Skip to main content

Advertisement

SpringerLink
Log in

Selection of reference genes for quantitative real-time PCR normalization in Ganoderma-infected oil palm (Elaeis guineensis) seedlings

  • Original Paper
  • Published:
Australasian Plant Pathology Aims and scope Submit manuscript

Abstract

African oil palm (Elaeis guineensis) is an important oil bearing tree commercially cultivated in Malaysia. Palm oil is an important product for local consumption, provides enormous socio-economic benefits of trade and employment opportunities, and fulfilling the growing global demand for vegetable oils. The monoculture system has fostered the outbreak of basal stem rot (BSR) disease caused by the fungus Ganoderma boninense. Quantitative real-time PCR (qRT-PCR) is a widely used molecular technique to examine the infection effect on gene expression in oil palm. The selection of appropriate reference genes is vital for accurate data normalization. In this study, the expression stability of six housekeeping genes- β-actin, cyclophilin, GAPDH, MSD, NAD and ubiquitin were validated in oil palm root tissue after fungal infection. NormFinder and BestKeeper algorithms were used to cross-validate the expression stability of the candidate reference genes. MSD, NAD and ubiquitin were shown to exhibit the highest expression stability. These genes were recommended as reference genes for gene expression studies of oil palm root tissue at early fungal infection stage.

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

Access this article

Log in via an institution

Subscribe and save

Springer+ Basic
$34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

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

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2

Similar content being viewed by others

References

  • Andersen C, Jensen J, Ørntoft T (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

    Article  PubMed  CAS  Google Scholar 

  • 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 55:4

    Article  CAS  Google Scholar 

  • 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, e36918

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Chung GF (2011) Management of Ganoderma diseases in oil palm plantations. Planter 87(1022):325–339

    Google Scholar 

  • Cooper RM, Flood J, Rees RW (2011) Ganoderma boninense in oil palm plantations: current thinking on epidemiology, resistance and pathology. The Planter 87:515–526

  • Czechowski T, Stitt M, Altman 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

    Article  PubMed  PubMed Central  CAS  Google Scholar 

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

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Gutierrez L, Mauriat M, Guénin S, Pelloux J, Lefebvre JF, Louvet R, Rusterucci C, Moritz T, Guerineau F, Bellini C, Wuytswinkel OV (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(6):609–618

    Article  PubMed  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  • Hushiarian R, Yusof NA, Dutse SW (2013) Detection and control of Ganoderma boninense: strategies and perspectives. SpringerPlus 2:555

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Idris AS, Kushairi D, Ariffin D, Basri MW (2006) Technique for inoculation of oil palm germinated seeds with Ganoderma. MPOB Information Series 314

  • Izzati NAMZ, Abdullah F (2008) Disease suppression in Ganoderma-infected oil palm seedlings treated with Trichoderma harzianum. Plant Prot Sci 44:101–107

    Article  Google Scholar 

  • Malaysian Palm Oil Board (MPOB) (2012) Overview of the Malaysian Oil Palm Industry 2012. http://bepi.mpob.gov.my. Assessed Dec 2012

  • Morgan MJ, Lehmann M, Schwarzländer M, Baxter CJ, Sienkiewicz-Porzucek A, Williams TCR, Schacver N, Fricker MD, Ratcliffe RG, Sweetlove LJ, Finkemeier I (2008) Decrease in manganese superoxide dismutase leads to reduced root growth and affects tricarboxylic acid cycle flux and mitochondrial redox homeostasis. Plant Physiol 147:101–114

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Naher L, Yusuf UK, Ismail A, Tan SG, Mondal MMA (2013) Ecological status of Ganoderma and basal stem rot disease of oil palms (Elaeis guineensis Jacq.). Aust J Crop Sci 7:1723–1727

    Google Scholar 

  • Nicot N, Hausman JF, 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  PubMed  CAS  Google Scholar 

  • Ooi S, Choo C, Ishak Z, Abdullah MO (2012) A candidate auxin-responsive expression marker gene, EgIAA9, for somatic embryogenesis in oil palm (Elaeis guineensis Jacq.). Plant Cell Tiss Org Cult 110(2):201–212

    Article  CAS  Google Scholar 

  • Paterson RRM, Sariah M, Lima N (2012) How will climate change affect oil palm fungal diseases? Crop Prot 46:113–120

    Article  Google Scholar 

  • Pfaffl M, Tichopad A, Prgomet C, Neuvians T (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

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Ransbotyn V, Reusch TB (2006) Housekeeping gene selection for quantitative real-time PCR assays in the seagrass Zostera marina subjected to heat stress. Limnol Oceanogr Methods 4:367–373

    Article  CAS  Google Scholar 

  • Rees RW, Flood J, Hasan Y, Cooper RM (2007) Effects of inoculum potential, shading and soil temperature on root infection of oil palm seedlings by the basal stem rot pathogen Ganoderma boninense. Plant Pathol 56:862–870

