Selection and validation of reference genes for qRT-PCR in cultivated octoploid strawberry

The rapid, reliable, and efficient characteristics of quantitative reverse transcription polymerase chain reaction (qRT-PCR) make it a highly advantageous method for assessing gene expression levels. The identification of stable reference genes is crucial for successful gene expression studies. Cultivated strawberry fruit has been extensively investigated as a model for studying the non-climacteric fruit ripening process. However, more research needs to be conducted on identifying suitable reference genes at different developmental stages of strawberry fruit. We selected the 'Yanli' and 'Chuliandeweidao' cultivars to screen potential reference genes in various tissues, organs, and developmental stages of strawberry fruit. Based on the analysis of high-quality haplotype-resolved genome and transcriptomic FPKM data, FaADPrf1 (ADP-ribosylation factor 1), FaGAPC2 (Glyceraldehyde-3-phosphate dehydrogenase), FaPPC1 (Peptidyl-prolyl cis-trans isomerase 1), and FaEF1-alpha (Elongation factor 1-alpha) were selected as candidate reference genes, along with the commonly used Fa26S rRNA, for normalization purposes. A qRT-PCR analysis showed 89.21% to 101.51% amplification efficiency for five candidate reference genes, with correlation coefficients (R2) exceeding 0.99. Reference genes' expression stability was assessed using GeNorm, NormFinder, BestKeeper, and Comparative delta-Ct method. RefFinder analysis determined that FaGAPC2 and FaADPrf1 were the most suitable reference genes, considering the results obtained from the abovementioned four methods. The calculated results were validated by studying the expression of FaMYB10, FaUGT1, and FaCHS in different developmental stages of 'Yanli' fruit. This validation confirmed that both FaGAPC2 and the combination of FaGAPC2 and FaADPrf1 could serve as suitable reference genes, with the combination of FaGAPC2 and FaADPrf1 being more suitable than the single FaGAPC2 in certain cases. In summary, we obtained suitable reference genes for research on cultivated strawberry fruit development, which will benefit further study on the ripening of non-climacteric fruits.