Abstract
The tree species, Pistacia vera (P. vera) is an important commercial product that is salt-tolerant and long-lived, with a possible lifespan of over one thousand years. Gene expression analysis is an efficient method to explore the possible regulatory mechanisms underlying these characteristics. Therefore, having the most suitable set of reference genes is required for transcript level normalization under different conditions in P. vera. In the present study, we selected eight widely used reference genes, ACT, EF1α, α-TUB, β-TUB, GAPDH, CYP2, UBQ10, and 18S rRNA. Using qRT-PCR their expression was assessed in 54 different samples of three cultivars of P. vera. The samples were collected from different organs under various abiotic treatments (cold, drought, and salt) across three time points. Several statistical programs (geNorm, NormFinder, and BestKeeper) were applied to estimate the expression stability of candidate reference genes. Results obtained from the statistical analysis were then exposed to Rank aggregation package to generate a consensus gene rank. Based on our results, EF1α was found to be the superior reference gene in all samples under all abiotic treatments. In addition to EF1α, ACT and β-TUB were the second best reference genes for gene expression analysis in leaf and root. We recommended β-TUB as the second most stable gene for samples under the cold and drought treatments, while ACT holds the same position in samples analyzed under salt treatment. This report will benefit future research on the expression profiling of P. vera and other members of the Anacardiaceae family.
Highlights
ObjectivesThe objective of this study was to identify the most stably expressed gene(s) across eight commonly used reference genes (ACT, α-TUB, β-TUB, 18S rRNA, CYP2, EF1α, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and UBQ10) in roots and leaves of three main pistachio rootstocks, P. vera L. cultivars Sarakhs, Badami, and Ghazvini under abiotic treatments
The results demonstrated that none of the selected reference genes is constantly expressed amongst all of the analysed samples
Data normalization using a set of reference genes is a crucial procedure during the evaluation of the expression level of target genes in various tissues or under different conditions using quantitative reverse transcription polymerase chain reaction (PCR) (qRT-PCR)
Summary
The objective of this study was to identify the most stably expressed gene(s) across eight commonly used reference genes (ACT, α-TUB, β-TUB, 18S rRNA, CYP2, EF1α, GAPDH, and UBQ10) in roots and leaves of three main pistachio rootstocks, P. vera L. cultivars Sarakhs, Badami, and Ghazvini under abiotic treatments
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