Identification of stable reference gene for transcript normalization in black pepper- 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 ( L.) leaf transcriptome were used as reference genes namely actin (), glyceraldehyde phosphate dehydrogenase (), β-tubulin (), ubiquitin conjugating enzyme (), 18sr and elongation factor-1-α () to identify the stable reference gene. We focused the selection of stable reference gene on important biotic stress () with different algorithms (geNorm, NormFinder and BestKeeper) along with Reffinder which resulted in identification of and 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 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 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 among resistant and susceptible genotypes showed as the suitable reference gene for . -. pathosystem.