Identification of reliable reference genes for quantitative real-time PCR normalization in pitaya

Area of Science:

• Plant molecular biology
• Gene expression analysis
• Biotechnology

Background:

• Accurate quantitative real-time PCR (qRT-PCR) relies on stable reference genes.
• Universality of reference genes varies across species, tissues, and developmental stages.
• No established reference genes exist for pitaya (Hylocereus) research.

Purpose of the Study:

• To identify the most stable reference genes for qRT-PCR normalization in pitaya.
• To evaluate candidate reference genes under diverse conditions including different tissues, temperature stresses, and fruit developmental stages.
• To provide a reliable reference gene for future molecular studies in pitaya.

Main Methods:

• Selection of six candidate reference genes: Actin(1), GAPDH, UBC(1), UBC(2), EF1-α(1), and histone(1).
• Assessment of gene expression stability using three statistical algorithms: GeNorm, NormFinder, and BestKeeper.
• Validation of reference gene stability by analyzing the expression profiles of betalain biosynthesis genes in pitaya fruit.

Main Results:

• Actin(1) and EF1-α(1) exhibited the highest expression stability among the six candidates.
• UBC(1) and UBC(2) displayed the lowest expression stability.
• Actin(1) was identified as the most reliable reference gene for qRT-PCR normalization in pitaya.

Conclusions:

• Actin(1) is the most stably expressed reference gene in pitaya across different tissues and fruit developmental stages.
• This study provides the first identification of reference genes for pitaya, facilitating future molecular biology and gene function studies.
• The findings will aid research in Hylocereus and related species.