Identification of suitable reference genes for real-time RT-PCR normalization in the grapevine-downy mildew...

Area of Science:

• Molecular Biology
• Plant Pathology
• Biotechnology

Background:

• Real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) is a sensitive method for gene expression quantification.
• RT-qPCR accuracy depends on proper normalization using stable reference genes.
• Universal reference genes are not always available, necessitating condition-specific validation.

Purpose of the Study:

• To identify the most suitable reference gene(s) for normalizing gene expression in grapevine during downy mildew interaction.
• To evaluate reference gene stability under induced resistance conditions using chemical elicitors.
• To determine the optimal number of reference genes for accurate gene expression analysis in grapevine.

Main Methods:

• Six candidate reference genes were evaluated in grapevine under experimental conditions.
• Gene expression stability was analyzed using geNorm and Normfinder software.
• Chitinase gene expression was studied to demonstrate the impact of reference gene selection.

Main Results:

• The study identified specific reference genes that are stably expressed in grapevine under downy mildew infection and induced resistance.
• Normfinder and geNorm analyses provided consistent recommendations for the most stable reference genes.
• The choice of reference genes significantly impacted the quantification of chitinase expression.

Conclusions:

• Accurate gene expression studies in grapevine require the selection of validated, condition-specific reference genes.
• Using multiple validated reference genes enhances the reliability of RT-qPCR data.
• This research provides a foundation for accurate gene expression profiling in grapevine-pathogen interactions.