Evaluation of suitable reference genes for qRT-PCR normalization in strawberry (Fragaria × ananassa) under different experimental conditions

Abstract

Background: Strawberry has received much attention due to its nutritional value, unique flavor, and attractive appearance. The availability of the whole genome sequence and multiple transcriptome databases allows the great possibility to explore gene functions, comprehensively. Gene expression profiles of a target gene can provide clues towards the understanding of its biological function. Quantitative real-time PCR (qRT-PCR) is a preferred method for rapid quantification of gene expression. The accuracy of the results obtained by this method requires the reference genes with consistently stable expression to normalize its data.

Results: In present study, the expression stability of seven candidate reference genes in diverse sample subsets of different tissues and fruit developmental stages, and plant subjected to light quality and low temperature treatments was evaluated using three statistical algorithms, geNorm, NormFinder, and BestKeeper. Our data indicated that the expression stability of reference genes varied under different experimental conditions. Overall, DBP, HISTH4, ACTIN1 and GAPDH expressed much more stably. PIRUV, ACTIN2 and 18S were not recommended for normalization in given experimental conditions due to low stability. In addition, the relative expression pattern of HY5 (ELONGATED HYPOCOTYL5) was conducted to further confirm the reliability of the reference genes, which demonstrated the correct adoption of reference genes was of great importance in qRT-PCR analysis.

Conclusions: Expression stability of reference genes from strawberry varied across selected experimental conditions. Systematic validation of reference genes prior to calculation of target gene expression level should be done to improve the accuracy and consistency of qRT-PCR analysis.

Keywords: Gene expression; Normalization; Reference gene; Strawberry; qRT-PCR.

Fig. 1

Melting curve analysis of seven candidate reference genes by quantitative real-time RT-PCR. A single peak indicated the specificity of primers

Fig. 2

Expression profiles of seven candidate reference genes in strawberry samples. Expression data for different tissues and fruit developmental stages (a), fruit treated with light quality (b) and fruit treated with low temperature (c) are displayed as Cq values for each reference gene. The box indicates the 25th and 75th percentiles. A line across the box represents the median. Whiskers are depicted as the maximum and minimum values and the black circles represent outliers. The higher boxes and whiskers mean the greater variations

Fig. 3

Pairwise variation (V) for determination of optimal number of reference genes for qRT-PCR normalization. Pairwise variation (V_n/V_n+1) was analyzed between the normalization factors NF_n and NF_n+1 by the geNorm software. The optimal number of genes for normalization was determined based on V less than 0.15

Fig. 4

Relative quantification of FaHY5 expression using validated reference genes for normalization in given experimental conditions. These experimental series include a different tissues, b different fruit developmental stages, c fruit treated with red light d fruit treated with low temperature. SG small green, LG large green, W white, TR turning red, HR half red, RR red ripe, FR full red. The relative expression levels are depicted as the mean ± standard error, which was calculated from three biological replicates