First report of sweepoviruses infecting Ipomeas batatas L. cultivars and landraces in Trinidad.

Root crops, referred to as ground provisions in the Caribbean, are traditional staples in Trinidad. One widely consumed example is sweet potato (Ipomeas batatas L.). The crop is mainly produced by subsistence farming which together with imports from neighboring Caribbean countries meet domestic demand (Singh et al. 2008). The Central Experiment Station, situated in the eastern part of Trinidad, maintains a sweet potato germplasm collection comprising both imported and locally-sourced landraces for cultivar development and distribution of propagules. In May 2017 chlorosis and leaf curling symptoms, typically associated with sweepoviruses, were observed on imported cultivars, Centennial, Jewel, 86 BM 31, TIB 313, TIB 8 21 1, and S128, and the landraces, Kick Up Jenny, John, and Carrot. Leaf samples from these nine symptomatic plants were collected for analysis, along with samples from the asymptomatic landrace, Chickenfoot. Total nucleic acids were extracted (Sharma et al. 2008) and the samples were assayed by PCR using degenerate primers SPG1 and SPG2 (Li et al. 2004) that target the replication associated protein gene (ORF C1), a highly conserved region of sweepoviruses. Amplicons of 912-bp were obtained from two of the nine symptomatic plants (TIB 8 21 1, Kick Up Jenny), but not from the asymptomatic Chickenfoot. The same samples were assayed by PCR amplification using primers SpvF and SpvR (Avelar 2015) which are specific to a highly conserved 632-bp region of the coat protein gene (ORF V1) of sweet potato leaf curl virus (SPLCV). All 10 samples tested positive for SPLCV, including the asymptomatic landrace, Chickenfoot. The ORF V1 PCR products from three of the 10 samples, namely Chickenfoot, TIB 8 21 1, and Kick Up Jenny, were cloned and sequenced (two clones per sample). Comparison of the sequences (GenBank accession nos. OR882007 [Chickenfoot], OR913125 [TIB 8 21 1] and OR913126 [Kick Up Jenny]) identified up to 4% nt sequence variability between samples. In BLASTn analysis, they were most closely related to the SPLCV isolate China:Sichuan (GenBank accession no. KJ013557), sharing 94 to 98% nt identity. Total nucleic extracts from one representative sample (TIB 8 21 1) was used as template for rolling circle amplification (RCA, TempliPhi Amplification Kit, GE Healthcare Life Sciences, Piscataway, NJ, USA). Digestion of the RCA product with StuI (Thermo Scientific, MA, USA) yielded ~2.8 kb DNA fragments indicative of monomeric full length genomes. Digested fragments were cloned, completely sequenced and deposited in GenBank under the accession nos. OR866202 (2,821 nts) and OR866203 (2,828 nts). Two species of sweepoviruses were detected. In BLASTn analysis, OR866202 showed 95% nt identity with sweet potato golden vein associated virus (SPGVaV) US:MS:1B-3 (GenBank accession no. HQ333143.1) which is a recombinant virus comprised of SPLCV and sweet potato leaf curl Georgia virus (SPLCGV) (Zhang and Ling 2011) and in BLASTx analysis OR866202 was most similar (92-99%) to SPLCV isolates from Brazil (GenBank accession nos. ACI23475.1, AGW16179.1, ACY79479.1), Peru (GenBank accession no. ACY79466.1) and China (GenBank accession nos. ACY79439.1). OR866203 shared 96% nt identity with SPLCV China:Henan25(8):2012 (GenBank accession no. KF040465.1) in BLASTn analysis and BLASTx analysis revealed ≥ 94% aa sequence identity with SPLCV from Brazil (GenBank accession nos. ACI23475.1, AGW16179.1, ADZ96559.1), Peru (GenBank accession no. ACY79479.1), China (GenBank accession no. ACY79466.1). and Spain (GenBank accession no. QWQ56365.1). Both Trinidad isolates also showed 90-96% nt identity with SPLCV from Korea (GenBank accession no.s KT992061.1, KT992064.1, unpublished). This is the first detection of sweepoviruses in Trinidad. SPGVaV has been reported in Brazil, the United States and Korea (Kil et al. 2014), while SPLCV has been described in other Caribbean islands, including Cuba, Jamaica, Puerto Rico, St. Vincent (Cuellar et al. 2015), and Barbados (Alleyne et al. 2019), as well as several countries in South America. Although Koch's postulates were not completed, our findings suggest that sweet potato crops in Trinidad harbor sweepoviruses, notwithstanding efforts to distribute pathogen-free materials and, in some instances, the apparent absence of visible symptoms on infected plants. Further studies on the management and impact of these viruses are necessary, including their prevalence in the sweet potato production regions of Trinidad.

