Identification and evaluation of internal control genes for gene expression studies by real-time quantitative PCR normalization in different tissues of Tuberose (Polianthes tuberosa)

Quantitative real time PCR has become the most popular method to study the gene expression due to its accuracy. However the expression level of the target gene may be misunderstood due to the unstable expression of the reference genes under different experimental conditions. Therefore, it is a prerequisite to identify and validate the reference genes in each species for gene expression analysis. Polianthes tuberosa, a very popular commercial flowering crop globally, lacks information on any such reference genes. In this study, we describe the first systematic evaluation of four conventional candidate reference genes; 18S ribosomal RNA (18S rRNA), Ribulose bisphosphate (RuBP), Glyceraldehyde 3 phosphate dehydrogenase (GAPDH), Actin and four novel genes; Coatomer subunit delta (CSD), Peptidyl-prolyl isomerase (PPI), Serine/threonine-protein phosphatase, (STPP) and ATP synthase E-subunit (ATP SE) in tuberose. The transcript abundance of these genes was analyzed in eleven different tissues like young leaf, leaf sheath, root, immature flower bud, mature flower bud, open flower, stamen, ovary, stigma, petals and flower tube. Three RT-qPCR statistical analysis methods, BestKeeper, NormFinder and geNorm were used to evaluate the stability of gene expression that indicated expression of PPI and CSD to be most stable across all the tested tissues. The stability of these two genes was also confirmed across four popular commercial varieties and under biotic and abiotic stresses. Utility of PPI in tuberose as a reference gene is a pioneer report in plants whereas, usefulness of CSD as a stable reference gene has been demonstrated for the first time in a crop besides the model plant, Arabidiopsis. (C) 2015 Elsevier B.V. All rights reserved.