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Proper cacao (Theobroma cacao L.) plant genotyping is mandatory for the conservation and use of the species genetic resources. A set of 15 international standard SSR markers was assumed as universal cacao genotyping system. Recently, different SNPs and SNP genotyping techniques have been exploited in cacao. However, a consensus on which to use has not been reached yet, driving the search for new approaches. To validate a new ddRADseq protocol for cacao genotyping, we compared the performances for population analysis of a dataset with 7,880 SNPs obtained from ddRADseq and the genotypic data from the aforementioned SSR set, using 158 cacao plants from productive farms and gene bank. Four genetic groups were identified with STRUCTURE and ADMIXTURE softwares using SSR and SNP data, respectively. Similarities of cacao ancestries among these groups allowed the identification of analogous pairs of groups of individuals, referred to as: G1SSR/G1SNP, G2SSR/G2SNP, G3SSR/G3SNP, G4SSR/G4SNP, whether SSRs or SNPs were used. Both marker systems identified Amelonado and Criollo as the most abundant cacao ancestries among all samples. Genetic distance matrices from both data types were significantly similar to each other according to Mantel test (p < 0.0001). PCoA and UPGMA clustering mostly confirmed the identified genetic groups. AMOVA and FST pairwise comparison revealed a moderate to very large genetic differentiation among identified groups from SSR and SNP data. Genetic diversity parameters from SSR (Hobs = 0.616, Hexp = 0.524 and PIC = 0.544) were higher than that from SNP data (0.288, 0.264, 0.230). In both cases, genetic groups carrying the highest Amelonado proportion (G1SSR and G1SNP) had the lowest genetic diversity parameters among the identified groups. The high congruence among population analysis results using both systems validated the ddRADseq protocol employed for cacao SNP genotyping. These results could provide new ways for developing a universal SNP-based genotyping system very much needed for cacao genetic studies.

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Humans have a long history of transporting and trading plants, contributing to the evolution of domesticated plants. Theobroma cacao originated in the Neotropics from South America. However, little is known about its domestication and use in these regions. In this study, ceramic residues from a large sample of pre-Columbian cultures from South and Central America were analyzed using archaeogenomic and biochemical approaches. Here we show, for the first time, the widespread use of cacao in South America out of its native Amazonian area of origin, extending back 5000 years, likely supported by cultural interactions between the Amazon and the Pacific coast. We observed that strong genetic mixing between geographically distant cacao populations occurred as early as the middle Holocene, in South America, driven by humans, favoring the adaptation of T. cacao to new environments. This complex history of cacao domestication is the basis of today's cacao tree populations and its knowledge can help us better manage their genetic resources.

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Water is a scarce, strategic resource and the most important input for economic development, especially in agricultural countries such as Brazil. Cocoa production is directly related to water availability, and, as climate changes, selecting drought-tolerant genotypes is vital to keep cacao crops sustainable. Here, we evaluated cacao genotypes under irrigated and water-stressed conditions and selected drought-tolerant ones based on nutritional and physiological traits. Thirty-nine genotypes were monitored for three years for agronomic traits and higher fruit yield. After this evaluation, the 18 most promising genotypes were evaluated in a randomized block design, under a 2 (with and without irrigation)  ×  18 (genotypes) factorial arrangement, with three replicates and five plants per plot. We evaluated seven physiological and 11 nutritional traits, selecting genotypes based on the Genotype-by-Trait Biplot approach. Significant effects (p < 0.05) were observed for the nutritional traits N, P, Mg, S, Zn, Cu, Mn and for the physiological traits CO2 assimilation rate (A), stomatal conductance (gs), transpiration (E), intercellular and atmospheric CO2 concentrations (Ci/Ca), intrinsic water use efficiency (A/gs), instantaneous water use efficiency (A/E), and instantaneous carboxylation efficiency (A/Ci), as determined by analysis of variance. The genotype  ×  irrigation treatment interaction was significant (p < 0.05) for the traits A, gs, and E. Genotypes CP 41, CP 43, and CCN 51 exhibited superior performance for both nutritional and physiological traits (A, gs, and E). In the irrigated environment, CP 41 showed superiority in traits such as P, A/E, A/gs, Mn, S, and Zn. Conversely, under non-irrigated conditions, CP 43 exhibited better performance in nutritional properties, specifically Mn, Mg, and Zn. Notably, in both irrigated and non-irrigated environments, CCN 51 excelled in key physiological traits, including A/Ci, A/E, and A/gs. This robust performance across diverse conditions suggests that these three genotypes possess physiological mechanisms to endure water-stressed conditions. Our research can generate valuable insights into these genotypes informing suitable choices for cocoa cultivation, especially in the context of global climate change.