From soil to cacao bean: Unravelling the pathways of cadmium translocation in a high Cd accumulating cultivar of Theobroma cacao L.

The research on strategies to reduce cadmium (Cd) accumulation in cacao beans is currently limited by a lack of understanding of the Cd transfer pathways within the cacao tree. Here, we elucidated the transfer of Cd from soil to the nib (seed) in a high Cd accumulating cacao cultivar. Here, we elucidated the transfer of Cd from soil to the nib (seed) in a high Cd accumulating cacao cultivar through Cd stable isotope fractionation, speciation (X-Ray Absorption Spectroscopy), and localization (Laser Ablation Inductively Coupled Plasma Mass Spectrometry). The plant Cd concentrations were 10-28 higher than the topsoil Cd concentrations and increased as placenta< nib< testa< pod husk< root< leaf< branch. The retention of Cd in the roots was low. Light Cd isotopes were retained in the roots whilst heavier Cd isotopes were transported to the shoots (Δ 114/110 Cd shoot-root = 0.27 ± 0.02 ‰ (weighted average ± standard deviation)). Leaf Cd isotopes were heavier than Cd in the branches (Δ 114/110 Cd IF3 leaves-branch = 0.18 ± 0.01 ‰), confirming typical trends observed in annual crops. Nibs and branches were statistically not distinguishable (Δ 114/110 Cd nib-branch = -0.08‰ ± 0.06 ‰), contrary to the leaves and nibs (Δ 114/110 Cd nib-IF3 leaves = -0.25‰ ± 0.05 ‰). These isotope fractionation patterns alluded to a more direct transfer from branches to nibs rather than from leaves to nibs. The largest fraction (57%) of total plant Cd was present in the branches where it was primarily bound to carboxyl-ligands (60-100%) and mainly localized in the phloem rays and phelloderm of the bark. Cadmium in the nibs was mainly bound to oxygen ligands (60-90%), with phytate as the most plausible ligand. The weight of evidence suggested that Cd was transferred like other nutrients from root to shoot and accumulated in the phloem rays and phelloderm of the branches to reduce the transfer to foliage. Finally, the data indicated that the main contribution of nib Cd was from the phloem tissues of the branch rather than from leaf remobilization. This study extended the limited knowledge on Cd accumulation in perennial, woody crops and revealed that the Cd pathways in cacao are markedly different than in annual crops.

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.

Proteomic and peptidomic UHPLC-ESI MS/MS analysis of cocoa beans fermented using the Styrofoam-box method.

This work characterises the peptide and protein profiles of Theobroma cacao beans of the genotype IMC 67 at different fermentation stages, using the Styrofoam-box fermentation method and employing UHPLC-ESI MS/MS for the analysis of peptides and proteins extracted from the beans. A total of 1058 endogenous peptides were identified and quantified over four fermentation time points. The majority of these peptides were formed after 2 and 4 days of fermentation, and originated predominantly from the proteolysis of two storage proteins - vicilin and a 21 kDa albumin. The changes in the peptide profile over fermentation were subsequently evaluated, and potential markers for assessing the degree of fermentation were identified. In particular, changes of the relative abundance of the major cocoa proteins detected can be proposed as potential markers for the fermentation stage. Furthermore, PCA of both the peptidomic and proteomic data has allowed differentiation of beans at different fermentation stages.

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.

The effectiveness of soil amendments, biochar and lime, in mitigating cadmium bioaccumulation in Theobroma cacao L.

