In this study, a double-mismatch allele-specific (DMAS) qPCR SNP genotyping method has been designed, tested and validated specifically for cacao, using 65 well annotated international cacao reference accessions retrieved from the Center for Forestry Research and Technology Transfer (CEFORTT) and the International Cocoa Quarantine Centre (ICQC). In total, 42 DMAS-qPCR SNP genotyping assays have been validated, with a 98.05% overall efficiency in calling the correct genotype. In addition, the test allowed for the identification of 15.38% off-types and two duplicates, highlighting the problem of mislabeling in cacao collections and the need for conclusive genotyping assays. The developed method showed on average a high genetic diversity (H
e
= 0.416) and information index (I = 0.601), making it applicable to assess intra-population variation. Furthermore, only the 13 most informative markers were needed to achieve maximum differentiation. This simple, effective method provides robust and accurate genotypic data which allows for more efficient resource management (e.g. tackling mislabeling, conserving valuable genetic material, parentage analysis, genetic diversity studies), thus contributing to an increased knowledge on the genetic background of cacao worldwide. Notably, the described method can easily be integrated in other laboratories for a wide range of objectives and organisms.
Despite the high economic importance of cacao beans, few RNA-based studies have been conducted on this plant material and hence no optimal RNA-extraction has been reported. Moreover, extraction of high-quality RNA from recalcitrant cacao bean tissue has shown many difficulties and requires optimization. Furthermore, cacao beans are mostly found at remote and under-resourced locations, which pressures the outsourcing of such analysis and thereby demands RNA-stable preservation and transportation of cacao beans. This study aims to select an appropriate RNA extraction and preservation/ transportation method for cacao beans. For this purpose, three sample homogenization and five extraction protocols on cacao beans were compared. In addition, 13 preservation conditions-differing in tissue crushing degree, preservation method, duration, and temperature-were compared and evaluated. A comparative analysis revealed that CTAB-based homogenization and extraction outcompeted all tested commercial protocols in RNA yield and integrity, respectively. Preservation at −80°C affected RNA quality the least, whereas freeze-drying was most suitable for transportation at room temperature for maximum 1 week. The cacao bean RNA obtained from the selected methods were compatible for downstream applications. The results of this study will facilitate on-field sampling and transportation of genetically sensitive cacao material prior to cacao bean transcriptomic studies. In addition, valuable insights on sample homogenization, extraction, preservation, and transportation have been provided, which is of interest to every plant geneticist.
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.
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