Effect of cocoa (Theobroma cacao L.) on platelet function testing profiles in patients with coronary artery disease: ECLAIR pilot study.

INTRODUCTION: This prospective pharmacodynamic nutraceutical study assessed the effect of a 1-week trial of 30 g/day of 65% cocoa (dark chocolate) (Theobroma cacao L.) consumption intervention on platelet reactivity. METHODS: Patients with stable coronary artery disease (CAD) (n=20) who were on maintenance dual antiplatelet therapy of aspirin (ASA) 81 mg/day and clopidogrel 75 mg/day were recruited. Platelet function was evaluated with the VerifyNow P2Y12 reaction unit (PRU) and aspirin reaction unit (ARU) assays (Werfen, Bedford, Massachusetts, USA) and assessed prior to initiation of and after a 1-week trial of 30 g/day of 65% cocoa consumption intervention. Results were compared with a paired t-test. RESULTS: Cocoa augmented the inhibitory effect of clopidogrel, demonstrated by a reduction of 11.9% (95% CI 5.7% to 18.0%, p value 0.001), significantly decreasing the PRU by 26.85 (95% CI 12.22 to 41.48, p value 0.001). The inhibitory effect of ASA was not impacted by cocoa, reflected by a non-significant reduction in ARU of 17.65 (95% CI 21.00 to 56.3, p value 0.351). No patients experienced any serious adverse events. CONCLUSIONS: Cocoa augmented the inhibitory effect of clopidogrel but not ASA. This nutraceutical study could be potentially informative and applicable for patients with stable CAD. Further long-term studies are required to confirm these exploratory findings. TRIAL REGISTRATION NUMBER: NCT04554901.

Comparative transcriptomic analysis reveals key components controlling spathe color in Anthurium andraeanum (Hort.).

Anthurium andraeanum (Hort.) is an important ornamental in the tropical cut-flower industry. However, there is currently insufficient information to establish a clear connection between the genetic model(s) proposed and the putative genes involved in the differentiation between colors. In this study, 18 cDNA libraries related to the spathe color and developmental stages of A. andraeanum were characterized by transcriptome sequencing (RNA-seq). For the de novo transcriptome, a total of 114,334,082 primary sequence reads were obtained from the Illumina sequencer and were assembled into 151,652 unigenes. Approximately 58,476 transcripts were generated and used for comparative transcriptome analysis between three cultivars that differ in spathe color ('Sasha' (white), 'Honduras' (red), and 'Rapido' (purple)). A large number of differentially expressed genes (8,324), potentially involved in multiple biological and metabolic pathways, were identified, including genes in the flavonoid and anthocyanin biosynthetic pathways. Our results showed that the chalcone isomerase (CHI) gene presented the strongest evidence for an association with differences in color and the highest correlation with other key genes (flavanone 3-hydroxylase (F3H), flavonoid 3'5' hydroxylase (F3'5'H)/ flavonoid 3'-hydroxylase (F3'H), and leucoanthocyanidin dioxygenase (LDOX)) in the anthocyanin pathway. We also identified a differentially expressed cytochrome P450 gene in the late developmental stage of the purple spathe that appeared to determine the difference between the red- and purple-colored spathes. Furthermore, transcription factors related to putative MYB-domain protein that may control anthocyanin pathway were identified through a weighted gene co-expression network analysis (WGCNA). The results provided basic sequence information for future research on spathe color, which have important implications for this ornamental breeding strategies.

Identification of Cacao Mild Mosaic Virus (CaMMV) and Cacao Yellow Vein-Banding Virus (CYVBV) in Cocoa (Theobroma cacao) Germplasm.

