Reference Gene Validation for Quantitative RT-PCR during Biotic and Abiotic Stresses in Vitis vinifera

Abstract

Grapevine is one of the most cultivated fruit crop worldwide with Vitis vinifera being the species with the highest economical importance. Being highly susceptible to fungal pathogens and increasingly affected by environmental factors, it has become an important agricultural research area, where gene expression analysis plays a fundamental role. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is currently amongst the most powerful techniques to perform gene expression studies. Nevertheless, accurate gene expression quantification strongly relies on appropriate reference gene selection for sample normalization. Concerning V. vinifera, limited information still exists as for which genes are the most suitable to be used as reference under particular experimental conditions. In this work, seven candidate genes were investigated for their stability in grapevine samples referring to four distinct stresses (Erysiphe necator, wounding and UV-C irradiation in leaves and Phaeomoniella chlamydospora colonization in wood). The expression stability was evaluated using geNorm, NormFinder and BestKeeper. In all cases, full agreement was not observed for the three methods. To provide comprehensive rankings integrating the three different programs, for each treatment, a consensus ranking was created using a non-weighted unsupervised rank aggregation method. According to the last, the three most suitable reference genes to be used in grapevine leaves, regardless of the stress, are UBC, VAG and PEP. For the P. chlamydospora treatment, EF1, CYP and UBC were the best scoring genes. Acquaintance of the most suitable reference genes to be used in grapevine samples can contribute for accurate gene expression quantification in forthcoming studies.

Identification of Reference Genes for qRT‐PCR Gene Expression Studies during Seed Development and under Abiotic Stresses in Cyamopsis tetragonoloba

“…These variations are caused by the differences in RNA quality, reverse transcription efficiency, and PCR efficiency. (Chandna et al, 2012), Arachis hypogea L. (Reddy et al, 2013), Vitis vinifera L. (Borges et al, 2014), Pennisetum glaucum (L.) R. Br. Such a method involves the use of a reference gene(s).…”

“…, Zea mays L. (Manoli et al, 2012), Brassica juncea (L.) Czern. (Chandna et al, 2012), Arachis hypogea L. (Reddy et al, 2013), Vitis vinifera L. (Borges et al, 2014), Pennisetum glaucum (L.) R. Br. (Reddy et al, 2015), and Apium graveolens L. (Li et al, 2016), have shown that the expression of the HKGs is not as stable as it was generally assumed.…”

Comparative metabolic profiling reveals the key role of amino acids metabolism in the rapamycin overproduction by Streptomyces hygroscopicus

“…Then, real-time PCR was carried out to detect gene relative transcriptional expression abundance according to the method of Borges et al [2]. cDNA was prepared using PrimeScript™ RT reagent Kit (Takara, Dalian, China) under instructions.…”

Selection and validation of reference genes for RT-qPCR analysis in Litchi chinensis Sonn. pericarp

“…Suitable reference genes for each experimental set should be identified independently. In grapevine, reference genes have been evaluated for different varieties [31], developmental stages [14], and physiological conditions [4], such as biotic and abiotic stress. This need to optimize reference genes for different conditions arises in studies of other plants, such as rice [18,30,45], potato [27,40], apple [5,21], and strawberry [12].…”