Cocoa (Theobroma cacao L.) is an important neotropical tree crop grown for its seeds or beans used in global chocolate and confectionary industries. Following studies showing ill effects of long-term dietary exposure of cadmium (Cd) on human health, a number of countries including the European Union (EU) have developed stringent regulations to protect consumers from exposure to cadmium. Cocoa is capable of bioaccumulating Cd in the cocoa beans when grown in soils high in cadmium and hence livelihood of cocoa farmers can be at risk if methods to mitigate the bioaccumulation of Cd are not developed. In vitro, greenhouse and field experiments were established with four, three and three replications respectively to evaluate the effectiveness of soil amendments, biochar and lime, at various application rates (0, 0.5×, 1×, 1.5× and 2× of the recommended rate), on soil pH, Cd phytoavailability and Cd bioaccumulation in Theobroma cacao L. For the in vitro study, Cd-containing soil was amended with 5 levels of biochar and lime, while for the greenhouse and field study four application rates were tested. The study showed that while lower rates were effective under in vitro conditions as you progressed from in vitro to greenhouse to field conditions the application rates and application frequency had to be increased, as the effectiveness and longevity of the treatments were compromised by environmental factors. Our study implies that the two amendments were complementary in their action and can be used in the recommended rated to reduce Cd bioaccumulation. However further studies are required on the placement of amendments to improve their effectiveness and longevity particularly under field conditions.

Morphological and genetic diversity of cacao (Theobroma cacao L.) in Uganda.

Cocoa is among the top foreign exchange earners in Uganda's agriculture sector and has benefitted the livelihood of farmers involved in production. Although cacao cultivation was adopted in the early 1900s, little is known about the on-farm diversity of the crop. A total of 125 cacao landraces were surveyed from eight districts in the Central and Western Regions to evaluate the morphological and genetic diversity of cacao in Uganda. Passport data included site, tree, fruit and seed information. Trees were genotyped using 96 single nucleotide polymorphism markers on a Fluidigm platform. Low heterozygosity was detected in the germplasm in both the Central [observed heterozygosity (H o ) = 0.295, expected heterozygosity (H e) = 0.334] and Western Regions (H o = 0.317, H e = 0.322). Genetic variation in both regions was generally comparable but the regions could be differentiated from each other. Inbreeding was noted in the Central Region while a greater sharing of genetic material was observed in the Western Region. The morphological and genetic data indicated that the Ugandan collection was an interspersed group with low to moderate variation with some separation of the Central from Western regions. Ancestry analysis indicated that the majority of the accessions were hybrids of Marañon lineage but also had Amelonado and Iquitos genetic backgrounds. These findings are consistent with the history of the movement of cacao into Uganda. A core collection of 18 individuals to represent the genetic diversity as well as 12 additional trees with possible advantageous traits is proposed.

Population genomic analyses of the chocolate tree, Theobroma cacao L., provide insights into its domestication process.

Domestication has had a strong impact on the development of modern societies. We sequenced 200 genomes of the chocolate plant Theobroma cacao L. to show for the first time to our knowledge that a single population, the Criollo population, underwent strong domestication ~3600 years ago (95% CI: 2481-13,806 years ago). We also show that during the process of domestication, there was strong selection for genes involved in the metabolism of the colored protectants anthocyanins and the stimulant theobromine, as well as disease resistance genes. Our analyses show that domesticated populations of T. cacao (Criollo) maintain a higher proportion of high-frequency deleterious mutations. We also show for the first time the negative consequences of the increased accumulation of deleterious mutations during domestication on the fitness of individuals (significant reduction in kilograms of beans per hectare per year as Criollo ancestry increases, as estimated from a GLM, P = 0.000425).

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.

Molecular characterization of previously elusive badnaviruses associated with symptomatic cacao in the New World.