Cocoa, Theobroma cacao, is an important tropical perennial crop grown widely in the humid tropics. The exchange of cocoa germplasm between germplasm collections and breeding centres is vital for varietal development. Intermediate quarantine facilities, such as the International Cocoa Quarantine Centre, Reading UK (ICQC-R) play a vital role in ensuring the transfer of germplasm whilst minimising the risk of spreading pests and diseases. Current screening procedures combine visual inspection and molecular techniques, which are effective in detecting Cocoa swollen shoot virus (CSSV), a badnavirus, which causes severe losses but are restricted to West Africa. However, the detection of latent or mild virus infections that produce no visual symptoms has been a challenge. Recently two badnavirus species of cocoa producing mild symptoms, cacao mild mosaic virus (CaMMV) and cacao yellow vein-banding virus (CYVBV), have been sequenced. Here, we report new assays for the detection of these two species, for the first time in non-symptomatic accessions. Evolutionary and bioinformatic analyses of the viruses suggest their most recent source was from Trinidad, though there is historic evidence that these viruses may have their origin in South America and then become widespread globally over the last century. We also report a novel colorimetric Loop-mediated isothermal amplification (LAMP) assay for the detection of CYVBV. This simple and accurate method could be employed in field virus testing.

Genetic variation in high light responses of Theobroma cacao L. accessions.

Cacao (Theobroma cacao L.) is a shade-tolerant tree species, but in recent years it has increasingly been cultivated under full sun conditions in an orchard system where photoinhibition is likely. Here we investigate the extent of photoinhibition in 17 cacao accessions from a range of genetic groups, growing under high light conditions. The ability of the photosynthetic systems to respond to high light was assessed using chlorophyll fluorescence parameters (diurnal F v /F m and instantaneous light response curves), and differences in photosynthetic pigment content were compared using biochemical assays. Damage due to photoinhibition was assessed using electrolyte leakage, lipid peroxidation, and reactive oxygen species scavenging systems were compared using biochemical assays (for APX, CAT and SOD). There was significant variation between the 17 accessions for photosynthetic parameters, although in all cases the light saturation points were well below the midday light levels. Light acclimation of photosynthetic pigments was evident and variation in the total chlorophyll to total carotenoid ratio was significantly correlated with electrolyte leakage. Significant genetic variation was observed across the 17 accessions in the activities of CAT, APX and SOD. Across all accessions, photoprotection appeared to be restricted by the ability of leaves to generate SOD. Significant negative correlations were observed between SOD activity and both APX activity and electrolyte leakage, while significant positive correlations were observed between electrolyte leakage and both APX and CAT activity. Accessions with higher light saturation points, as well as high carotenoid and high SOD concentrations were able to tolerate the moderately high light, however, none of the accessions were clearly superior to the commonly grown Amelonado accession. The results imply that screening for SOD activity, total carotenoid content and light saturation point can aid in selection of genotypes with better tolerance to high light.

Plant metal concentrations in Theobroma cacao as affected by soil metal availability in different soil types.

Beans of cacao (Theobroma cacaoL.) are used to produce a variety of chocolate products. Bioaccumulation of metals at toxic levels through the consumption of contaminated products has been identified as a health concern in humans. Both metal diversity and concentration as well as their interactions in the soil influence essential and non-essential metal uptake in plants; but the effects of these on bioaccumulation of metals in cacao is not understood across diverse soil types. In this study eight metals (Cd, Cr, Cu, Fe, Mn, Ni, Pb, and Zn) were investigated in 12 soil subgroups belonging to four soil orders across 15 locations in Trinidad, with the aim to investigate the effect of soil metal diversity and concentration on metal bioaccumulation in cacao. Soil metals were extracted using five methods (aqua regia, DTPA, Mehlich 3, nitric acid, and water). Cacao leaf metal concentrations were determined using the USEPA 3052 method. Metal extraction efficiency ranged between methods with aqua regia ≥ nitric acid > Mehlich 3 ≥ DTPA ≥ water across all metals. The soil extraction method that best predicted cacao leaf metal concentrations varied with the metal - Mehlich 3 or DTPA for Cd, Ni, Zn; aqua regia, Mehlich 3, or nitric acid for Pb, and water for Mn. A stepwise regression analysis showed that plant metal concentration can be predicted using soil physicochemical characteristics as well as the concentration of metals in the soil. The importance of soil type on cacao leaf metal bioaccumulation is discussed.

Genetic diversity and ancestry of cacao (Theobroma cacao L.) in Dominica revealed by single nucleotide polymorphism markers.

