Identification of stable reference gene for transcript normalization in black pepper-Phytophthora capsici...

Area of Science:

• Plant Molecular Biology
• Genomics and Transcriptomics
• Plant Pathology

Background:

• Accurate gene expression studies rely on stable reference genes for normalizing transcript levels.
• Black pepper (Piper nigrum) is susceptible to biotic stresses, necessitating reliable gene expression analysis tools.
• Previous studies often lack systematic validation of reference genes in Piper nigrum.

Purpose of the Study:

• To systematically identify and validate the most stable reference genes in black pepper (Piper nigrum) for gene expression studies.
• To evaluate the impact of reference gene selection on the expression analysis of a target gene (PnBGLU) during Phytophthora stress.
• To determine the most suitable reference gene for the Piper nigrum-Phytophthora capsici pathosystem.

Main Methods:

• Utilized annotated black pepper leaf transcriptome sequences for candidate reference genes: actin (PnACT), glyceraldehyde phosphate dehydrogenase (PnGAPDH), β-tubulin (PnTUB), ubiquitin conjugating enzyme (PnUBCE), 18srRNA, and elongation factor-1-α (PnElF).
• Employed multiple algorithms (geNorm, NormFinder, BestKeeper, Reffinder) and NormqPCR (R package) to assess reference gene stability under Phytophthora biotic stress.
• Analyzed the expression patterns of the target gene 1,3 beta glucanase (PnBGLU) using both the most stable and least stable identified reference genes.

Main Results:

• Identified PnGAPDH and PnUBCE as the most stable reference genes across different algorithms.
• Demonstrated that using the least stable reference gene led to significant overestimation and misinterpretation of PnBGLU gene expression levels.
• PnGAPDH was confirmed as the most suitable reference gene for analyzing PnBGLU expression dynamics in resistant and susceptible black pepper genotypes challenged with Phytophthora capsici.

Conclusions:

• Systematic validation of reference genes is essential for reliable gene expression studies in black pepper.
• The choice of reference gene significantly impacts the accuracy of gene expression data, particularly for target genes like PnBGLU.
• PnGAPDH is recommended as the optimal reference gene for studies involving the Piper nigrum-Phytophthora capsici pathosystem.