Australasian Plant Pathology

, Volume 45, Issue 3, pp 261–268 | Cite as

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

  • Yee-Min Kwan
  • Sariah Meon
  • Chai-Ling Ho
  • Mui-Yun WongEmail author
Original Paper


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.


Reference genes Gene expression qRT-PCR Oil palm Ganoderma boninense 



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.


  1. 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 64:5245–5250CrossRefPubMedGoogle Scholar
  2. 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 55:4CrossRefGoogle Scholar
  3. 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, e36918CrossRefPubMedPubMedCentralGoogle Scholar
  4. Chung GF (2011) Management of Ganoderma diseases in oil palm plantations. Planter 87(1022):325–339Google Scholar
  5. Cooper RM, Flood J, Rees RW (2011) Ganoderma boninense in oil palm plantations: current thinking on epidemiology, resistance and pathology. The Planter 87:515–526Google Scholar
  6. 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 139:5–17CrossRefPubMedPubMedCentralGoogle Scholar
  7. 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 8:131CrossRefPubMedPubMedCentralGoogle Scholar
  8. 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):609–618CrossRefPubMedGoogle Scholar
  9. Hugget J, Dheda K, Bustin S, Zumla A (2005) Real-time RT-PCR normalization; strategies and considerations. Genes Immun 6:279–284CrossRefGoogle Scholar
  10. Hushiarian R, Yusof NA, Dutse SW (2013) Detection and control of Ganoderma boninense: strategies and perspectives. SpringerPlus 2:555CrossRefPubMedPubMedCentralGoogle Scholar
  11. Idris AS, Kushairi D, Ariffin D, Basri MW (2006) Technique for inoculation of oil palm germinated seeds with Ganoderma. MPOB Information Series 314Google Scholar
  12. Izzati NAMZ, Abdullah F (2008) Disease suppression in Ganoderma-infected oil palm seedlings treated with Trichoderma harzianum. Plant Prot Sci 44:101–107CrossRefGoogle Scholar
  13. Malaysian Palm Oil Board (MPOB) (2012) Overview of the Malaysian Oil Palm Industry 2012. Assessed Dec 2012
  14. 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 147:101–114CrossRefPubMedPubMedCentralGoogle Scholar
  15. 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 7:1723–1727Google Scholar
  16. 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 56:2907–2914CrossRefPubMedGoogle Scholar
  17. 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):201–212CrossRefGoogle Scholar
  18. Paterson RRM, Sariah M, Lima N (2012) How will climate change affect oil palm fungal diseases? Crop Prot 46:113–120CrossRefGoogle Scholar
  19. 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 26:509–515CrossRefPubMedGoogle Scholar
  20. 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 30:1237–1252CrossRefPubMedPubMedCentralGoogle Scholar
  21. 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 4:367–373CrossRefGoogle Scholar
  22. 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 56:862–870CrossRefGoogle Scholar
  23. Rozen S, Skaletsky H (2000) Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol 132:365–86PubMedGoogle Scholar
  24. 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 500:335–339CrossRefPubMedPubMedCentralGoogle Scholar
  25. 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 170:1455–1460CrossRefPubMedGoogle Scholar
  26. 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), e0117569CrossRefPubMedPubMedCentralGoogle Scholar
  27. Udvardi MK, Czechowski T, Scheible WR (2008) Eleven golden rules of quantitative RT-PCR. Plant Cell 20:1736–1737CrossRefPubMedPubMedCentralGoogle Scholar
  28. 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
  29. 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 184:63–73CrossRefPubMedGoogle Scholar
  30. 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 5:13908CrossRefPubMedPubMedCentralGoogle Scholar
  31. 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 31:1829–1843CrossRefPubMedGoogle Scholar
  32. 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 169:1565–70CrossRefPubMedGoogle Scholar
  33. 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 10:127–132Google Scholar
  34. 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 30:638–645CrossRefPubMedGoogle Scholar

Copyright information

© Australasian Plant Pathology Society Inc. 2016

Authors and Affiliations

  • Yee-Min Kwan
    • 1
  • Sariah Meon
    • 1
    • 2
  • Chai-Ling Ho
    • 1
    • 3
  • Mui-Yun Wong
    • 1
    • 2
    Email author
  1. 1.Laboratory of Plantation Crops, Institute of Tropical AgricultureUniversiti Putra MalaysiaSerdangMalaysia
  2. 2.Department of Plant Protection, Faculty of AgricultureUniversiti Putra MalaysiaSerdangMalaysia
  3. 3.Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular SciencesUniversiti Putra MalaysiaSerdangMalaysia

Personalised recommendations