Cacao (Theobroma cacao L.), an introduced tree crop in Dominica, is important for foreign exchange earnings from fine or flavour cocoa. The genetic structure of farmed cacao in Dominica was examined to identify varieties for conservation, breeding, and propagation to improve their cocoa industry. Cacao trees (156) from 73 sites over seven geographical regions were genotyped at 192 single nucleotide polymorphism (SNP) markers. Identity, regional differentiation, phylogenetic, multi-variate, ancestry, and core collection analyses were performed. Farmed cacao germplasm had moderate gene diversity (He = 0.320 ± 0.005) from generally unique trees, but cocoa growing regions were genetically similar. Synonymous matching (16.3%) showed that some clonal material was supplied to farmers. Cacao trees were mainly mixed from Amelonado, Criollo, Iquitos, Contamana, and Marañon ancestries, with predominantly Amelonado-Criollo hybrids. Criollo ancestry, linked to fine or flavour cocoa, was found at more than 30% in 28 unique trees. Forty-five trees, containing the SNP diversity of cacao in Dominica, are recommended as a core germplasm collection. This study identifies promising trees for improving cocoa quality; provides genetic evidence that community, regional, or country-wide pooling would not compromise the exclusive fine or flavour cocoa industry; and discusses other implications towards improving the Dominican cocoa industry.

Development of a core SNP panel for cacao (Theobroma cacao L.) identity analysis.

Single nucleotide polymorphisms (SNPs) are preferred markers for DNA fingerprinting and diversity studies in cacao (Theobroma cacao L.). Yet, a consensus SNP panel with a minimum number of SNPs for optimal identity analysis is unavailable for cacao. An initial set of 146 SNP panels of varying sizes were assembled based on heterozygosity, linkage disequilibrium (LD), linkage group (LG) distribution, major allele frequency, minor allele frequency (MiAF), polymorphism information content (PIC), and random distribution. These panels were assessed to determine their ability to distinguish among a training set of 155 accessions. The panels with the best separation ability were supplemented with additional SNPs to create 16 designer panels, which separated all 155 accessions. The 16 designer SNP panels were then assessed on a dataset of 1220 accessions coming from 10 ancestral groups. Increasing the number of SNPs generally yielded improved resolution of genetic identities with concomitant reduction of synonymous groups. The number and choice of SNPs were critical factors with LD, MiAF, and PIC being important selection attributes but an even LG distribution was unnecessary. A robust set of 96 SNPs is recommended as a minimal core SNP panel for cacao DNA fingerprinting to the international cacao community.

Genetic variation in bioaccumulation and partitioning of cadmium in Theobroma cacao L.

Cadmium (Cd) is a non-essential heavy metal that is toxic to both plants and animals and chocolates have been identified as a contributor to the human dietary Cd intake. One hundred accessions representing the various genetic groups and hybrid populations in Theobroma cacao L. held at the International Cocoa Genebank, Trinidad were evaluated for leaf and bean cadmium levels with three tree replications. Representative samples of soil from the drip zone around each tree were evaluated for bioavailable cadmium. Although there were significant differences (P ≤ 0.05) among genetic groups for leaf and bean Cd much of the variation was between accessions. There was a 13-fold variation in bean Cd and a 7-fold variation in leaf Cd between accessions despite the bioavailable Cd in the soil being uniform. There were differences in the level of partitioning into beans evident by significant variation (P ≤ 0.05) in bean Cd as a percentage of the cumulative leaf and bean Cd concentration (15-52%) between accessions. Although in general there was a higher concentration of cadmium in the testa than the cotyledon of the cocoa bean there was considerable genetic variation. These results point to the potential of using a genetic strategy to mitigate cadmium within cocoa beans either through breeding or through the use of low cadmium uptake rootstocks in grafting. The results will fuel further work into the understanding of mechanisms and genetics of cadmium uptake and partitioning in cocoa.

Association mapping of seed and disease resistance traits in Theobroma cacao L.

