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. nigrum–P. capsici pathosystem.
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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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Agarose gel showing the amplification of reference genes: (1) PnACT (2) PnGAPDH (3) PnTUB (4) Pn18S, (5) PnUBCE (6) PnE1F (PNG 127 kb)
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). https://doi.org/10.1007/s12298-019-00653-9
- Biotic stress
- Gene expression
- Housekeeping genes