Assessing the Suitability of Reference Genes for Normalising Gene Expression in Ganoderma boninense-Infected Oil Palm
Keywords:
Oil palm reference genes, qPCR, Tissue culture, Basal stem rotAbstract
Oil palm is a major agricultural commodity in Southeast Asia, particularly in Malaysia and Indonesia. Its productivity, however, is being jeopardised by climate change and disease outbreaks, particularly basal stem rot (BSR) caused by Ganoderma boninense. Monoculture practices exacerbate disease spread, calling for innovative and sustainable solutions. Beyond current integrated plant management (IPM) practices, developing disease-tolerant plant material could enhance disease management. For that purpose, efforts have been initiated to identify potential genetic markers associated with G. boninense tolerance; include in vitro screening (IVS) using oil palm clonal material is employed, where artificial infection is conducted under a controlled environment. However, the reliability of such studies depend on accurate quantification of gene expression, which requires the use of reliable reference genes (RGs). Quantitative PCR (qPCR) is a powerful tool for measuring gene expression levels; however, reliable normalisation depends on RGs that maintain consistent expression across tissues and experimental conditions. Consistent RG expression is essential for minimising technical and biological variation, ensuring that observed changes in target gene expression reflect true biological responses rather than variation arising from the normalisation process. Therefore, this project evaluated ten potential RGs selected from the literature for gene expression analysis in young oil palm ramets under controlled G. boninense-infected conditions. Among the ten RGs evaluated, ubi and fbox were consistently identified as the most stable and reliable RGs for in vitro expression analysis under controlled disease-stressed conditions, based on geNorm, NormFinder, BestKeeper, and RefFinder analyses. In contrast, gapdh was unsuitable as an RG under these conditions. The RGs identified and validated in this study are anticipated to enhance the accuracy of gene expression analysis in G. boninense-infected oil palm ramets under controlled in vitro conditions. This approach will be instrumental in identifying potential defence-related genes associated with G. boninense tolerance through comprehensive gene expression profiling.
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