Selection of Reference Genes for Gene Expression Studies in Zucchini (Cucurbita pepo) Using qPCR

Abstract

The zucchini (Cucurbita pepo) is an important food crop, the transcriptomics of which are a fundamental tool to accelerate the development of new varieties by breeders. However, the suitability of reference genes for data normalization in zucchini has not yet been studied. The aim of this study was to assess the suitability of 13 genes for their potential use as reference genes in quantitative real-time PCR. Assays were performed on 34 cDNA samples representing plants under different stresses and at different developmental stages. The application of geNorm and NormFinder software revealed that the use of a combination of UFP, EF-1A, RPL36aA, PP2A, and CAC genes for the different experimental sets was the best strategy for reliable normalization. In contrast, 18S rRNA and TUA were less stable and unsuitable for use as internal controls. These results provide the possibility to allow more accurate use of qPCR in this horticultural crop.

Reference gene selection in Artemisia annua L., a plant species producing anti-malarial artemisinin

“…Under temperature treatment, EF1a was the most stably expressed gene in A. annua, and the results were consistent with the previously reported results from chrysanthemum, a plant species belonging to the family of Compositae (Gu et al 2011). EF1a was also recommended to be the suitable reference gene under the treatment of plant growth regulators in zucchini (Obrero et al 2011) and Catharanthus roseus (Wei 2010). In MeJA or ABA treatment, EF1a was the most stably expressed gene among the ten candidate reference genes.…”

Systematic selection and validation of appropriate reference genes for gene expression studies by quantitative real-time PCR in pear

“…These advantages allow a straightforward comparison and make it become the most appropriate choice for gene expression analysis (Garson et al 2005;Li et al 2010;Martin et al 2008). Despite its numerous advantages, several serious flaws have been reported, including the lack of reliable reference genes for normalization (Obrero et al 2011). To obtain reliable results, normalization is crucial for RT-qPCR studies to control for sample differences (Tong et al 2009).…”

“…Thus, these genes are commonly used for normalization without any validation (Brunner et al 2004;Chi et al 2012;Marum et al 2012). However, a few recent studies have shown that the widely used reference genes vary considerably in response to some specific experimental conditions (Marino et al 2008;Morgante et al 2011;Nicot et al 2005), that if not chosen properly, the reference genes may be unsuitable for normalization purposes owing to large measurement errors (Dekkers et al 2012;Obrero et al 2011). Therefore, to ensure proper normalization, it must be demonstrated that the reference genes of choice have high expression stability independent of the varying experimental conditions.…”

Reference gene selection for gene expression studies in lily using quantitative real-time PCR

“…Previous studies on the selection of reference genes in tomato, Fagopyrum esculentum developmental stages, and soybean under varied light regimes (Expósito-Rodríguez et al, 2008;Hu et al, 2009;Demidenko et al, 2011) also identified TIP as the most stable reference gene for vegetative samples. In previous studies, Clathrin has also been identified as the best candidate for normalization (Expósito-Rodríguez et al, 2008;Obrero et al, 2011).…”