RNA-seq-based selection of reference genes for RT-qPCR analysis of pitaya

Area of Science:

• Plant molecular biology
• Gene expression analysis
• Hylocereus undatus (pitaya) research

Background:

• Accurate gene expression analysis relies on stable reference genes.
• Pitaya (Hylocereus undatus) lacks systematic reference gene evaluation.
• RT-qPCR requires validated reference genes for reliable results.

Purpose of the Study:

• To systematically evaluate candidate reference genes in pitaya.
• To identify the most stable reference genes for RT-qPCR under various conditions.
• To provide guidelines for pitaya gene expression studies.

Main Methods:

• Evaluated five candidate reference genes: HuEF1-α, Hu18S rRNA, HuUBQ, HuACT, and HuUQT.
• Assessed gene expression stability using geNorm and NormFinder software.
• Validated reference gene stability by normalizing target genes (HuPGK, HuERF).

Main Results:

• The combination of HuUBQ and HuUQT demonstrated the highest stability across all tested conditions.
• HuEF1-α, HuUBQ, and HuUQT were the most stable under abiotic stresses (salt, drought, heat) and across cultivars.
• HuEF1-α, HuACT, and HuUQT showed stable expression across different pitaya tissues.

Conclusions:

• The combined use of HuUBQ and HuUQT is recommended as the optimal reference for pitaya gene expression studies.
• Specific reference genes (HuEF1-α, HuUBQ, HuUQT, HuACT) are suitable for particular experimental conditions in pitaya.
• This study provides essential data for selecting appropriate reference genes in pitaya research.