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Identification of stable reference gene for transcript normalization in black pepper-Phytophthora capsici pathosystem


A systematic validation of reference genes is a pre-requisite for the proper normalization of gene transcripts. In the present study, the annotated sequences from black pepper (Piper nigrum L.) leaf transcriptome were used as reference genes namely actin (PnACT), glyceraldehyde phosphate dehydrogenase (PnGAPDH), β-tubulin (PnTUB), ubiquitin conjugating enzyme (PnUBCE), 18srRNA and elongation factor-1-α (PnElF) to identify the stable reference gene. We focused the selection of stable reference gene on important biotic stress (Phytophthora) with different algorithms (geNorm, NormFinder and BestKeeper) along with Reffinder which resulted in identification of PnGAPDH and PnUBCE as stable genes. Norm qPCR (R package) was also used to estimate the stability of the selected genes. We elucidated the expression patterns of a target gene PnBGLU which codes for 1,3 beta glucanase with most stable as well as least stable reference genes by which the importance of selecting the stable gene for gene expression studies in this system was emphasized. The mean expression levels of PnBGLU was significantly overestimated and misinterpreted when least stable reference gene was used as normalizer. The selected reference genes on further analysis of the expression dynamics of PnBGLU among resistant and susceptible genotypes showed PnGAPDH as the suitable reference gene for P. nigrumP. capsici pathosystem.

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  1. Anandaraj M, Sharma YR (1990) A simple baiting technique to detect and isolate Phytophthora capsici (P. palmivora MF4) from soil. Mycol Res 94:1003–1004

    Article  Google Scholar 

  2. 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

    Article  CAS  PubMed  Google Scholar 

  3. Bustin SA, Benes V, Garson JA, Hellemans J, Kubista M, Mueller R, Nolan T, Pfaffl M, 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

    Article  CAS  PubMed  Google Scholar 

  4. Egea C, Dickinson MJ, Candela M, Candela ME (1999) β-1,3-glucanase isoenzymes and genes in resistant and susceptible pepper (Capsicum annuum) cultivars infected with Phytophthora capsici. Physiol Plant 107:312–318

    Article  CAS  Google Scholar 

  5. Gordo SMC, Pinheiro DG, Moreira ECO, Rodrigues SM, Poltronieri MC, DeLemos OF, Da Silva IT, Ramos RTJ, Silva A, Schneider H, Silva WA, Sampaio I, Darnet S (2012) High throughput sequencing of black pepper root transcriptome. BMC Plant Biol 12:168

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Hao C, Xia Z, Fan R, Tan L, Hu L, Wu B, Wu H (2016) De novo transcriptome sequencing of black pepper (Piper nigrum L.) and an analysis of genes involved in phenylpropanoid metabolism in response to Phytophthora capsici. BMC Genom 17:822

    Article  CAS  Google Scholar 

  7. Hu L, Hao C, Fan R, Wu B, Tan L, Wu H (2015) De novo assembly and characterization of fruit transcriptome in black pepper (Piper nigrum). PLoS ONE 10(6):e0129822.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Jacob F, Guertler R, Naim S, Nixdorf S, Fedier A, Hacker NF, Heinzelmann-Schwarz V (2013) Careful selection of reference genes is required for reliable performance of RT-qPCR in human normal and cancer cell lines. PLoS ONE 8:e5918

    Article  Google Scholar 

  9. Jebakumar SR, Anandaraj M, Sarma YR (2001) Induction of PR proteins and defense related enzymes in black pepper due to inoculation with Phytophthora capsici. Ind Phytopathol 54:23–28

    CAS  Google Scholar 

  10. Johnson KG, Vijeshkumar IP, Anandaraj M (2012) Transcriptomics approaches for gene discovery in plants—a case study in Piper. Agrotechnology 1:2

    Google Scholar 

  11. Joy N, Asha S, Mallika V, Soniya EV (2013) De novo transcriptome sequencing reveals a considerable bias of simple sequence repeats towards the downstream of ‘Pre-miRNAs’ of black pepper. PLoS ONE 8:e56694

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Mahadevan C, Krishnan A, Saraswathy GG, Surendran A, Jaleel A, Sakuntala M (2016) Transcriptome-assisted label-free quantitative proteomics analysis reveals novel insights into Piper nigrumPhytophthora capsici Phytopathosystem. Front Plant Sci 7:785

    Article  PubMed  PubMed Central  Google Scholar 

  14. Nazeem PA, Achuthan CR, Babu TD, Parabu GV, Girija D, Keshavachandran R, Samiyappan R (2008) Expression of pathogenesis related proteins in black pepper (Piper nigrum L.) in relation to Phytophthora foot rot disease. J Trop Agric 46:45–51

    CAS  Google Scholar 

  15. Pawłowicz I, Kosmala A, Rapacz M (2012) Expression pattern of the psbO gene and its involvement in acclimation of the photosynthetic apparatus during abiotic stresses in Festuca arundinacea and F. pratensis. Acta Physiol Plant 34:1915–1924

    Article  CAS  Google Scholar 

  16. Perkins JR, McMahon SB, Bennett DLH, Orengo C, Kohl M (2012) ReadqPCR and NormqPCR: R packages for the reading, quality checking and normalisation of RT-qPCR quantification cycle (Cq) data. BMC Genom 13:296

    Article  CAS  Google Scholar 

  17. Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29:e45

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. 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

    Article  CAS  PubMed  Google Scholar 

  19. Saikia R, Singh BP, Kumar R, Arora DK (2005) Detection of pathogenesis-related proteins–chitinase and beta-1, β-glucanase in induced chickpea. Curr Sci 89:659–663

    CAS  Google Scholar 

  20. Sou YY, Leung DWM (2001) Elevation of extracellular β-1,3-glucanase and chitinase activities in rose in response to treatment with acibenzolar-S-methyl and infection by D. rosae. J Plant Physiol 158:971–976

    Article  Google Scholar 

  21. 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 Bio 3:RESEARCH0034

    Google Scholar 

  22. Wakelin AM, Leung DWM (2009) β-1,3-glucanase activity in the stigma of healthy petunia flowers. Biol Plant 53:69–74

    Article  CAS  Google Scholar 

  23. Zhang YL, Li DW, Gong ZH, Wang JE, Yin YX, Ji JJ (2013) Genetic determinants of the defense response of resistant and susceptible pepper (Capsicum annuum) cultivats infected with Phytophthora capsici (Oomycetes; Pythiaceae). Genet Mol Res 12:3605–3621

    Article  CAS  PubMed  Google Scholar 

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The authors are thankful to Mrs. Rosana Babu for primer designing, Mr. Jayarajan for statistical analysis. The authors extend their gratitude to Indian Council of Agriculture Research (ICAR) for financial support through Outreach programme on Phytophthora, Fusarium and Ralstonia diseases of horticultural and field crops (PhytoFuRa).

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Correspondence to P. Umadevi.

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Supplementary Figure 1

Agarose gel showing the amplification of reference genes: (1) PnACT (2) PnGAPDH (3) PnTUB (4) Pn18S, (5) PnUBCE (6) PnE1F (PNG 127 kb)

Supplementary Figure 2

Box plot graphs of Cq values for each reference genes tested at different time points after inoculation with P. capsici (TIFF 239 kb)

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Umadevi, P., Suraby, E.J., Anandaraj, M. et al. Identification of stable reference gene for transcript normalization in black pepper-Phytophthora capsici pathosystem. Physiol Mol Biol Plants 25, 945–952 (2019).

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  • Biotic stress
  • Gene expression
  • Housekeeping genes
  • Validation