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As part of a long-term study aiming to isolate and identify yeast species that inhabit the surface of leaves and fruits of native fine-aroma cacao in the department of Amazonas, Peru, we obtained multiple isolates of Hannaella species. Yeasts of the genus Hannaella are common inhabitants of the phyllosphere of natural and crop plants. On the basis of morphological, and physiological characteristics, and sequence analysis of the D1/D2 domains of the large subunit rRNA gene (LSU) and the internal transcribed spacer region (ITS), we identified five species of Hannaella from the phyllosphere of Peruvian cacao. Four have been previously described: H. phyllophila (isolates KLG-073, KLG-091), H. pagnoccae (KLG-076), H. sinensis (KLG-121), and H. taiwanensis (KLG-021). A fifth, represented by eight isolates (KLG-034, KLG-063, KLG-074, KLG-078, KLG-79, KLG-082, KLG-084, KLG-085), is not conspecific with any previously described Hannaella species, and forms the sister clade to H. surugaensis in the phylogenetic analysis. It has 2.6-3.9% (18-27 substitutions, 2-4 deletions, and 1-3 insertions in 610-938 bp-long alignments), and 9.8-10.0% nucleotide differences (37 substitutions and 14 insertions in 511-520 bp-long alignments) in the LSU and ITS regions, respectively, to H. surugaensis type strain, CBS 9426. Herein, the new species Hannaella theobromatis sp. nov. is described and characterised. The species epithet refers to its epiphytic ecology on its host Theobroma cacao.

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BACKGROUND: This study explored the impact of Leucothyreus femoratus, a previously unreported folivorous pest in cacao cultivation, on cacao tree survival, development, and yield. The study was conducted in an experimental cacao plot in the Colombian plains, it featured 20 cacao genotypes in an agroforestry system, with plantain and Mexican sunflower providing temporary shade, and yopo offering permanent shade. RESULTS: We found an infestation rate of 2.9 ± 0.3 adult beetles per cacao tree. L. femoratus larvae were discovered in association with the roots of all plants within the agroforestry arrangement; however, yopo and plantain exhibited the highest incidence of root-feeding larvae among these associated plants. Interestingly, male and female L. femoratus displayed distinct leaf consumption patterns in the laboratory, with females consuming more foliage relative to their body weight. Moreover, field observations highlighted the detrimental impact of L. femoratus herbivory on cacao tree survival and growth, leading to leaf skeletonization, reduced plant height, and stem diameter. Trees with over 50% leaf consumption suffered more than 20% mortality. Additionally, herbivory negatively affected cacao yield, correlating higher leaf surface damage with a decrease in harvested pods. The study also identified varying antixenotic resistance in different cacao genotypes, with some consistently displaying resistance while others showed variable levels during tree establishment and production stages. CONCLUSION: This research underscores the significant role of L. femoratus as a cacao pest, emphasizing its adverse effects on cacao tree survival, development, and yield. Consequently, implementing effective control measures is vital for ensuring sustainable cacao cultivation. © 2023 Society of Chemical Industry.

