Search

Diseases of Theobroma cacao L. (Malvaceae) disrupt cocoa bean supply and economically impact growers. Vascular streak dieback (VSD), caused by Ceratobasidium theobromae, is a new encounter disease of cacao currently contained to southeast Asia and Melanesia. Resistance to VSD has been tested with large progeny trials in Sulawesi, Indonesia, and in Papua New Guinea with the identification of informative quantitative trait loci (QTLs). Using a VSD susceptible progeny tree (clone 26), derived from a resistant and susceptible parental cross, we assembled the genome to chromosome-level and discriminated alleles inherited from either resistant or susceptible parents. The parentally phased genomes were annotated for all predicted genes and then specifically for resistance genes of the nucleotide-binding site leucine-rich repeat class (NLR). On investigation, we determined the presence of NLR clusters and other potential disease response gene candidates in proximity to informative QTLs. We identified structural variants within NLRs inherited from parentals. We present the first diploid, fully scaffolded, and parentally phased genome resource for T. cacao L. and provide insights into the genetics underlying resistance and susceptibility to VSD., (© 2024 The Author(s). The Plant Genome published by Wiley Periodicals LLC on behalf of Crop Science Society of America.)

Microorganisms are critical indicators of soil quality due to their essential role in maintaining ecosystem services. However, anthropogenic activities can disrupt the vital metabolic functions of these microorganisms. Considering that soil biology is often underestimated and traditional assessment methods do not capture its complexity, molecular methods can be used to assess soil health more effectively. This study aimed to identify the changes in soil microbial diversity and activity under different cocoa agroforestry systems, specially focusing on taxa and functions associated to carbon and nitrogen cycling. Soils from three different cocoa agroforestry systems, including a newly established agroforestry with green fertilization (GF), rubber ( Hevea brasiliensis )-cocoa intercropping (RC), and cocoa plantations under Cabruca (cultivated under the shave of native forest) (CAB) were analyzed and compared using metagenomic and metaproteomic approaches. Samples from surrounding native forest and pasture were used in the comparison, representing natural and anthropomorphic ecosystems. Metagenomic analysis revealed a significant increase in Proteobacteria and Basidiomycota and the genes associated with dissimilatory nitrate reduction in the RC and CAB areas. The green fertilization area showed increased nitrogen cycling activity, demonstrating the success of the practice. In addition, metaproteomic analyses detected enzymes such as dehydrogenases in RC and native forest soils, indicating higher metabolic activity in these soils. These findings underscore the importance of soil management strategies to enhance soil productivity, diversity, and overall soil health. Molecular tools are useful to demonstrate how changes in agricultural practices directly influence the microbial community, affecting soil health.

A recently published untargeted metabolomics approach toward marker compounds of cocoa germination revealed and identified 12-hydroxyjasmonic acid sulfate, (+)-catechin, and (-)-epicatechin as the most downregulated compounds and two hydroxymethylglutaryl glucosides (HMG gluc) A and B, among others, as the decisive upregulated compounds in the germinated material. These findings were quantitatively evaluated using ultrahigh-performance liquid chromatography-tandem mass spectrometry not only in previously examined sample material but also in a vastly expanded array of cocoa samples of different provenience and process and in cocoa products such as cocoa liquor and chocolate. Hereby, yields of newly identified HMG gluc derivatives could be determined in raw, fermented, germinated, and alternatively processed cocoa, and isomers of HMG gluc A and B could be established as key process indicators. Based on unsupervised clustering and supervised classification, models could identify germinated samples in testing sets consisting of raw, fermented, and germinated samples.

Background: Theobroma cacao, the cocoa tree, is a tropical crop grown for its highly valuable cocoa solids and fat which are the basis of a 200-billion-dollar annual chocolate industry. However, the long generation time and difficulties associated with breeding a tropical tree crop have limited the progress of breeders to develop high-yielding disease-resistant varieties. Development of marker-assisted breeding methods for cacao requires discovery of genomic regions and specific alleles of genes encoding important traits of interest. To accelerate gene discovery, we developed a gene atlas composed of a large dataset of replicated transcriptomes with the long-term goal of progressing breeding towards developing high-yielding elite varieties of cacao., Results: We describe the creation of the Cacao Transcriptome Atlas, its global characterization and define sets of genes co-regulated in highly organ- and temporally-specific manners. RNAs were extracted and transcriptomes sequenced from 123 different tissues and stages of development representing major organs and developmental stages of the cacao lifecycle. In addition, several experimental treatments and time courses were performed to measure gene expression in tissues responding to biotic and abiotic stressors. Samples were collected in replicates (3-5) to enable statistical analysis of gene expression levels for a total of 390 transcriptomes. To promote wide use of these data, all raw sequencing data, expression read mapping matrices, scripts, and other information used to create the resource are freely available online. We verified our atlas by analyzing the expression of genes with known functions and expression patterns in Arabidopsis (ACT7, LEA19, AGL16, TIP13, LHY, MYB2) and found their expression profiles to be generally similar between both species. We also successfully identified tissue-specific genes at two thresholds in many tissue types represented and a set of genes highly conserved across all tissues., Conclusion: The Cacao Gene Atlas consists of a gene expression browser with graphical user interface and open access to raw sequencing data files as well as the unnormalized and CPM normalized read count data mapped to several cacao genomes. The gene atlas is a publicly available resource to allow rapid mining of cacao gene expression profiles. We hope this resource will be used to help accelerate the discovery of important genes for key cacao traits such as disease resistance and contribute to the breeding of elite varieties to help farmers increase yields., (© 2024. The Author(s).)

