Comprehensive validation of reference genes in Solanum nigrum under abiotic stresses: Identifying...

Black nightshade (Solanum nigrum L.), a globally invasive weed of the Solanaceae family, exhibits remarkable adaptability to diverse abiotic stresses. Despite its ecological and agricultural significance, validated reference genes (RGs) for reliable RT-qPCR normalization in this species remain undefined. This study systematically evaluated eight candidate RGs under six abiotic stresses, including cold, drought, salinity, heat, cadmium (Cd) exposure and pendimethalin treatment, using four computational algorithms (Delta Ct, geNorm, NormFinder and BestKeeper) integrated through RefFinder. Stress-specific optimal RG pairs were identified: TUB and EF-2 (cold), ACT and TUA (drought), GAPDH and TUA (salt), TUA and EIF4A (Cd), TUA and EF-2 (heat), TUB and ACT (pendimethalin). We prioritised the investigation of candidate genes involved in S. nigrum response to pendimethalin using RNA-seq due to its escalating agricultural challenges. By using optimal RG pairs, we validated the candidate genes via RT-qPCR, including five transcription factors (WRKY56, MYB93, bHLH96-1, bHLH96-2, ARR2), three herbicide-metabolizing enzymes (CYP71A4, CYP735A1, 2OG-Fe(II) oxygenase), and a peroxidase (POD) gene (PER64), which may be associated with adaptive evolution to pendimethalin. These findings establish the first standardized framework for gene expression normalization in S. nigrum leaves, providing a basis for the reliable validation of transcriptomic data and the molecular mechanisms underlying weed adaptation to herbicides.