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Significant scientific advances to elucidate the Moniliophthora perniciosa pathosystem have been achieved in recent years, but the molecular biology of this pathogen-host interaction is still a field with many unanswered questions. In order to present insights at the molecular level, we present the first systematic review on the theme. All told, 1118 studies were extracted from public databases. Of these, 109 were eligible for the review, based on the inclusion and exclusion criteria. The results indicated that understanding the transition from the biotrophic-necrotrophic phase of the fungus is crucial for control of the disease. Proteins with strong biotechnological potential or that can be targets for pathosystem intervention were identified, but studies regarding possible applications are still limited. The studies identified revealed important genes in the M. perniciosa-host interaction and efficient molecular markers in the search for genetic variability and sources of resistance, with Theobroma cacao being the most common host. An arsenal of effectors already identified and not explored in the pathosystem were highlighted. This systematic review contributes to the understanding of the pathosystem at the molecular level, offering new insights and proposing different paths for the development of new strategies to control witches' broom disease.

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Investigations of the compatibility between cacao genotypes of the population of the Parinari series (Pa), resulting from the reciprocal crossing of Pa 30 × Pa 169 and Pa 121 × Pa 169, allowed the verification of the occurrence of the recessive lethal single character called Luteus-Pa. These genotypes have this gene in heterozygosity, which when intercross or self-fertilize, segregate in a 3:1 ratio. Normal (NS) and mutant (MS) seedlings grow normally and, after a period of approximately 30 days of age, MS leaves begin to show a metallic yellow color, followed by necrotic spots, and death of the entire seedling, approximately 40 days after the emergency. The work evaluate the molecular, biochemical and micromorphological responses in NS and MS, with and without cotyledons, resulting from the crossing of the Pa 30 × Pa 169 cacao genotypes, aiming to elucidate the possible lethal mechanisms of the homozygous recessive Luteus-Pa. The presence of the lethal gene Luteus-Pa in the seedlings of the cacao genotypes of the population of the Parinari (Pa), with and without cotyledons, resulting from the crossing of Pa 30 × Pa 169, in addition to regulating the synthesis of proteins related to the photosynthetic and stress defense processes, promoted an increase in the synthesis of proteins involved in the glycolic pathway, induced oxidative stress, altered the mobilization of cotyledonary reserves, the integrity of cell membranes, leaf micromorphology and induced the death of seedlings, soon after depletion of protein and carbohydrate reserves, especially in the absence of cotyledons.

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Cocoa currently faces differentiation processes toward niches of specialty products, leading to greater competitiveness for producers who must compete with products differentiated by their integral quality regarding their organoleptic characteristics, such as fine-flavor cocoa and their functional characteristics. Quality is influenced by the genetic variety of the cultivars on the one hand, and the correct postharvest processing operations of cocoa seeds, on the other. During the transformation operations, the native chemical compounds of the seeds, especially proteins, carbohydrates, and polyphenols, are transformed and generate other compounds called flavor precursors, which are responsible for defining the product quality. In this sense, the analysis of the most relevant chemical compounds in cocoa is essential to guarantee higher overall quality. Similarly, understanding the fundamental aspects that affect fine-flavor cocoa production is crucial for improving transformation processes. Therefore, reliable and robust analytical techniques are required to detect and quantify these chemical compounds. This review highlights the main techniques used to analyze essential cocoa metabolites and derived products throughout all postharvest transformation stages: from cocoa seeds to chocolate bar, offering an overview of the sample preparation methods and the analytical and imaging methodologies often employed to characterize qualifying cocoa products.

