The development of a novel SNP genotyping assay to differentiate cacao clones

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 (He = 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

A combined RNA preservation and extraction protocol for gene expression studies in cacao beans

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 degrees 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

From genetics to chemical characterisation and flavour profiling of Vietnamese cocoa

Theobroma cacao L. is the source of chocolate, one of the world’s most renowned sweets. Chocolate quality vastly depends on the quality of the key ingredient, cocoa beans. Cocoa quality is a complex parameter influenced by several factors, including others origin (genetic and geographical), processing, and associated flavour potential. Little is known about Vietnamese cocoa, while this might be an interesting source for single-origin chocolate, possibly with unique flavours. Thus, an interdisciplinary study was conducted, including genetics, chemical characterisation and flavour profiling, to unravel the quality potential of Vietnamese cocoa. Genetic analysis appeared essential for correct varietal determination, and mainly classified Vietnamese cocoa as Trinitario hybrids, suggesting fine flavour potential. The quality evaluation of cultivar-specific dried fermented cocoa beans, via instrumental (un)targeted analyses, illustrated differences at cultivar level, while the need for a proper fermentation was clearly emphasized. Cultivar-specific cocoa liquors sensorially differed in sourness, along with bitterness and astringency. Several trends were observed between the chemical composition, via instrumental (un)targeted analyses, and these sensory attributes. Finally, two Vietnamese cultivars were suggested as promising fine flavour cultivars, with potential profitable uniqueness. In general, the obtained results might aid the rejuvenation of the Vietnamese cocoa industry and lead Vietnam to gain a spot in the world market

Unraveling the genetic background of the Yangambi Research Center cacao germplasm collection, DR Congo

The Democratic Republic of the Congo (DR Congo or DRC) has some of the most fertile soils in Africa to cultivate cacao, an important cash crop and source of income for many smallholder farmers. Although cacao is currently produced there on small scale, DRC has the potential to grow as a cacao-producing country, thereby supplying the increasing cacao demand in the global market. Since the introduction of cacao varieties in the late nineteenth century, selection and breeding experiments have been carried out based on phenotype, without any knowledge on the genetic background of the cultivars. Therefore, this study analyzes 62 Congolese accessions of the Centre de Recherche de Yangambi (CRY) and 51 accessions of international collections representing 10 reference groups and Trinitario cultivars using 14 microsatellite markers. Descriptive statistics revealed a high gene diversity and polymorphic information content (PIC) for most of the markers, of which mTcCIR 12, 37, and 60 were the most discriminative. Both Bayesian clustering and principal coordinate analysis (PCoA) revealed high-admixed ancestry of the CRY cultivars. The collection was divided in two clusters, of which the first formed a hybrid population, linked to Amelonado, Trinitario, MaraIOEn, and Nanay, and the second was assigned to predominantly Amelonado, followed by Nanay, Contamana, and Nacional. This high admixture level resulted from numerous hybridization and recombination events that took place in the previous century. The obtained knowledge is essential for efficient conservation, utilization, and selection of high-quality cacao cultivars, which are agronomical favored, disease resistant, and of superior flavor quality

Discrimination of cocoa liquors based on their odor fingerprint : a fast GC electronic nose suitability study

With the rising interest by consumers for high-quality cocoa products from a clear geographical origin, a rapid analytical method for quality control, authenticity and traceability assessment is of paramount importance. However, the complex mixture of volatiles present in cocoa liquor, the main ingredient for the chocolate production, complicates reaching this purpose. Hence, an analytical fingerprint approach using advanced electronic nose (E-nose) technology may offer a suitable technique. This study aimed to verify the suitability of an E-nose based on ultra-fast gas chromatography (GC) for the rapid discrimination between cocoa liquors from different origins. Fourteen cocoa liquors, produced of cocoa beans from ten different geographical origins, were analyzed. The obtained odor fingerprints were investigated using principal component analysis (PCA) which successfully discriminated most cocoa liquors, within one continent, according to their geographical origin. Besides, discriminant factor analysis (DFA) showed the possibility to differentiate between bulk and fine cocoa. Further tentative identification of predominant volatile compounds allowed the detection of compounds within a wide range of chemical classes occurring in cocoa products, such as acids, alcohols, aldehydes, ketones, esters, pyrazines, pyrones, and pyrroles. Most odorant compounds were previously described in literature as key volatiles in cocoa flavor, notable examples are acetic acid, 2-heptanol, 2/3-methylbutanal, acetophenone, isoamyl acetate, tetramethylpyrazine, maltol, and 2-acetyl-1-pyrroline. This study proves for the first time the usefulness of the GC E-nose for effective and rapid aroma profiling and discrimination between single origin cocoa liquors, which can be easily applied in the cocoa industry

Availability of prostate cancer exercise rehabilitation resources and practice patterns in Belgium : results of a cross-sectional study

Exercise is recommended for prostate cancer (PCa) patients treated with androgen deprivation therapy. The goal of the study was to assess the availability of hospital-based rehabilitation resources and national practice patterns for PCa in Belgium. A questionnaire was conducted with rehabilitation physical therapists in all Belgian hospital with urology and rehabilitation departments. Practice patterns were compared with the American College of Sports Medicine guidelines. PCa prevalence data were obtained from the Belgian Cancer Registry and attitude of physicians towards physical activity was documented. We included 98 Belgian hospitals. Only 25% of the PCa population had access to PCa-specific programmes. The occupancy rate of PCa-specific rehabilitation slots was 69%. The main perceived barriers to organise PCa-specific rehabilitation were existence of general programmes (40%) and low referrals (18%). All PCa programmes consisted of aerobic and resistance exercise and 62% included flexibility. Minimal criteria for frequency and duration per session were followed in 83%. The majority (89%) of physicians believed in the positive effects of supervised exercise programmes. The majority of PCa programmes follow the evidence-based guidelines except for flexibility exercises. The minority of PCa patients has access to specific programmes, although not all treatment slots are occupied

Phylogenetic relationships among Vietnamese cocoa accessions using a non-coding region of the chloroplast DNA

Cocoa (Theobroma cacao L.) cultivation has increased in tropical areas around the world, including Vietnam, due to the high demand of cocoa beans for chocolate production. The genetic diversity of cocoa genotypes is recognized to be complex, however, their phylogenetic relationships need to be clarified. The present study aimed to classify the cocoa genotypes that are imported and cultivated in Vietnam based on a chloroplast DNA region. Sixty-three Vietnamese Cocoa accessions were collected from different regions in Southern Vietnam for this research. Their phylogenetic relationships were identified using the universal primers c-B49317 and d-A49855 from the chloroplast DNA region. The sequences were situated in the trnL intron genes among the illustration terrestrial plant species of the chloroplast genome. DNA sequences were determined and subjected to an analysis of the phylogenetic relationship using the maximum evolution method. The genetic analysis showed clustering of 63 cocoa accessions in three groups: the domestically cultivated Trinitario group, the Indigenous cultivars, and the cultivations from Peru. The analyzed sequencing data also illustrated that the TD accessions and CT accessions were closely phylogenetically related. Based on these results the PA and NA accessions were established as the hybrid origins of the TD and CT accessions. Some foreign accessions, including UIT, SCA and IMC accessions were confirmed of their genetic relationship. The present study is the first report of phylogenetic relationships of Vietnamese cocoa collections. The cocoa program in Vietnam has been in development for thirty years