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

ROSLI, HERNÁN GUILLERMO; ZHEN, YI; MARTIN, GREGORY; POMBO, MARINA ALEJANDRA; FEI, ZHANGJUN; ROSLI, HERNÁN GUILLERMO; ZHEN, YI; MARTIN, GREGORY; POMBO, MARINA ALEJANDRA; FEI, ZHANGJUN

Scientific Reports

Nature Publishing Group

Año: 2017

2045-2322

The agronomical relevant tomato-Pseudomonas syringae pv. tomato pathosystem is widely usedto explore and understand the underlying mechanisms of the plant immune response. Transcriptabundance estimation, mainly through reverse transcription-quantitative PCR (RT-qPCR), is a commonapproach employed to investigate the possible role of a candidate gene in certain biological processunder study. The accuracy of this technique relies heavily on the selection of adequate reference genes.Initially, genes derived from other techniques (such as Northern blots) were used as reference genes inRT-qPCR experiments, but recent studies in different systems suggest that many of these genes are notstably expressed. The development of high throughput transcriptomic techniques, such as RNA-seq,provides an opportunity for the identification of transcriptionally stable genes that can be adopted asnovel and robust reference genes. Here we take advantage of a large set of RNA-seq data originatingfrom tomato leaves infiltrated with different immunity inducers and bacterial strains. We assessed andvalidated 9 genes that are much more stable than two traditional reference genes. Specifically, ARD2and VIN3 were the most stably expressed genes and consequently we propose they be adopted for RTqPCRexperiments involving this pathosystem.