Selection and validation of reference genes for quantitative real-time PCR of Quercus mongolica Fisch. ex Ledeb under abiotic stresses

Abstract

Quercus mongolica Fisch. ex Ledeb is the main species of coniferous and broadleaved mixed forests in northeast and north China, which has high ornamental, economic, and ecological value. The appropriate reference genes must be selected for quantitative real-time PCR to reveal the molecular mechanisms of stress responses and their contribution to breeding of Q. mongolica. In the present study, we chose 11 candidate reference genes (TUA, CYP18, HIS4, RPS13, ACT97, TUB1, UBQ10, UBC5, SAND, PP2A, and SAMDC) and used four programs (GeNorm, NormFinder, BestKeeper, and RefFinder) to assess the expression stability of the above genes in roots, stems, and leaves under five abiotic stress factors (cold, salt, drought, weak light, and heavy metal). The findings revealed that under various experimental environments, the most stable genes were different; CYP18, ACT97, and RPS13 ranked the highest under most experimental environments. Moreover, two genes induced by stress, CMO and P5CS2, were chosen to demonstrate the reliability of the selected reference genes in various tissues under various stress conditions. Our research provides a significant basis for subsequent gene function studies of Q. mongolica.

Fig 1. The distribution of Ct values of 11 candidate reference genes of Q. mongolica leaves, stem, and roots under various abiotic stress conditions.

The line across the box and the lower and upper boundaries of the box are the 50th, 25th and 75th percentile, respectively. Whisker caps represents the maximum and minimum values.

Fig 2. The stability measurement (M) of the expression of 11 candidate reference genes in Q. mongolica leaves, stem, and root samples under the five abiotic stress environments analyzed by GeNorm software.

CR: Cold-treated roots; CS: Cold-treated stem; CL: Cold-treated leaves; SR: Salt-treated roots; SS: Salt-treated stem; SL: Salt-treated leaves; DR: Drought-treated roots; DS: Drought-treated stem; DL: Drought-treated leaves; CdR: Cd-treated roots; CdS: Cd-treated stem; CdL: Cd-treated leaves; WR: Weak light-treated roots; WS: Weak light-treated stem; WL: Weak light-treated leaves; DT: Different tissues; Total: All samples.

Fig 3. Pairwise variation (V) analysis of the 11 candidate reference genes using GeNorm under various experimental conditions.

CR: Cold-treated roots; CS: Cold-treated stem; CL: Cold-treated leaves; SR: Salt-treated roots; SS: Salt-treated stem; SL: Salt-treated leaves; DR: Drought-treated roots; DS: Drought-treated stem; DL: Drought-treated leaves; CdR: Cd-treated roots; CdS: Cd-treated stem; CdL: Cd-treated leaves; WR: Weak light-treated roots; WS: Weak light-treated stem; WL: Weak light-treated leaves; DT: Different tissues; Total: All samples.

Fig 4. Relative gene expression levels of utilizing the chosen reference genes.

The outcomes were normalized through two of the most unstable reference genes together with two most stable genes (in combination or alone) following stress treatment after 0 h, 24 h, 48 h, and 72 h. (A) The relative expression levels of P5CS2 in SL samples. (B) The relative expression levels of CMO in SL samples. (C) The relative expression levels of P5CS2 in CS samples. (D) The relative expression levels of CMO in CS samples. (E) P5CS2 and CMO expression levels in DR samples. (F) P5CS2 and CMO expression levels in DR samples. The error bar represents the standard deviation of three bio-replicates. Different letters indicate that the expression of the reference gene expression in each condition was significantly different (P < 0.05, Duncan’s test).