Identification of reference genes for RT-qPCR analysis in peach genotypes with contrasting chilling requirements

Abstract

ABSTRACT. Selecting and validating reference genes are the first steps in studying gene expression by reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR). The present study aimed to evaluate the stability of five reference genes for the purpose of normalization when studying gene expression in various cultivars of Prunus persica with different chilling requirements. Flower bud tissues of nine peach genotypes from Embrapa's peach breeding program with different chilling requirements were used, and five candidate reference genes based on the RT-qPCR that were useful for studying the relative quantitative gene expression and stability were evaluated using geNorm, NormFinder, and bestKeeper software packages. The results indicated that among the genes tested, the most stable genes to be used as reference genes are Act and UBQ10. This study is the first survey of the stability of reference genes in peaches under chilling stress and provides guidelines for more accurate RT-qPCR results.

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

“…In addition, GADPH , TEF2, and PLA2 were ranked as the last three in TRV-infected fruits, with GADPH , TEF2, and 18S in different genotypes ( Table 3 ). Although most authors used only a single reference gene as a standardized internal control, it has been suggested that RT-qPCR studies using two or more reference genes may contribute to more reliable results [ 12 , 47 , 48 , 49 ]. According to pairwise variation, two reference genes were required for gene expression level normalization in this study since the V2/3 was lower than 0.15 ( Figure 3 ).…”

“…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.…”

Advancements in Reference Gene Selection for Fruit Trees: A Comprehensive Review

“…According to the study of Wang et al [91], the reference genes for persimmon under low and high temperature stresses are UBC, RPII and Tua. The most stable genes are ACT and UBQ10 in peaches under chilling stress, thus providing guidelines for more accurate RT-qPCR results [92].…”