Identification of suitable reference genes for gene expression normalization in qRT-PCR analysis in watermelon

Area of Science:

• Plant Molecular Biology
• Genomics
• Biotechnology

Background:

• Quantitative real-time reverse transcriptase PCR (qRT-PCR) is essential for gene expression studies in watermelon.
• Accurate normalization using validated reference genes is critical for reliable qRT-PCR results.
• A systematic validation of reference genes in watermelon has been lacking.

Purpose of the Study:

• To systematically validate and identify the most stable reference genes for qRT-PCR in watermelon across various conditions.
• To compare the performance of candidate reference genes using geNorm and NormFinder algorithms.
• To provide reliable reference gene recommendations for watermelon gene expression studies.

Main Methods:

• Quantification of 15 candidate reference gene transcripts using qRT-PCR in watermelon.
• Stability analysis of candidate reference genes using geNorm and NormFinder.
• Validation of identified reference genes using catalase family genes under low-temperature stress.

Main Results:

• geNorm identified optimal gene pairs for different conditions (e.g., ClTUA/ClACT, ClEF1α/ClACT, ClCAC/ClTUA).
• NormFinder identified best single reference genes (e.g., ClYLS8, ClUBCP, ClCAC) and optimal combinations (ClYLS8/ClPP2A).
• The commonly used 18SrRNA was found to be unstable, leading to overestimated gene expression, particularly for catalase genes under stress.

Conclusions:

• ClYLS8 and ClPP2A are recommended as the best reference genes across all tested samples.
• The number of reference genes required for reliable normalization varies from two to nine, depending on experimental conditions.
• This study provides essential guidance for selecting appropriate reference genes, enhancing the accuracy of qRT-PCR in watermelon research.