalisation; strategies and considerations. Genes Immun 6(4):279–284

    Article  CAS  Google Scholar 

  • Ke X, Huang L, Han Q, Gao X, Kang Z (2013) Histological and cytological investigations of the infection and colonization of apple bark by Valsa mali var. mali. Australas Plant Pathol 42(1):85–93

    Article  Google Scholar 

  • Lee JM, Roche JR, Donaghy DJ, Thrush A, Sathish P (2010) Validation of reference genes for quantitative RT-PCR studies of gene expression in perennial ryegrass (Lolium perenne L.). BMC Mol Biol 11(1):8

    Article  Google Scholar 

  • Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25(4):402–408

    Article  CAS  Google Scholar 

  • Mariño-Ramírez L, Demidenko NV, Logacheva MD, Penin AA (2011) Selection and validation of reference genes for quantitative real-time PCR in buckwheat (Fagopyrum esculentum) based on transcriptome sequence data. PLoS ONE 6(5):e19434

    Article  Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15(3):473–497

    Article  CAS  Google Scholar 

  • Nailis H, Coenye T, Van Nieuwerburgh F, Deforce D, Nelis HJ (2006) Development and evaluation of different normalization strategies for gene expression studies in Candida albicans biofilms by real-time PCR. BMC Mol Biol 7(1):25

    Article  Google Scholar 

  • 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):11

    Article  Google Scholar 

  • Ruan W, Lai M (2007) Actin, a reliable marker of internal control? Clin Chim Acta 385(1–2):1–5

    Article  CAS  Google Scholar 

  • Sakuma T (1990) Valsa canker. In: Jones AL, Aldwinckle HS (eds) Compendium of apple and pear disease. American Phytopathological Society Press, St Paul, MN, USA, pp 39–40

    Google Scholar 

  • Steiger MG, Mach RL, Mach-Aigner AR (2010) An accurate normalization strategy for RT-qPCR in Hypocrea jecorina (Trichoderma reesei). J Biotechnol 145(1):30–37

    Article  CAS  Google Scholar 

  • Svircev A, Biggs A, Miles N (1991) Isolation and partial purification of phytotoxins from liquid cultures of Leucostoma cincta and Leucostoma persoonii. Can J Bot 69(9):1998–2003

    Article  CAS  Google Scholar 

  • Tamura O, Saito I (1982) Histopathological changes of apple bark infected by Valsa ceratosperma (Tode ex Fr.) maire during dormant and growing periods. Ann Phytopath Soc Japan 48(4):490–498

    Article  Google Scholar 

  • Teste M-A, Duquenne M, François JM, Parrou J-L (2009) Validation of reference genes for quantitative expression analysis by real-time RT-PCR in Saccharomyces cerevisiae. BMC Mol Biol 10(1):99

    Article  Google Scholar 

  • Thellin O, Zorzi W, Lakaye B, De Borman B, Coumans B, Hennen G, Grisar T, Igout A, Heinen E (1999) Housekeeping genes as internal standards: use and limits. J Biotechnol 75(2):291–295

    Article  CAS  Google Scholar 

  • Tong Z, Gao Z, Wang F, Zhou J, Zhang Z (2009) Selection of reliable reference genes for gene expression studies in peach using real-time PCR. BMC Mol Biol 10(1):71

    Article  Google Scholar 

  • Traquair J (1987) Oxalic acid and calcium oxalate produced by Leucostoma cincta and L. persoonii in culture and in peach bark tissues. Can J Bot 65(9):1952–1956

    Article  CAS  Google Scholar 

  • Udvardi MK, Czechowski T, Scheible WR (2008) Eleven golden rules of quantitative RT-PCR. Plant Cell 20(7):1736–1737

    Article  CAS  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(7):research0034

    Article  Google Scholar 

  • Wang X, Wei J, Huang L, Kang Z (2011) Re-evaluation of pathogens causing valsa canker on apple in China. Mycologia 103(2):317–324

    Article  Google Scholar 

Download references

Acknowledgments

This study was financially supported by the National Natural Science Foundation of China (NO.31171796 and NO.31101476), the Program for Agriculture (nyhyzx201203034-03) and the 111 Project (B07049). Special thanks are due to Dr. H. Buchenauer and Dr. Xiaoyu Qiang for correcting the manuscript.

Author information

Authors and Affiliations

  1. State Key Laboratory of Crop Stress Biology for Arid Areas, Northwest A&F University, Yangling, 712100, China

    Zhiyuan Yin, Xiwang Ke, Dingxuan Huang, Xiaoning Gao, Zhensheng Kang & Lili Huang

  2. Innovation Experimental College, Northwest A&F University, Yangling, 712100, China

    Zhiyuan Yin

  3. College of Plant Protection, Northwest A&F University, Yangling, 712100, China

    Xiwang Ke, Dingxuan Huang, Xiaoning Gao, Zhensheng Kang & Lili Huang

  4. Institut für Phytomedizin, Universität Hohenheim, Stuttgart, Germany

    Ralf T. Voegele

Authors
  1. Zhiyuan Yin
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Xiwang Ke
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Dingxuan Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Xiaoning Gao
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ralf T. Voegele
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Zhensheng Kang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Lili Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Lili Huang.

Additional information

Zhiyuan Yin and Xiwang Ke contributed equally to this work.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Yin, Z., Ke, X., Huang, D. et al. Validation of reference genes for gene expression analysis in Valsa mali var. mali using real-time quantitative PCR. World J Microbiol Biotechnol 29, 1563–1571 (2013). https://doi.org/10.1007/s11274-013-1320-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11274-013-1320-6

Keywords

Search

Navigation