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Unlike the chloroplast genomes (ptDNA), the plant mitochondrial genomes (mtDNA) are much more plastic in structure and size but maintain a conserved and essential gene set related to oxidative phosphorylation. Moreover, the plant mitochondrial genes and mtDNA are good markers for phylogenetic, evolutive, and comparative analyses. The two most known species in Theobroma L. (Malvaceae s.l.) genus are T. cacao, and T. grandiflorum. Besides the economic value, both species also show considerable biotechnology potential due to their other derived products, thus, aggregating additional economic value for the agroindustry. Here, we assembled and compared the mtDNA of Theobroma cacao and T. grandiflorum to generate a new genomics resource and unravel evolutionary trends. Graph-based analyses revealed that both mtDNA exhibit multiple alternative arrangements, confirming the dynamism commonly observed in plant mtDNA. The disentangled assembly graph revealed potential predominant circular molecules. The master circle molecules span 543,794 bp for T. cacao and 501,598 bp for T. grandiflorum, showing 98.9% of average sequence identity. Both mtDNA contains the same set of 39 plant mitochondrial genes, commonly found in other rosid mitogenomes. The main features are a duplicated copy of atp4, the absence of rpl6, rps2, rps8, and rps11, and the presence of two chimeric open-reading frames. Moreover, we detected few ptDNA integrations mainly represented by tRNAs, and no viral sequences were detected. Phylogenomics analyses indicate Theobroma spp. are nested in Malvaceae family. The main mtDNA differences are related to distinct structural rearrangements and exclusive regions associated with relics of Transposable Elements, supporting the hypothesis of dynamic mitochondrial genome maintenance and divergent evolutionary paths and pressures after species differentiation.

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The thread blight disease (TBD) of cacao ( Theobroma cacao) in the department of Amazonas, Peru was recently reported to be caused by Marasmius tenuissimus (sect. Neosessiles). This same species is known to be the main causal agent of TBD in West Africa. However, some morphological characteristics, such as the presence of rhizomorphs, the almost exclusively white color, and pileus sizes less than 5 mm, among others, differ to the description of M. tenuissimus. Therefore, we aimed to conduct a taxonomic revision of the cacao-TBD causal agent in Peru, by using thorough micro and macro morphological, phylogenetic, and nuclear and mitochondrial genomic approaches. We showed that the causal agent of TBD of cacao in Amazonas, Peru, belongs to a new species, Marasmius infestans sp. nov. This study enriches our knowledge of species in the sect. Neosessiles, and strongly suggests that the M. tenuissimus species complex is highly diverse.

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Cacao is a globally important crop with a long history of domestication and selective breeding. Despite the increased use of elite clones by cacao farmers, worldwide plantations are established mainly using hybrid progeny material derived from heterozygous parents, therefore displaying high tree-to-tree variability. The deliberate development of hybrids from advanced inbred lines produced by successive generations of self-pollination has not yet been fully considered in cacao breeding. This is largely due to the self-incompatibility of the species, the long generation cycles (3-5 years) and the extensive trial areas needed to accomplish the endeavor. We propose a simple and accessible approach to develop inbred lines based on accelerating the buildup of homozygosity based on regular selfing assisted by genome-wide SNP genotyping. In this study we genotyped 90 clones from the Brazilian CEPEC´s germplasm collection and 49 inbred offspring of six S1 or S2 cacao families derived from self-pollinating clones CCN-51, PS-13.19, TSH-1188 and SIAL-169. A set of 3,380 SNPs distributed across the cacao genome were interrogated on the EMBRAPA multi-species 65k Infinium chip. The 90 cacao clones showed considerable variation in genome-wide SNP homozygosity (mean 0.727± 0.182) and 19 of them with homozygosity ≥90%. By assessing the increase in homozygosity across two generations of self-pollinations, SNP data revealed the wide variability in homozygosity within and between S1 and S2 families. Even in small families (<10 sibs), individuals were identified with up to ~1.5 standard deviations above the family mean homozygosity. From baseline homozygosities of 0.476 and 0.454, offspring with homozygosities of 0.862 and 0.879 were recovered for clones TSH-1188 and CCN-51 respectively, in only two generations of selfing (81-93% increase). SNP marker assisted monitoring and selection of inbred individuals can be a practical tool to optimize and accelerate the development of inbred lines of outbred tree species. This approach will allow a faster and more accurate exploitation of hybrid breeding strategies in cacao improvement programs and potentially in other perennial fruit and forest trees.