Suspected virus-like symptoms were observed in cacao plants in Trinidad during 1943, and the viruses associated with these symptoms were designated as strains A and B of cacao Trinidad virus (CTV). However, viral etiology has not been demonstrated for either phenotype. Total DNA was isolated from symptomatic cacao leaves exhibiting the CTV A and B phenotypes and subjected to Illumina HiSeq and Sanger DNA sequencing. Based on de novo assembly, two apparently full-length badnavirus genomes of 7,533 and 7,454 nucleotides (nt) were associated with CTV strain A and B, respectively. The Trinidad badnaviral genomes contained four open reading frames, three of which are characteristic of other known badnaviruses, and a fourth that is present in only some badnaviruses. Both badnaviral genomes harbored hallmark caulimovirus-like features, including a tRNAMet priming site, a TATA box, and a polyadenylation-like signal. Pairwise comparisons of the RT-RNase H region indicated that the Trinidad isolates share 57-71% nt sequence identity with other known badnaviruses. Based on the system for badnavirus species demarcation in which viruses with less than 80% nt sequence identity in the RT-RNase gene are considered members of separate species, these isolates represent two previously unidentified badnaviruses, herein named cacao mild mosaic virus and cacao yellow vein banding virus, making them the first cacao-infecting badnaviruses identified thus far in the Western Hemisphere.

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.

Fast and neat--determination of biochemical quality parameters in cocoa using near infrared spectroscopy.

The qualitative heterogeneity and increasing consumption of cocoa products require fast and efficient methods for quality assessment of fermented cocoa with regard to fermentation quality and flavor potential. To date, quality control is achieved by visual inspection (e.g., "cut test") and sensory testing. Chromatographic methods for quantification of flavor relevant substances are limited in their applicability in standard quality control due to laborious isolation and purification steps. Therefore, the aim of this study was the development of a near infrared spectroscopic (NIRS) method for routine analytical prediction of biochemical quality parameters. Different compound classes like phenolic substances (R(2)=0.93) or organic acids (R(2)=0.88) as well as individual substances like epicatechin (R(2)=0.93) or lactic acid (R(2)=0.87) could be precisely determined just as fermentation time (R(2)=0.92) and pH value (R(2)=0.94) presenting NIRS as fast and reliable alternative in routine quality assessment.

A molecular assessment of the genetic model of spathe color inheritance in Anthurium andraeanum (Hort.).

Past genetic studies have shown three independent loci designated O, R and M control spathe color in Anthurium andraeanum (Hort.). To evaluate the genetic model and to understand the control of anthocyanin biosynthesis at the molecular level, the expression of the anthocyanin biosynthetic genes, CHS, F3H, DFR, ANS and F3'H, was examined at the mRNA and protein levels and correlated to anthocyanin content and spathe color in eight genetically characterized anthurium cultivars representing different states of the O, R and M loci. The results showed that the expression of F3H and ANS was co-regulated by a putative transcription factor encoded by the R locus, and the expression of DFR was regulated by a putative transcription factor encoded by the O locus. White cultivars, which were in the homozygous recessive state for either O or R or both, exhibited reduced expression of the anthocyanin biosynthetic genes and hence had negligible levels of anthocyanin. Cultivars that were mm displayed reduced expression of F3'H suggesting that it may either encode a defective form of the F3'H gene or a regulator that controls its expression. Additionally, a correlation between anthocyanin abundance and the expression of F3'H in the red cultivars suggested that F3'H expression may be a key control point in the regulation of anthocyanin biosynthesis in anthurium and hence plays a major role in influencing the shade intensity in red cultivars. These findings provide evidence in support of the genetic model for color inheritance in the spathe.

Discovery and mapping of a new expressed sequence tag-single nucleotide polymorphism and simple sequence repeat panel for large-scale genetic studies and breeding of Theobroma cacao L.

Theobroma cacao is an economically important tree of several tropical countries. Its genetic improvement is essential to provide protection against major diseases and improve chocolate quality. We discovered and mapped new expressed sequence tag-single nucleotide polymorphism (EST-SNP) and simple sequence repeat (SSR) markers and constructed a high-density genetic map. By screening 149 650 ESTs, 5246 SNPs were detected in silico, of which 1536 corresponded to genes with a putative function, while 851 had a clear polymorphic pattern across a collection of genetic resources. In addition, 409 new SSR markers were detected on the Criollo genome. Lastly, 681 new EST-SNPs and 163 new SSRs were added to the pre-existing 418 co-dominant markers to construct a large consensus genetic map. This high-density map and the set of new genetic markers identified in this study are a milestone in cocoa genomics and for marker-assisted breeding. The data are available at http://tropgenedb.cirad.fr.

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.