Optimization and identification of stable reference genes for RT-qPCR analysis in Capsicum annuum L.

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As a fundamental methodology in gene expression analysis, reverse transcription quantitative PCR (RT-qPCR) requires rigorous normalization using endogenous reference genes demonstrating expression stability across diverse experimental conditions. Although RT-qPCR has become ubiquitous in plant biotechnology, the rapid evolution of genomic technologies and burgeoning omics datasets have imposed stringent requirements for reference gene optimization, particularly in non-model organisms like pepper, in which high-quality genome assemblies have only recently been generated. Leveraging the chromosome-level Capsicum annuum 'Zunla-1' genome assembly, the study developed a reference gene validation framework that integrates five complementary algorithms or evaluation parameters (GeNorm, NormFinder, BestKeeper, ΔCt, and Cq values). Through systematic evaluation of nine evolutionarily conserved candidate genes (CaGAPDH1/2, CaUBIQUITIN1/2, CaTUBULIN1/2, CaActin1-3) across six tissue types (root, stem, leaf, flower, ovary, and fruit), and five experimental treatment conditions (IAA, GA3, waterlogging, drought, and pathogen), CaGAPDH2 and CaActin1 were validated as optimal reference genes in pepper. Specifically, CaActin1 exhibited exceptional stability under phytohormone treatments, establishing it as the optimal reference gene for hormonal response studies. CaGAPDH2 demonstrated robust performance across multiple stress conditions, validating its utility for stress-related expression profiling.