Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Nov 8;11(11):e0166216.
doi: 10.1371/journal.pone.0166216. eCollection 2016.

Identification of Reference Genes for Quantitative Real-Time PCR in Date Palm (Phoenix dactylifera L.) Subjected to Drought and Salinity

Himanshu V Patankar  1 , Dekoum V M Assaha  1 , Rashid Al-Yahyai  2 , Ramanjulu Sunkar  3 , Mahmoud W Yaish  1
Affiliations

Identification of Reference Genes for Quantitative Real-Time PCR in Date Palm (Phoenix dactylifera L.) Subjected to Drought and Salinity

Himanshu V Patankar et al. PLoS One. .

Abstract

Date palm is an important crop plant in the arid and semi-arid regions supporting human population in the Middle East and North Africa. These areas have been largely affected by drought and salinity due to insufficient rainfall and improper irrigation practices. Date palm is a relatively salt- and drought-tolerant plant and more recently efforts have been directed to identifying genes and pathways that confer stress tolerance in this species. Quantitative real-time PCR (qPCR) is a promising technique for the analysis of stress-induced differential gene expression, which involves the use of stable reference genes for normalizing gene expression. In an attempt to find the best reference genes for date palm's drought and salinity research, we evaluated the stability of 12 most commonly used reference genes using the geNorm, NormFinder, BestKeeper statistical algorithms and the comparative ΔCT method. The comprehensive results revealed that HEAT SHOCK PROTEIN (HSP), UBIQUITIN (UBQ) and YTH domain-containing family protein (YT521) were stable in drought-stressed leaves whereas GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE (GAPDH), ACTIN and TUBULIN were stable in drought-stressed roots. On the other hand, SMALL SUBUNIT RIBOSOMAL RNA (25S), YT521 and 18S ribosomal RNA (18S); and UBQ, ACTIN and ELONGATION FACTOR 1-ALPHA (eEF1a) were stable in leaves and roots, respectively, under salt stress. The stability of these reference genes was verified by using the abiotic stress-responsive CYTOSOLIC Cu/Zn SUPEROXIDE DISMUTASE (Cyt-Cu/Zn SOD), an ABA RECEPTOR, and a PROLINE TRANSPORTER 2 (PRO) genes. A combination of top 2 or 3 stable reference genes were found to be suitable for normalization of the _target gene expression and will facilitate gene expression analysis studies aimed at identifying functional genes associated with drought and salinity tolerance in date palm.

PubMed Disclaimer

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Variation in Cq values for the selected reference genes in date palm leaves and roots under drought and salinity.
A) Cq for drought-stressed leaves, B) Cq for drought-stressed roots, C) Cq for salinity-stressed leaves, D) Cq for salinity-stressed roots, E) control leaves, and, F) control roots. Dots on the graph represent median Cq values and the bars are standard deviations.
Fig 2
Fig 2. Expression stability analysis of candidate reference genes in drought- and salinity-stressed date palm leaves and roots.
A) drought-stressed leaves, B) drought-stressed roots, C) salinity-stressed leaves, and, D) salinity-stressed roots. The gene expression stability graph is based on expression stability values (M-values) obtained with the geNorm algorithm. As per this, lower the M-value, higher the stability of the gene. The arrow direction indicates the order of most stable and least stable reference genes.
Fig 3
Fig 3. Expression stability analysis of candidate reference genes in drought- and salinity- stressed date palm leaves and roots.
A) drought-stressed leaves, B) drought-stressed roots, C) salinity-stressed leaves, and, D) salinity-stressed roots. The gene expression stability graph is based on stability values obtained from the NormFinder algorithm. The lower the stability value, the higher the stability of the gene. The arrow direction indicates the order of most stable and least stable reference genes.
Fig 4
Fig 4. Expression stability analysis of candidate reference genes in drought- and salinity-stressed date palm leaves and roots.
A) drought-stressed leaves, B) drought-stressed roots, C) salinity-stressed leaves, and, D) salinity-stressed roots. The gene expression stability graph is based on coefficient of variation (CV) and standard deviation (SD). The lower the CV, the higher the stability of the gene. The arrow direction indicates the order of the most stable and least stable reference genes.
Fig 5
Fig 5. Expression stability analysis of candidate reference genes in drought- and salinity-stressed date palm leaves and roots.
A) drought-stressed leaves, B) drought-stressed roots, C) salinity-stressed leaves, and, D) salinity-stressed roots. The gene expression stability graph is based on average standard deviation of variability in Cq values, calculated with the ΔCT method.
Fig 6
Fig 6. Expression stability analysis of candidate reference genes in drought- and salinity-stressed date palm leaves and roots.
A) drought-stressed leaves, B) drought-stressed roots, C) salinity-stressed leaves, and, D) salinity-stressed roots. The gene expression stability graph is based on the geometric mean calculated by RefFinder based on the comprehensive results from the four algorithms. The lower the geometric mean, the higher the stability of the gene.
Fig 7
Fig 7. The effect of single reference gene and two reference genes on normalization of most stable gene in each of the four samples (HSP for drought-stressed leaves, GAPDH for drought-stressed roots, 25S for salinity-stressed leaves, and, UBQ for salinity-stressed roots).
Bars represent mean gene relative quantity (RQ) ± SE (n = 3).
Fig 8
Fig 8
The effect of the most stable and the least stable reference genes on the expression level (fold change) of Cyt-Cu/ZnSOD (SOD), abscisic acid receptor (ABA), and proline transporter 2 (PRO) obtained using the qPCR in drought-stressed leaves (A), drought-stressed roots (B), salinity-stressed leaves (C) and salinity-stressed roots (D). Bars represent mean log 2 fold change ± SE (n = 3), lower case letters indicates significant difference at P ≤ 0.05.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Chao CT, Krueger RR. The date palm (Phoenix dactylifera L.): Overview of biology, uses, and cultivation. HortScience. 2007; 42: 1077–1082.
    1. Al-Yahyai R, Khan MM. Date Palm Status and Perspective in Oman In: Al-Khayri JM, Jain SM, Johnson DV, editors. Date palm genetic resources and utilization. Netherlands: Springer Science and Business Media; 2015. pp. 207–240. 10.1007/978-94-017-9707-8_6 - DOI
    1. Yaish MW, Kumar PP. Salt Tolerance Research in Date Palm Tree (Phoenix dactylifera L.), Past, Present and Future Perspectives. Front Plant Sci. 2015; 6 10.3389/fpls.2015.00348 - DOI - PMC - PubMed
    1. El-Juhany LI. Degradation of date palm trees and date production in Arab countries: causes and potential rehabilitation. Aust J Basic Appl Sci. 2010; 4: 3998–4010.
    1. Munns R, Tester M. Mechanisms of salinity tolerance. Annu Rev Plant Biol. 2008; 59: 651–681. 10.1146/annurev.arplant.59.032607.092911 - DOI - PubMed

Substances

Grants and funding

Support was provided by The Research Council of Oman number 151.
  NODES
twitter 2