Selection and validation reference genes for qRT-PCR normalization in different cultivars during fruit ripening and softening of peach (Prunus persica)

Abstract

Quantitative real-time PCR (qRT-PCR) has been emerged as an effective method to explore the gene function and regulatory mechanisms. However, selecting appropriate reference gene (s) is a prerequisite for obtaining accurate qRT-PCR results. Peach is one of important fruit in Rosaceae and is widely cultivated worldwide. In this study, to explore reliable reference gene (s) in peach with different types during fruit ripening and softening (S1–S4), nine candidate reference genes (EF-1α, GAPDH, TBP, UBC, eIF-4α, TUB-A, TUB-B, ACTIN, and HIS) were selected from the whole-genome data. Then, the expression levels of the nine selected genes were detected using qRT-PCR in three peach types, including ‘Hakuho’ (melting type), ‘Xiacui’ (stony hard type), ‘Fantasia’ and ‘NJC108’ (non-melting type) cultivars were detected using qRT-PCR. Four software (geNorm, NormFinder, BestKeeper and RefFinder) were applied to evaluate the expression stability of these candidate reference genes. Gene expression was characterized in different peach types during fruit ripening and softening stages. The overall performance of each candidate in all samples was evaluated. The Actin gene (ACTIN) was a suitable reference gene and displayed excellent stability in ‘Total’ set, ‘Hakuho’ samples, S3 and S4 fruit developmental stages. Ubiquitin C gene (UBC) showed the best stability in most independent samples, including ‘Fantasia’, ‘NJC108’, S2 sets. Elongation factor-1α gene (EF-1α) was the most unstable gene across the set of all samples, ‘NJC108’ and S2 sets, while showed the highest stability in ‘Xiacui’ samples. The stability of candidate reference genes was further verified by analyzing the relative expression level of ethylene synthase gene of Prunus persica (PpACS1) in fruit ripening and softening periods of ‘Hakuho’. Taken together, the results from this study provide a basis for future research on the mining of important functional genes, expression patterns and regulatory mechanisms in peach.

Identification of Suitable Reference Genes for qRT-PCR Normalization in Kiwifruit

“…Our results provided further evidence that the expression of reference genes varied between varieties and that there was a need to conduct reference gene screening for different trials. Meanwhile, we also found that in addition to GAPDH, ACT1 and ACT2 also presented good stability between different varieties of kiwifruit, which was in agreement with recent studies that ACT was the most stable gene between different varieties of peach [34] and jujuba [35]. Both ACT1 and UBQ maintained stable transcript levels in kiwifruit stimulated by different hormones (or pollen polysaccharide), which was similar to the results of a previous study about bananas [36].…”

Selection and Validation of Reliable Reference Genes for Gene Expression Studies in Different Genotypes and TRV-Infected Fruits of Peach (Prunus persica L. Batsch) during Ripening

“…These results show that TEF2 is not the best normalization factor for different genotypes. It is possible that the different varieties of peach and experimental conditions have led to the discrepancy between the two assays [ 13 ]. The poor stability of TEF2 was also pointed out in peach and other species.…”

“…Many studies have reported the selection and validation of reference genes in plants [ 9 , 10 , 11 , 12 , 13 , 14 , 15 ]. However, these reference genes exhibit constant expression patterns under certain experimental conditions, but their expression levels vary widely under other experimental conditions.…”

“…Reference genes are generally stable expressed genes that maintain minimal vital functions of the cell, such as ubiquitin ( UBQ ) and RNA polymerase subunit ( RP ) [ 7 ]. In the current study, actin protein, tublin and 18S ribosomal RNA ( 18S ) are the most commonly used as internal reference genes for gene expression analysis in peach [ 13 , 15 , 28 ]. Many stably expressed reference genes reported in other species, such as glyceraldehyde-3-phosphate dehydrogenase ( GADPH ), translation enlongation factor ( TEF ), and cyclophilin ( CYP ), were also included as candidate genes for subsequent analysis [ 14 , 17 ].…”

Variation of Fruit Quality Traits in Apricot as Sources for Nutraceutical Breeding

“…PCR reaction specificity was assessed by the presence of a single peak in the dissociation curve after amplification and through size estimation of the amplified products by agarose electrophoresis. Normalized gene expression levels were measured by the relative standard curve procedure using the geometric mean of two reference genes, Actin and Sand-like (Lloret et al, 2017;You et al, 2021). Results were the average of 3 technical replicates each one.…”