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A genome-wide association study (GWAS) was undertaken to unravel marker-trait associations (MTAs) between SNP markers and phenotypic traits. It involved a subset of 421 cacao accessions from the large and diverse collection conserved ex situ at the International Cocoa Genebank Trinidad. A Mixed Linear Model (MLM) in TASSEL was used for the GWAS and followed by confirmatory analyses using GAPIT FarmCPU. An average linkage disequilibrium (r2) of 0.10 at 5.2 Mb was found across several chromosomes. Seventeen significant (P ≤ 8.17 × 10-5 (-log10 (p) = 4.088)) MTAs of interest, including six that pertained to yield-related traits, were identified using TASSEL MLM. The latter accounted for 5 to 17% of the phenotypic variation expressed. The highly significant association (P ≤ 8.17 × 10-5) between seed length to width ratio and TcSNP 733 on chromosome 5 was verified with FarmCPU (P ≤ 1.12 × 10-8). Fourteen MTAs were common to both the TASSEL and FarmCPU models at P ≤ 0.003. The most significant yield-related MTAs involved seed number and seed length on chromosome 7 (P ≤ 1.15 × 10-14 and P ≤ 6.75 × 10-05, respectively) and seed number on chromosome 1 (P ≤ 2.38 × 10-05), based on the TASSEL MLM. It was noteworthy that seed length, seed length to width ratio and seed number were associated with markers at different loci, indicating their polygenic nature. Approximately 40 candidate genes that encode embryo and seed development, protein synthesis, carbohydrate transport and lipid biosynthesis and transport were identified in the flanking regions of the significantly associated SNPs and in linkage disequilibrium with them. A significant association of fruit surface anthocyanin intensity co-localised with MYB-related protein 308 on chromosome 4. Testing of a genomic selection approach revealed good predictive value (genomic estimated breeding values (GEBV)) for economic traits such as seed number (GEBV = 0.611), seed length (0.6199), seed width (0.5435), seed length to width ratio (0.5503), seed/cotyledon mass (0.6014) and ovule number (0.6325). The findings of this study could facilitate genomic selection and marker-assisted breeding of cacao thereby expediting improvement in the yield potential of cacao planting material.

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Propagation by somatic embryogenesis in Theobroma cacao has some issues to be solved, as many morphologically abnormal somatic embryos that do not germinate into plants are frequently observed, thus hampering plant production on a commercial scale. For the first time the methylome landscape of T. cacao somatic embryogenesis was examined, using whole-genome bisulfite sequencing technique, with the aim to understand the epigenetic basis of somatic embryo abnormalities. We identified 873 differentially methylated genes (DMGs) in the CpG context between zygotic embryos, normal and abnormal somatic embryos, with important roles in development, programmed cell death, oxidative stress, and hypoxia induction, which can help to explain the morphological abnormalities of somatic embryos. We also identified the role of ethylene and its precursor 1-aminocyclopropane-1-carboxylate in several biological processes, such as hypoxia induction, cell differentiation and cell polarity, that could be associated to the development of abnormal somatic embryos. The biological processes and the hypothesis of ethylene and its precursor involvement in the somatic embryo abnormalities in cacao are discussed.

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Cocoa (Theobroma cacao L.) is the only tree that can produce cocoa. Cocoa beans are highly sought after by chocolate makers to produce chocolate. Cocoa can be fine aromatic, characterized by floral and fruity notes, or it can be described as standard cocoa with a more pronounced cocoa aroma and bitterness. In this study, the genetic and biochemical determinants of sensorial notes and nonvolatile compounds related to bitterness, astringency, fat content, and protein content will be investigated in two populations: a cultivated modern Nacional population and a population of cocoa accessions collected recently in the Ecuadorian South Amazonia area of origin of the Nacional ancestral variety. For this purpose, a genome-wide association study (GWAS) was carried out on both populations, with results of biochemical compounds evaluated by near-infrared spectroscopy (NIRS) assays and with sensory evaluations. Twenty areas of associations were detected for sensorial data especially bitterness and astringency. Fifty-three areas of associations were detected linked to nonvolatile compounds. A total of 81 candidate genes could be identified in the areas of the association.

