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.
Highlights
Date palm (Phoenix dactylifera L.) is one of the most important socio-economic plants in arid and semi-arid regions, where fresh water is very limited
Gene expression data is only meaningful when it is normalized with an appropriate stable reference gene. Quantitative real-time PCR (qPCR) is a decisive technique, which is routinely used in the rapid and accurate quantification of gene expression
The efficiency of the qPCR results depends mainly on the reference gene used for normalizing target gene expression
Summary
Date palm (Phoenix dactylifera L.) is one of the most important socio-economic plants in arid and semi-arid regions, where fresh water is very limited. It is of great spiritual and cultural significance to the people in these areas [1]. Because the availability of fresh water is very limited in this region, the growth and development of date palm is frequently affected by drought stress, which together with salinity, adversely affects plant productivity [3]. These common strategies include upregulation of dehydration-response element-binding proteins [14,15], enhanced water uptake and hydraulic conductivity (aquaporins) [16,17], accumulation of osmolytes (such as sugars, polyols, proline and glycine betaine) and late embryogenesis abundant protein (dehydrins) [18] as well as enhanced antioxidant systems [19]
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