Identification of stable reference gene for transcript normalizationin black pepper-Phytophthora capsicipathosystem

Abstract

A systematic validation of reference genes is apre-requisite for the proper normalization of gene trancscripts. In the present study, the annotated sequences from black pepper (Piper nigrum L.) leaf transcriptome were used as reference genes namely actin (PnACT), glycer-aldehyde phosphate dehydrogenase (PnGAPDH), b-tubulin (PnTUB), ubiquit in conjugating enzyme (PnUBCE),18srRNAand elongation factor-1-a(PnElF) to identify the stable reference gene. We focused the selection ofstable reference gene on important biotic stress (Phytophthora) with different algorithms (geNorm, Norm Finder and Best Keeper) along with Reffinder which resulted in identification of PnGAPDH and PnUBCEas stable genes.Norm qPCR (R package) was also used to estimate thestability of the selected genes. We elucidated the expression patterns of a target gene PnBGLU which codes for 1,3beta glucanase with most stable as well as least stable reference genes by which the importance of selecting thestable gene for gene expression studies in this system wasemphasized. 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 PnBGLUamong resistant and susceptible genotypes showed Pn GAPDHas the suitable reference gene for P.nigrum – P.capsici pathosystem.