Evaluation of reference genes for transcript normalization in Fragaria chiloensis fruit and vegetative tissues

Abstract

Quantitative real-time PCR (RT-qPCR) is used extensively in gene expression studies. For adequate comparisons, the identification and use of reliable reference genes are crucial. Nevertheless, the availability of such genes in strawberry species is limited and has yet to be described for the Chilean strawberry, Fragaria chiloensis. In this study, the expression dynamics of a set of 10 candidate reference genes were analyzed in various F. chiloensis vegetative tissues (root, runners, stem, leaf, and flower), and fruits at different ripening stages or subjected to different hormonal treatments (ABA, auxin). The expression stability of candidate genes was examined by a series of algorithms, such as geNorm, NormFinder, BestKeeper, and ΔCt, for comparisons and rankings. Finally, by using RefFinder, a comprehensive and comparative ranking of the four methods was achieved. The results highlight that the expression stability of candidate reference genes fluctuates depending on tissue type, fruit stage, and hormonal treatment. As reference genes, the use of FcCHP2 and FcACTIN1 is recommended for F. chiloensis vegetative tissues; FcDBP and FcCHP1 for fruit ripening stages; FcGAPDH and FcDBP for fruit subjected to ABA and NDGA treatments; FcCHP1 and FcCHP2 for fruit under AUXIN and TIBA treatments; and FcDBP and FcCHP2 when all fruit stages and hormonal treatments are compared. If just one reference gene is employed as a normalizer, FcDBP should be chosen as it is the most stable internal control in most conditions. Therefore, the present study delivers a set of reliable reference genes for RT-qPCR expression analysis in F. chiloensis tissues and fruits subjected to several hormonal treatments.

Supplementary information: The online version contains supplementary material available at 10.1007/s12298-022-01227-y.

Keywords: Fragaria chiloensis; Fruit ripening; Normalization; RT-qPCR; Reference genes.

Fig. 1

Expression stability of ten candidate reference genes in F. chiloensis samples. From qPCR analyses performed using different tissues or fruit subjected to several treatments, the quantification cycle (Cq) values for each reference gene were plotted. In A Cq values for fruit at different ripening stages (C1-C4), in B Cq values for a set of vegetative tissues (leaves, roots, runners, stems, and flowers), in C Cq values for fruit treated with ABA and NDGA, and in D Cq values for fruit treated with auxin and TIBA. The genes evaluated are glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ACTIN1, 18S ribosomal RNA (18S and 18S2), pyruvate decarboxylase (PDC), DNA binding protein (DBP), chloroplastic BYPASS (CHP1 and CHP2), endoplasmin 1 (ENP1), and histone H4 (HISTH4). Each box indicates the 25th and 75th percentiles, the line across the box represents the median, the whiskers describe the maximum and minimum values, whilst bigger boxes and whiskers represent expression values with the greatest variations. Each qPCR reaction was carried out using cDNA samples obtained from three independent RNA extractions prepared from pools of tissue/fruit samples, and three technical replicates. Water was used as a negative control in each qPCR run

Fig. 2

Relative expression profiles of FcRGL1, FcEXPA1, FcMYB1, and FcNAC1 in F. chiloensis fruit samples. qPCR reactions were carried out in triplicate using fruit samples from four ripening stages (C1, C2, C3, and C4). Relative expression data correspond to the means of three biological replicates (± SE) normalized against the previously-used FcGAPDH, the top-ranked reference genes FcDBP, FcCHP1, and FcCHP2, and finally, the two lowest-ranked reference genes (FcPDC and Fc18S)