Identification of two putative reference genes from grapevine suitable for gene expression analysis in berry and related tissues derived from RNA-Seq data

Abstract

BackgroundData normalization is a key step in gene expression analysis by qPCR. Endogenous control genes are used to estimate variations and experimental errors occurring during sample preparation and expression measurements. However, the transcription level of the most commonly used reference genes can vary considerably in samples obtained from different individuals, tissues, developmental stages and under variable physiological conditions, resulting in a misinterpretation of the performance of the target gene(s). This issue has been scarcely approached in woody species such as grapevine.ResultsA statistical criterion was applied to select a sub-set of 19 candidate reference genes from a total of 242 non-differentially expressed (NDE) genes derived from a RNA-Seq experiment comprising ca. 500 million reads obtained from 14 table-grape genotypes sampled at four phenological stages. From the 19 candidate reference genes, VvAIG1 (AvrRpt2-induced gene) and VvTCPB (T-complex 1 beta-like protein) were found to be the most stable ones after comparing the complete set of genotypes and phenological stages studied. This result was further validated by qPCR and geNorm analyses.ConclusionsBased on the evidence presented in this work, we propose to use the grapevine genes VvAIG1 or VvTCPB or both as a reference tool to normalize RNA expression in qPCR assays or other quantitative method intended to measure gene expression in berries and other tissues of this fruit crop, sampled at different developmental stages and physiological conditions.

Selection and Validation of Appropriate Reference Genes for Quantitative Real-Time PCR Normalization in Staminate and Perfect Flowers of Andromonoecious Taihangia rupestris

“…For non-model plant species, the reference genes are mainly identified by the search for orthologous sequences of common housekeeping genes reported in model plant species, due to limited genetic and sequence information (Gonzalezaguero et al, 2013). However, numerous studies have reported that the expression levels of traditional housekeeping genes vary considerably alterations in different biological sample and variable experimental condition (Hu et al, 2009; Zhang et al, 2017).…”

Use of RNA-seq data to identify and validate RT-qPCR reference genes for studying the tomato-Pseudomonas pathosystem

“…In the past years, RNA-seq has emerged as a powerful high-throughput technology used for transcriptome analysis in different organisms and treatments3940414243. In spite of being a technique used for many years, RNA-seq data has been used in the plant research field for the selection and validation of new and more robust RT-qPCR reference genes only in grape, soybean and Lycoris 444546.…”

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].…”