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Witches' broom disease of cacao is caused by the pathogenic fungus Moniliophthora perniciosa. By using tomato (Solanum lycopersicum) cultivar Micro-Tom (MT) as a model system, we investigated the physiological and metabolic consequences of M. perniciosa infection to determine whether symptoms result from sink establishment during infection. Infection of MT by M. perniciosa caused reductions in root biomass and fruit yield, a decrease in leaf gas exchange, and down-regulation of photosynthesis-related genes. The total leaf area and water potential decreased, while ABA levels, water conductance/conductivity, and ABA-related gene expression increased. Genes related to sugar metabolism and those involved in secondary cell wall deposition were up-regulated upon infection, and the concentrations of sugars, fumarate, and amino acids increased. 14C-glucose was mobilized towards infected MT stems, but not in inoculated stems of the MT line overexpressing CYTOKININ OXIDASE-2 (35S::AtCKX2), suggesting a role for cytokinin in establishing a sugar sink. The up-regulation of genes involved in cell wall deposition and phenylpropanoid metabolism in infected MT, but not in 35S::AtCKX2 plants, suggests establishment of a cytokinin-mediated sink that promotes tissue overgrowth with an increase in lignin. Possibly, M. perniciosa could benefit from the accumulation of secondary cell walls during its saprotrophic phase of infection.

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Many decades of improvement in cacao have aided to obtain cultivars with characteristics of tolerance to diseases, adaptability to different edaphoclimatic conditions, and higher yields. In Ecuador, as a result of several breeding programs, the clone CCN 51 was obtained, which gradually expanded through the cacao-production regions of Ecuador, Colombia, Brazil and Peru. Recognized for its high yield and adaptability to different regions and environments, it has become one of the most popular clones for breeding programs and cultivation around the world. This review aims to summarize the current evidence on the origin, genetics, morphological, volatile compounds, and organoleptic characteristics of this clone. Physiological evidence, production dynamics, and floral biology are also included to explain the high yield of CCN 51. Thus, characteristics such as osmotic adjustment, long pollen longevity, and fruit formation are further discussed and associated with high production at the end of the dry period. Finally, the impact of this popular clone on the current and future cacao industry will be discussed highlighting the major challenges for flavor enhancement and its relevance as a platform for the identification of novel genetic markers for cultivar improvement in breeding programs.

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The cultivation of cocoa is of great socio-economic importance worldwide. Cocoa beans are the essential raw material for chocolate production. The variability of cacao studied presents only a small fraction of the existing genetic diversity, mainly in the Amazon region. Furthermore, just a small part of this variability has been exploited in cocoa breeding. Thus, the present study processed the genetic evaluation and selection of cocoa clones, based on morpho-agronomical traits. For this, we evaluated 145 clones, during 37 harvests from, four consecutive years. The following traits were evaluated: total number of fruits collected (TNFC), total number of healthy fruits (TNHF), weight of wet seeds from healthy fruits (WWSHF), average weight of wet seeds per healthy fruit (AWWSHF), weight of dry seeds from healthy fruit (WDSHF), average weight of dry seeds per healthy fruit (AWDSHF), percentage of fruits with witches' broom disease (PFWB), percentage of fruits with borer (PFBR), percentage of fruits with germinated seeds (PFGS), number of branches with witches' broom disease (NBWB), and number of inflorescence with witches' broom disease (NIWB). Significant differences (P < 0.05) among the clones were observed for all traits, which reveal an expressive variability and possibility of gains with selection. The highest significant correlations (P < 0.05) occurred between traits TNFC and TNHF (0.94), TNHF and AWWSHF (0.86), and TNHF and AWDSHF (0.86). Based on the selection index, the clones POUND 12 and CAB 12, 228, 253, 257, 258, and 422 were the most suitable for selection.

