Selection and validation of reference genes for accurate RT-qPCR gene expression normalization in cacao beans during fermentation

Abstract

To date, little attention has been paid to the genotypic plasticity and influence of the fermentation process on gene functions and biological processes in cacao beans. The primary tools for such analyses are gene expression studies with reverse transcription quantitative PCR (RT-qPCR). While this is a well-appreciated technique, it is only reliable when considering the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines, which is unfortunately barely applied in plant sciences and non-existent in cacao-related studies. In this study, an appropriate from bean to RT-qPCR protocol was developed. In total, sixty-five candidate reference gene (RG) assays were validated. These assays were either adopted from literature (traditional "housekeeping" genes) or based on RNA-sequencing data (novel). After validation, three novel reference genes (SUGP1, NAP1, SGT1) were recommended for normalization of gene expression within fermented cacao beans. The suitability of the novel candidates surpassed the traditional housekeeping genes. In addition, these assays seemed largely unaffected by RNA integrity. This is the first study to establish a standardized RT-qPCR workflow on cacao beans during fermentation, facilitating future studies. We recommend similar MIQE-based approaches for future gene expression studies on other organisms for miscellaneous objectives.

The GASA Gene Family in Cacao (Theobroma cacao, Malvaceae): Genome Wide Identification and Expression Analysis

“…This high expression may reveal the conserved function of these genes in the development of the bean and cacao flavor. Gene expression analysis of tcGASA genes in cacao beans in disease resistance (bulk) and disease susceptibility (fine flavor cacao) could be of interest [87,88]. The role of tcGASAs was also stated in the development of grapevine seeds [13].…”

Magnesium transporter Gene Family: Genome-Wide Identification and Characterization in Theobroma cacao, Corchorus capsularis, and Gossypium hirsutum of Family Malvaceae

“…The black rod disease caused by the genus Phytophthora is a production limiting factor, reducing cacao production by around 20-25% [79]. On the other hand, expression studies of genes in beans of bulk cultivars, like disease resistance and fine flavor cocoa (disease susceptibility), may be of interest [80,81]. In the current study, the expression level of TcMGT genes was investigated using RNA sequencing data responses to P. megakarya in two contrasting cultivars of T. cacao, susceptible and fungal-resistant cultivars.…”