Selection and validation of reference genes for gene expression normalization in coconut (Cocos nucifera L.) under biotic stress and hormone stimuli

Abstract

Coconut (Cocos nucifera L.), is as important tree crop of the tropical regions with multifarious uses. Coconut palms are highly susceptible to various biotic and abiotic stresses. Gene expression analysis in coconut would enable screening of stress responsive genes and accelerate the breeding process through identification of stress tolerant coconut genotypes. Investigations of gene expression entail accurate measurements of specific mRNA transcripts. Real-time quantitative PCR (RT-qPCR) is a reliable assay extensively utilized to quantify gene expression. To avoid any bias in RT-qPCR analysis, normalization with internal control genes, that show uniform expression pattern under diverse biological conditions, is mandatory. Identification of stable reference genes in coconut will enable enhanced accuracy of assays for selection of stress-responsive genes in this species. In the present study, four commonly used algorithms were utilized to analyze the expression stabilities of seven candidate reference genes (CnGAPDH, CnACT, CneIF-4γ, CnUBC10, Cn18S rRNA, CnEF-1α and Cn25S rRNA, under various conditions, viz., salicylic acid and methyl jasmonate stimuli and biotic stress (Phytophthora palmivora infection). Results showed that Cn18S rRNA and CnEF-1α possessed stable expression patterns during biotic stress, while CnEF-1α, Cn18S rRNA and CneIF-4γ displayed stability under hormone stimuli. Also, Cn18S rRNA and CnEF-1α were stable genes of choice among all the tested samples while CnUBC possessed the least stability. Analysis of gene expression profiles of CnWRKY was undertaken to corroborate the selected reference genes. The data generated through this study offers a basis for selection of reference genes and useful guidelines for further gene expression studies in coconut, in addition to benefitting other species of the Arecaceae.