Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 14, 110
eCollection

Reference Genes Identification for Normalization of qPCR Under Multiple Stresses in Hordeum brevisubulatum

Affiliations

Reference Genes Identification for Normalization of qPCR Under Multiple Stresses in Hordeum brevisubulatum

Lili Zhang et al. Plant Methods.

Abstract

Background: Real-time quantitative PCR has been widely used as the most reliable method to measure gene expression, due to its high accuracy and specificity. Wild barley (Hordeum brevisubulatum (Trin.) Link) is a wild relative species in Triticeae that has strong tolerance to abiotic stresses and extremely wide adaptation. However, suitable references gene have not been documented for standardization of gene expression in wild barley under abiotic stress.

Results: Here we report the first systematic and comprehensive analysis of reference genes for quantitative real-time PCR standardization in wild barley. We selected 11 genes, including ACT (Actin), ADP (ADP-ribosylation factor 1), CYP2 (Cyclophilin 2), EF- (Elongation factor 1-alpha), GAPDH (Glyceraldehyde 3-phosphate dehydrogenase), HSP90 (Heat shock protein 90), TUBα (Alpha-tubulin), TUBβ6 (Beta-tubulin 6), UBI (Ubiquitin), 18SrRNA-1 (guanine1575-N7-methyltransferase) and 18SrRNA-3 (adenine1779-N6-dimethyltransferase) from a wild barley transcriptome database and analyzed their expression stabilities in shoots and roots of wild barley seedling under various stress conditions using comparative ΔCt, BestKeeper, Normfinder and geNorm software. The results demonstrated that ADP was the most suitable reference gene in salt stress while UBI showed peak stability under mannitol and ABA stress; EF- was the most appropriate reference gene for PEG, GA3, ethylene and heat stress; 18SrRNA-3 was the best choice for cold stress; and TUBα was the first stable gene across different tissues.

Conclusions: Our main contribution was to identify reference genes with suitable and stable expression in wild barley under various stress conditions and in different tissues to provide a useful resource for future studies. The results demonstrate the importance of transcriptome data as a useful resource for the screening of candidate reference genes and highlight the need for specific reference genes for specific conditions. Furthermore, these findings will provide valuable information for wild barley and relative species for future research.

Keywords: Abiotic stress; Hordeum brevisubulatum; Quantitative real-time PCR; Reference gene.

Figures

Fig. 1
Fig. 1
Specificity of each candidate reference gene primer pair. a Confirmation of the specificity of qPCR primer amplification of candidate reference genes by agarose gel electrophoresis. b Melting curve analysis of quantitative real-time PCR (qRT-PCR) amplification of 11 candidate reference genes in H. brevisubulatum
Fig. 2
Fig. 2
Comparative analysis of Ct values of 11 reference genes in shoot and root tissue under normal conditions. The data represent the mean ± SD
Fig. 3
Fig. 3
Boxplot showing the variation in CT values of candidate reference genes in different treatments and tissues. a 350 mM NaCl treatment, b 10% PEG6000 treatment, c 350 mM mannitol treatment, d 20 μM ABA treatment, e 100 μM GA3 treatment, f 100 μM ethylene treatment, g 4 °C cold stress, and h 42 °C heat stress. The boxes indicate the first and third quartile, while the middle line marks the median, and points represent the average. The whisker caps show the distribution of the highest and lowest CT values, and the farther points represent outliers
Fig. 4
Fig. 4
Aggregation of four algorithmic rankings of 11 candidate reference genes under various stress treatments and tissues in H. brevisubulatum. a 350 mM NaCl treatment, b 10% PEG6000 treatment, c 350 mM mannitol treatment, d 20 μM ABA treatment, e 100 μM GA3 treatment, f 100 μM ethylene treatment, g 4 °C cold stress, h 42 °C heat stress, and i shoot and root tissues
Fig. 5
Fig. 5
Ranking values of comprehensive gene stability of 11 candidate reference genes under various stress treatments and tissues in H. brevisubulatum based on the Geomean method of RefFinder and measured across a 350 mM NaCl treatment, b 10% PEG6000 treatment, c 350 mM mannitol treatment, d 20 μM ABA treatment, e 100 μM GA3 treatment, f 100 μM ethylene treatment, g 4 °C cold stress, h 42 °C heat stress, and i shoot and root tissues
Fig. 6
Fig. 6
Pairwise variation (Vn/Vn + 1) analysis of the number of candidate reference genes in H. brevisubulatum under various stress treatments and tissues. Pairwise variation was analyzed by geNorm software that determined the optimal number of control genes for normalization. A value < 0.15 indicates that the normalization could not be dramatically changed by additional reference genes

Similar articles

See all similar articles

Cited by 1 PubMed Central articles

References

    1. Li RF, Zhang JW, Wu GY, Wang HZ, Chen YJ, Wei JH. HbCIPK2 a novel CBL-interacting protein kinase from halophyte Hordeum brevisubulatum, confers salt and osmotic stress tolerance. Plant Cell Environ. 2012;35:1582–1600. doi: 10.1111/j.1365-3040.2012.02511.x. - DOI - PubMed
    1. Wang CM, Xia ZR, Wu GQ, Yuan HJ, Wang XR, Li JH, Tian FP, Zhang Q, Zhu XQ, He JJ, Kumar T, Wang XL, Zhang JL. The coordinated regulation of Na+ and K+ in Hordeum brevisubulatum responding to time of salt stress. Plant Sci. 2016;252:358–366. doi: 10.1016/j.plantsci.2016.08.009. - DOI - PubMed
    1. Lü SY, Jing YX, Shen SH, Zhao HY, Ma LQ, Zhou XJ, Ren Q, Li YF. Antiporter gene from Hordum brevisubulatum (Trin.) link and its overexpression in transgenic tobaccos. J Integr Plant Boil. 2005;47:343–349. doi: 10.1111/j.1744-7909.2005.00027.x. - DOI
    1. Bustin SA. Quantification of mRNA using real-time reverse transcription PCR (RTPCR): trends and problems. J Mol Endocrinol. 2002;29:23–39. doi: 10.1677/jme.0.0290023. - DOI - PubMed
    1. Bustin SA, Nolan T. Pitfalls of quantitative real-time reverse-transcription polymerase chain reaction. J Biomol Tech. 2004;15:155–166. - PMC - PubMed

LinkOut - more resources

Feedback