MAIN CONCLUSION: Microsatellite and single nucleotide polymorphism markers that could be used in marker assisted breeding of cacao were identified for number of filled seeds, black pod resistance and witches' broom disease resistance. An association mapping approach was employed to identify markers for seed number and resistance to black pod and witches' broom disease (WBD) in cacao (Theobroma cacao L.). Ninety-five microsatellites (SSRs) and 775 single nucleotide polymorphisms (SNPs) were assessed on 483 unique trees in the International Cocoa Genebank Trinidad (ICGT). Linkage disequilibrium (LD) and association mapping studies were conducted to identify markers to tag the phenotypic traits. Decay of LD occurred over an average 9.3 cM for chromosomes 1-9 and 2.5 cM for chromosome 10. Marker/trait associations were generally identified based on general linear models (GLMs) that incorporated principal components from molecular information on relatedness factor. Seven markers (mTcCIR 8, 66, 126, 212; TcSNP368, 697, 1370) on chromosomes 1 and 9 were identified for number of filled seeds (NSEED). A single marker was found for black pod resistance (mTcCIR280) on chromosome 3, whereas six markers on chromosomes 4, 5, 6, 8, and 10 were detected for WBD (mTcCIR91, 183; TcSNP375, 720, 1230 and 1374). It is expected that this association mapping study in cacao would contribute to the knowledge of the genetic determinism of cocoa traits and that the markers identified herein would prove useful in marker assisted breeding of cacao.

Partial 16S rRNA gene sequence diversity and numerical taxonomy of slow growing pigeonpea (Cajanus cajan L Millsp) nodulating rhizobia.

An investigation was carried out to determine the diversity of 30 isolates of slow growing pigeonpea nodulating rhizobia based on variations in partial sequences of the 16S rRNA gene and numerical analysis of 80 phenotypic traits. Phylogenetic analysis using molecular sequences of 23 isolates showed that ARPE1 separated from the other isolates at an average distance of >14% divergence level. The other isolates were all within 5% divergence from each other but separated into four main groups, with group 1 containing 16 of the 23 isolates. Comparisons to sequences of reference strains revealed that the group 1 isolates were phylogenetically closely related to the slow growing soybean nodulating rhizobia belonging to Bradyrhizobium elkanii, although only three of these isolates were able to nodulate soybean. Numerical analysis of phenotypic data of 19 isolates showed that 14 isolates clustered together in one branch of the phenogram, which included the group 1, group 2 and group 4 isolates from the phylogenetic analysis. The group 3 isolates were highly variable in the phenogram with similarity levels lower than 50% among these isolates.

Distribution and diversity of geminiviruses in Trinidad and Tobago.

Seven crop and eight weed species from 12 agricultural locations in Trinidad and Tobago were assayed for the presence of whitefly-transmitted geminiviruses (WTGs) by using dot blot hybridization and polymerase chain reaction (PCR) amplification of the N-terminal coat protein sequence with degenerate primers. The amplified fragments were cloned and analyzed by restriction enzyme digestion to determine fragment length polymorphism among the cloned fragments. Representative clones were then sequenced and subjected to phylogenetic analysis to determine the sequence similarity to known WTGs. WTGs were found in every location sampled and in 10 of the 15 species investigated: Lycopersicon esculentum(tomato), Capsicum annuum (pepper), Capsicum frutescens (sweet pepper), Abelmoschus esculentus (okra), Phaseolus vulgaris (beans), Alternanthera tenella, Desmodium frutescens, Euphorbia heterophylla, Malva alceifolia, and Sida acuta. The geminiviruses infecting these plants were closely related to potato yellow mosaic virus from Venezuela (PYMV-VE) and tomato leaf curl virus from Panama (ToLCV-PA). However, in pepper, sweet pepper, okra, Alternanthera tenella, Euphorbia heterophylla, Des-modium frutescens, and in one sample of tomato, a PYMV-VE-related virus was found in mixed infections with a virus related to pepper huasteco virus. Full-length infectious DNA-A and DNA-B of a tomato-infecting geminivirus from Trinidad and Tobago were cloned and sequenced. DNA-A appears to be a recombinant derived from PYMV-VE or ToLCV-PA, and Sida golden mosaic from Honduras. The implications of these findings in the control of WTGs are discussed.