Transcriptome-based identification and validation of optimal reference genes for quantitative real-time PCR normalization in Psathyrostachys huashanica

Abstract

Background: P. huashanica ( Psathyrostachys huashanica ), known as an important resistance resource reservoir, is a rare and endangered plant growing suitably in Huashan mount region and would be urgently exploited in wheat genetic improvements sooner. During the utilization process, different IRGs (internal reference genes) need to be appropriately selected as standards based on biotic and abiotic stress conditions. It is crucial that Real-time RT-qPCR with combination of bioinformatics were adopted to explore the reliable IRGs from transcriptome of P . huashanica.Results: The present work reported new 3 species of IRGs, UBC2 , UBC17, 18S rRNA , which were screened from transcriptome of P. huashanica under biotic and abiotic stress conditions, using RT-qPCR and four algorithms, including geNorm, NormFinder, BestKeeper, and RefFinder, to analyse expression of sixteen candidate reference genes. These genes appear as following 18S rRNA (18S ribosomal RNA), EF1-α (eukaryotic elongation factor 1 alpha), UBC2 (ubiquitin-conjugating enzyme E2-2), UBC17 (ubiquitin-conjugating enzyme E2-17), α-TUB2A (alpha tubulin-2A), β-TUB3 (beta tubulin 3), ADF4 (Actin-depolymerising factor 4), ACTIN (actin), GAPDH (Glyceraldehyde-3-phosphate dehydrogenase), 60SARP (60S acidic ribosomal protein), UBQ (polyubiquitin), SamDC (S-Adenosylmethionine decarboxylase), EIF4A (eukaryotic initiation factor 4A), ARF (ADP-ribosylation factor), HIS1 (histone H1), and HIS2B (histone H2B). Analysis of gene expression demonstrated that the expression of UBC2 gene was most stable under ABA hormone stress, low temperature stress and high temperature stress, similarly, UBC17 gene under IAA hormone stress, salinity stress and drought stress, both UBC17 genes and 18S rRNA genes under abiotic and biotic stress, respectively. The most stable gene was UBC2 gene in the root, UBC17 gene in stem and leaf. In this study, α-TUB2A , UBC and ACTIN genes were verified as the suitable reference genes across all tested samples. To further validate the suitability of the selected reference genes, we evaluated the relative expression of PsaCPK3 (Calcium-dependent protein kinase) and PsaHSP70-1 (heat shock protein 70-1), which are stress-related genes that may be involved in response to adversity.Conclusions: This study has identified a set of the most stable IRGs suiting for RT-qPCR detection of a few target gene expressions from P . huashanica in different experimental conditions. In addition, this study should provide the accuracy information for gene expression analysis in P . huashanica .