Selection of reference genes for quantitative real-time PCR in Cocos nucifera during abiotic stress

Reverse transcription quantitative real-time polymerase chain reaction (PCR) is a widely used and reproducible method for studying gene expression changes. However, its accuracy and reliability is highly dependent on the normalization step. Cocos nucifera L., a perennial palm with a long productive life span, is frequently exposed to soil and atmospheric drought and other stresses. In applying gene expression analysis to understand coconut stress responses, validation of suitable reference genes is an important first step. In this study, seven putative reference genes were identified from coconut transcriptome data. The stability of these putative reference genes was assessed in a diverse set of 18 coconut samples subject to cold, drought, and high-salinity treatment and representing different endosperm developmental stages. Using statistical algorithms geNorm, NormFinder, and BestKeeper, eEF1-alpha and UBC10 genes were identified as stable reference genes in all stress treatments and endosperm development stages, consistent with validated reference genes in rice (Oryza sativa L.), potato (Solanum tuberosum L.), and other species. Further validation of analyzed reference genes by normalization of a C-repeat binding factor (CBF)-like gene in cold-treatment samples indicated that the less stable reference genes produced different results from the more stable reference genes, with weaker variation or higher expression levels of a target gene. Our results will be beneficial for further research on molecular mechanisms of stress resistance.