    Article  Google Scholar 

  • Rozen S, Skaletsky H (2000) Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol 132:365–86

    PubMed  CAS  Google Scholar 

  • Singh R, Ong-Abdullah M, Low ET, Manaf MA, Rosli R, Nookiah R, Ooi LC, Ooi SE, Chan KL, Halim MA, Azizi N, Nagappan J, Bacher B, Lakey N, Smith SW, He D, Hogan M, Budiman MA, Lee EK, Desalle R, Kudrna D, Goicoechea JL, Wing RA, Wilson RK, Fulton RS, Ordway JM, Martienssen RA, Sambanthamurthi R (2013) Oil palm genome sequence reveals divergence of interfertile species in old and new worlds. Nature 500:335–339

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Tan YC, Yeoh KA, Wong MY, Ho CL (2013) Expression profiles of putative defense-related proteins in oil palm (Elaeis guineensis) colonized by Ganoderma boninense. J Plant Physiol 170:1455–1460

    Article  PubMed  CAS  Google Scholar 

  • Tian C, Jiang Q, Wang F, Wang GL, Xu ZS, Xiong AS (2015) Selection of suitable reference genes for qPCR normalization under abiotic stresses and hormone stimuli in carrot leaves. PLoS ONE 10(2), e0117569

    Article  PubMed  PubMed Central  CAS  Google Scholar 

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

    Article  PubMed  PubMed Central  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:research0034.1-0034.11. doi:10.1186/gb-2002-3-7-research0034

  • Xia W, Mason AS, Xiao Y, Liu Z, Yang Y, Lei X, Wu X, Ma Z, Peng M (2014) Analysis of multiple transcriptomes of the African oil palm (Elaeis guineensis) to identify reference genes for RT-qPCR. J Biotechnol 184:63–73

    Article  PubMed  CAS  Google Scholar 

  • Yamori W, Shikanai T, Makino A (2015) Photosystem I cyclic electron flow via chloroplast NADH dehydrogenase-like complex performs a physiological role for photosynthesis at low light. Sci Rep 5:13908

    Article  PubMed  PubMed Central  Google Scholar 

  • Yeap WC, Ooi TEK, Namasivayam P, Kulaveerasingam H, Ho CL (2012) EgRBP42 encoding an hnRNP-like RNA-binding protein from Elaeis guineensis Jacq. is responsive to abiotic stresses. Plant Cell Rep 31:1829–1843

    Article  PubMed  CAS  Google Scholar 

  • Yeoh KA, Othman A, Meon S, Abdullah F, Ho CL (2012) Sequence analysis and gene expression of putative exo- and endo-glucanases from oil palm (Elaeis guineensis) during fungal infection. J Plant Physiol 169:1565–70

    Article  PubMed  CAS  Google Scholar 

  • Zaiton S, Sariah M, Zainal AMA (2008) Effect of endophytic bacteria on growth and suppression of Ganoderma infection in oil palm. Int J Agric Biol 10:127–132

    Google Scholar 

  • Zheng W, Sun L (2011) Evaluation of housekeeping genes as references for quantitative real time RT-PCR analysis of gene expression in Japanese flounder (Paralichthys olivaceus). Fish Shellfish Immunol 30:638–645

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

The authors would like to acknowledge the Ministry of Science, Technology and Innovation, Malaysia for funding this research via ScienceFund (Project number 02-01-04-SF1769). Yee-Min Kwan was supported by scholarship from the Ministry of Higher Education, Malaysia. We would also like to thank the GanoDROP Laboratory of Malaysian Palm Oil Board (MPOB) for providing Ganoderma boninense PER71 culture.

Author information

Authors and Affiliations

  1. Laboratory of Plantation Crops, Institute of Tropical Agriculture, Universiti Putra Malaysia, Selangor Darul Ehsan, Serdang, 43400, Malaysia

    Yee-Min Kwan, Sariah Meon, Chai-Ling Ho & Mui-Yun Wong

  2. Department of Plant Protection, Faculty of Agriculture, Universiti Putra Malaysia, Selangor Darul Ehsan, Serdang, 43400, Malaysia

    Sariah Meon & Mui-Yun Wong

  3. Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, Selangor Darul Ehsan, Serdang, 43400, Malaysia

    Chai-Ling Ho

Authors
  1. Yee-Min Kwan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sariah Meon
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Chai-Ling Ho
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Mui-Yun Wong
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Mui-Yun Wong.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kwan, YM., Meon, S., Ho, CL. et al. Selection of reference genes for quantitative real-time PCR normalization in Ganoderma-infected oil palm (Elaeis guineensis) seedlings. Australasian Plant Pathol. 45, 261–268 (2016). https://doi.org/10.1007/s13313-016-0417-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s13313-016-0417-4

Keywords

Search

Navigation