O cultivo do cacau tem grande importância socioeconômica mundial. Os amêndoas de cacau são a matéria-prima essencial para a produção de chocolate. A variabilidade do cacaueiro avaliada representa apenas uma pequena fração da diversidade genética existente, principalmente na região amazônica. Além disso, apenas uma pequena parte dessa variabilidade foi explorada no melhoramento de cacau. Dessa forma, o presente estudo teve como objetivo realizar a avaliação genética e seleção de clones de cacau, com base em características morfo-agronômicas. Para isso, foram avaliados 145 clones, durante 37 colheitas, em quatro anos consecutivos. As seguintes características de frutos e sementes foram avaliadas: número total de frutos colhidos (NTFC), número total de frutos sadios (NTFS), peso de sementes úmidas dos frutos sadios (PSUFS), peso médio de sementes úmidas dos frutos sadios (PMSUFS), peso de sementes secas dos frutos sadios (PSSFS), peso médio de sementes secas dos frutos sadios (PMSSFS), porcentagem de frutos com vassoura-de-bruxa (PFVB), porcentagem de frutos com broca (PFBR), porcentagem de frutos com sementes germinadas (PFSG), número de ramos com vassoura-de-bruxa (NRVB) e número de almofadas florais com vassoura-de-bruxa (NAFVB). Diferenças significativas (P < 0,05) entre os clones foram observadas para todas as características, o que revela uma expressiva variabilidade e possibilidade de ganhos com a seleção. As maiores correlações significativas (P < 0,05) ocorreram entre os caracteres NTFC e NTFS (0,94), NTFS e PMSUFS (0,86) e, NTFS e PMSSFS (0,86). Com base no índice de seleção, os clones POUND 12 e CAB 12, 228, 253, 257, 258 e 422 foram considerados os mais adequados para a seleção.

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BACKGROUND: Theobroma cacao is a major source of flavonoids such as catechins and their monomers proanthocyanidins (PAs), widely studied for their potential benefits in cardiovascular diseases. Light has been shown to promote plant secondary metabolite production in vitro. In this study, cacao cells cultured in 7.5 L stirred tank photobioreactors (STPs) were exposed to a change of white to blue LED lights for 28 days (d). RESULTS: Transcriptomic analyses were performed in three time points comparing changing expression patterns, after cell exposure to white light (d0-VS-d14), after a shift from white to blue light (d14-VS-d15), and after an extended period of blue light for the following 15 days (d15-VS-d28). Under white light, there was enrichment in metabolic pathways associated with cell growth (carbon, glycolysis, and amino acid biosynthesis) accompanied by a significant increase in the PAs content. In the shift to blue light, further increase in PAs content was observed concomitantly with the significant expression of TWO-COMPONENT RESPONSE REGULATOR genes involved in the early stress responses via circadian clock and hormone pathways. Under blue light exposure, we observed a depletion of PAs content associated with ROS-mediated stress pathways. CONCLUSIONS: Light effects on large-scale cell cultures in photobioreactors are complex and pleiotropic; however, we have been able to identify key regulatory players upstream cacao flavonoid biosynthesis in STPs, including TWO-COMPONENT SYSTEM and ROS-signaling genes. The crosstalk between flavonoid biosynthesis and regulatory networks led to understand the dynamics of flavonoid production and degradation in response to light-driven ROS signals. This can be used to optimize the time, and the yield of in vitro targeted metabolites in large-scale culture systems.

