Evaluation of the Use of the Polyubiquitin Genes, Ubi4 and Ubi10 as Reference Genes for Expression Studies in Brachypodium distachyon

Abstract

Background Brachypodium distachyon is emerging as the model plant for temperate grass research and the genome of the community line Bd21 has been sequenced. Additionally, techniques have been developed for Agrobacterium-mediated transformation for the generation of T-DNA insertional lines. Recently, it was reported that expression of the polyubiquitin genes, Ubi4 and Ubi10 are stable in different tissues and growth hormone-treated plant samples, leading to the conclusion that both Ubi4 and Ubi10 are good reference genes for normalization of gene expression data using real-time, quantitative PCR (qPCR).Principal FindingsMining of the Joint Genome Institute (JGI) 8X Brachypodium distachyon genome assembly showed that Ubi4 and Ubi10 share a high level of sequence identity (89%), and in silico analyses of the sequences of Ubi4 (Bradi3g04730) and Ubi10 (Bradi1g32860) showed that the primers used previously exhibit multiple binding sites within the coding sequences arising from the presence of tandem repeats of the coding regions. This can potentially result in over-estimation of steady-state levels of Ubi4 and Ubi10. Additionally, due to the high level of sequence identity between both genes, primers used previously for amplification of Ubi4 can bind to Ubi10 and vice versa, resulting in the formation of non-specific amplification products.ConclusionsThe results from this study indicate that the primers used previously were not sufficiently robust and specific. Additionally, their use would result in over-estimation of the steady-state expression levels of Ubi4. Our results question the validity of using the previously proposed primer sets for qPCR amplification of Ubi4 and Ubi10. We demonstrate that primers designed to target the 3′-UTRs of Ubi4 and Ubi10 are better suited for real-time normalization of steady-state expression levels in Brachypodium distachyon.

Expression profiling of marker genes responsive to the defence-associated phytohormones salicylic acid, jasmonic acid and ethylene in Brachypodium distachyon

“…Gene expression analyses were performed by qRT-PCR using a KAPA SYBR Fast qPCR Kit (KAPA BIOSYSTEMS, Woburn, MA, USA) with a GVP-9600 real-time PCR instrument (Shimadzu, Kyoto, Japan). The quantification of target transcripts was performed using the GVP-9600 internal software GVP gene detection system, and the data were normalised to the BdUbi4 gene ( Bradi3g04730 ), which has been established as a reference gene for expression studies in B. distachyon [ 59 ]. Primers used in this study are listed in Additional file 2 : Table S1.…”

Identification and validation of quantitative real-time reverse transcription PCR reference genes for gene expression analysis in teak (Tectona grandis L.f.)

“…New genes have been studied as internal controls in model or commercial plants, such as Arabidopsis [15], Populus [16] and Brachypodium [17]. Tests for the selection of reference genes for qRT-PCR in teak have not been published yet.…”

Genome-wide selection of superior reference genes for expression studies in Ganoderma lucidum

“…distachon (Chambers et al, 2012). Based on the G. lucidum genome and transcriptome database, 38 candidate reference genes including 28 novel genes were systematically selected and evaluated for qRT-PCR normalization.…”

“…We firstly collected 21 targeted genes based on the common reference genes in previous studies (Cao et al, 2012;Chambers et al, 2012;Chao et al, 2012;Czechowski et al, 2005;Han et al, 2012;Hong et al, 2008;Hu et al, 2009;Lin et al, 2013;Marum et al, 2012;Xu et al, 2012;Xu et al, 2014;Zhu et al, 2012;). Then, candidate reference genes, including homologous genes, were selected from the G. lucidum genome and transcriptome database using the Tblastx algorithm with e-value cut-off (10-5) (Chen et al, 2012).…”

“…However, the unsuitability of the reference gene in qRT-PCR has yielded unverified gene analysis results that can generate confusion. The high sequence homology and poor primer specificity of reference genes, such as the polyubiquitin genes (Ubi4 and Ubi10) in Brachypodium distachon, resulted in unsuitability for qRT-PCR (Chambers et al, 2012). Although many evaluation studies have been performed in animals, plants, and fungi, such as Arabidopsis thaliana (Czechowski et al, 2005), B. distachon (Hong et al, 2008), Carica papaya L. (Zhu et al, 2012), Quercus suber (Marum et al, 2012), Vernicia fordii Hemsl.…”