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This study evaluated the genotoxicity of lyophilized glycolic extract of Theobroma cacao Linné seeds (TCL), using the micronucleus assay in bone marrow of mice. The interaction between TCL and doxorubicin (DXR) was also analyzed. Experimental groups were evaluated 24-48 h after treatment with N-Nitroso-N-ethylurea (NEU: 50 mg/kg), DXR (5 mg/kg), NaCl (145 mM), TCL (0.5-2 g/kg), and TCL (2 g/kg) in combination with DXR (antigenotoxic assays). Analysis of micronucleated polychromatic erythrocytes (MNPCEs) showed no significant differences between all the treatment doses of TCL and NaCl control. Mice experimentally treated with DXR and NEU significantly induced MNPCEs. However, a significant reduction of MNPCEs was also observed when TCL was administered in combination with the chemotherapeutic agent DXR. The analysis of the PCE/NCE ratio revealed no significant differences between the NaCl control, all doses of TCL, and DXR. However, there were significant differences in the PCE/NCE ratio between positive NEU control and all other treatments. The PCE/NCE ratio observed after treatment with TCL and DXR showed significant differences and intermediate values to controls (NaCl and NEU). This study suggests absence of genotoxicity and cytotoxicity of TCL, regardless of dose, sex, and time. TCL reduced genotoxic effects induced by DXR, suggesting potential antigenotoxic effects.(AU)

Este estudo avaliou a genotoxicidade do extrato glicólico liofilizado de sementes de Theobroma cacao Linné (TCL), usando o ensaio do micronúcleo em medula óssea de camundongos. A interação entre TCL e doxorrubicina (DXR) foi também analisada. Grupos experimentais foram avaliados 24-48 h após tratamento com N-Nitroso-N-etilureia (NEU: 50 mg/kg), DXR (5 mg/kg), NaCl (145 mM), TCL (0,5-2 g/kg), e TCL (2 g/kg) em combinação com DXR (ensaio antigenotóxico). As análises de eritrócitos policromáticos micronucleados (EPCMNs) não mostraram diferenças significantes entre todas as doses de tratamento do TCL e o controle NaCl. Camundongos experimentalmente tratados com DXR e NEU induziram significativamente EPCMNs. Contudo, uma redução significante de EPCMNs foi também observada quando TCL foi administrada em combinação com o agente quimioterapêutico DXR. As análises da relação EPC/ENC (eritrócito policromático/eritrócito normocromático) revelaram ausência de diferenças significantes entre o controle NaCl, todas as doses de TCL e DXR. Contudo, houve diferenças significantes na relação EPC/ENC entre o controle positivo NEU e todos os outros tratamento. A relação ECP/ENC observada após o tratamento com TCL e DXR mostrou diferenças significantes e valores intermediários aos controles (NaCl e NEU). Este estudo sugere ausência de genotoxicidade e citotoxicidade de TCL, independentemente da dose, sexo e tempo. TCL reduziu os efeitos genotóxicos induzidos por DXR, sugerindo potencial efeitos antigenotóxicos.(AU)

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Lactobacillaceae presents potential for interspecific Quorum Sensing (QS) in spontaneous cocoa fermentation, correlated with high abundance of luxS. Three Brazilian isolates from cocoa fermentation were characterized by Whole Genome Sequencing and luxS gene was surveyed in their genomes, in comparison with public databases. They were classified as Lactiplantibacillus plantarum, Limosilactobacillus fermentum and Pediococcus acidilactici. LuxS genes were conserved in core genomes of the novel isolates, but in some non-cocoa related Lactic Acid Bacteria (LAB) it was accessory and plasmid-borne. The conservation and horizontal acquisition of luxS reinforces that QS is determinant for bacterial adaptation in several environments, especially taking into account the luxS has been correlated with modulation of bacteriocin production, stress tolerance and biofilm formation. Therefore, in this paper, new clade and species-specific primers were designed for future application for screening of luxS gene in LAB to evaluate the adaptive potential to diverse food fermentations.

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