Durian (Durio zibethinus) is a tropical fruit that has a unique flavor and aroma. It occupies a significant phylogenetic position within the Malvaceae family. Extant core-eudicot plants are reported to share seven ancestral karyotypes that have undergone reshuffling, resulting in an abundant genomic diversity. However, the ancestral karyotypes of the Malvaceae family, as well as the evolution trajectory leading to the 28 chromosomes in durian, remain poorly understood. Here, we report the high-quality assembly of the durian genome with comprehensive comparative genomic analyses. By analyzing the collinear blocks between cacao and durian, we inferred 11 Malvaceae ancestral karyotypes. These blocks were present in a single-copy form in cacao and mainly in triplicates in durian, possibly resulting from a recent whole genome triplication (WGT) event that led to hexaploidization of the durian genome around 20 (17-24) million years ago. A large proportion of the duplicated genes in durian, such as those involved in the lignin biosynthesis module for phenylpropane biosynthesis, are derived directly from whole genome duplication, which makes it an important force in reshaping its genomic architecture. Transcriptome studies have revealed that genes involved in feruloyl-CoA formations were highly preferentially expressed in fruit peels, indicating that the thorns produced on durian fruit may comprise guaiacyl and syringyl lignins. Among all the analyzed transcription factors (TFs), members of the heat shock factor family (HSF) were the most significantly upregulated under heat stress. All subfamilies of genes encoding heat shock proteins (HSPs) in the durian genome appear to have undergone expansion. The potential interactions between HSF Dzi05.397 and HSPs were examined and experimentally verified. Our study provides a high-quality durian genome and reveals the reshuffling mechanism of ancestral Malvaceae chromosomes to produce the durian genome. We also provide insights into the mechanism underlying lignin biosynthesis and heat stress tolerance., (© 2024. Science China Press.)

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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Ramirez-Ramirez et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The cocoa pod borer (CPB) Conopomorpha cramerella (Snellen) (Lepidoptera: Gracillaridae) is one of the major constraints for cocoa production in South East Asia. In addition to cultural and chemical control methods, autocidal control tactics such as the Sterile Insect Technique (SIT) could be an efficient addition to the currently control strategy, however SIT implementation will depend on the population genetics of the targeted pest. The aim of the present work was to search for suitable microsatellite loci in the genome of CPB that is partially sequenced. Twelve microsatellites were initially selected and used to analyze moths collected from Indonesia, Malaysia, and the Philippines. A quality control verification process was carried out and seven microsatellites found to be suitable and efficient to distinguish differences between CPB populations from different locations. The selected microsatellites were also tested against a closely related species, i.e. the lychee fruit borer Conopomorpha sinensis (LFB) from Vietnam and eight loci were found to be suitable. The availability of these novel microsatellite loci will provide useful tools for the analysis of the population genetics and gene flow of these pests, to select suitable CPB strains to implement the SIT., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Purificacion et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Theobroma cacao, the chocolate tree, is indigenous to the Amazon basin, the greatest biodiversity hotspot on earth. Recent advancement in plant genomics highlights the importance of de novo sequencing of multiple reference genomes to capture the genome diversity present in different cacao populations. In this study, three high-quality chromosome-level genomes of wild cacao were constructed, de novo assembled with HiFi long reads sequencing, and scaffolded using a reference-free strategy. These genomes represent the three most important genetic clusters of cacao trees from the Upper Amazon region. The three wild cacao genomes were compared with two reference genomes of domesticated cacao. The five cacao genetic clusters were inferred to have diverged in the early and middle Pleistocene period, approximately 1.83-0.69 million years ago. The results shown here serve as an example of understanding how the Amazonian biodiversity was developed. The three wild cacao genomes provide valuable resources for studying genetic diversity and advancing genetic improvement of this species., (© 2024. The Author(s).)

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., (© 2023 Society of Chemical Industry.)

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 CO 2 assimilation rate (A), stomatal conductance (gs), transpiration (E), intercellular and atmospheric CO 2 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., (© 2024. The Author(s).)

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., (© 2024. The Author(s).)

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., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Background: Plants have complex and dynamic immune systems that have evolved to resist pathogens. Humans have worked to enhance these defenses in crops through breeding. However, many crops harbor only a fraction of the genetic diversity present in wild relatives. Increased utilization of diverse germplasm to search for desirable traits, such as disease resistance, is therefore a valuable step towards breeding crops that are adapted to both current and emerging threats. Here, we examine diversity of defense responses across four populations of the long-generation tree crop Theobroma cacao L., as well as four non-cacao Theobroma species, with the goal of identifying genetic elements essential for protection against the oomycete pathogen Phytophthora palmivora., Results: We began by creating a new, highly contiguous genome assembly for the P. palmivora-resistant genotype SCA 6 (Additional file 1: Tables S1-S5), deposited in GenBank under accessions CP139290-CP139299. We then used this high-quality assembly to combine RNA and whole-genome sequencing data to discover several genes and pathways associated with resistance. Many of these are unique, i.e., differentially regulated in only one of the four populations (diverged 40 k-900 k generations). Among the pathways shared across all populations is phenylpropanoid biosynthesis, a metabolic pathway with well-documented roles in plant defense. One gene in this pathway, caffeoyl shikimate esterase (CSE), was upregulated across all four populations following pathogen treatment, indicating its broad importance for cacao's defense response. Further experimental evidence suggests this gene hydrolyzes caffeoyl shikimate to create caffeic acid, an antimicrobial compound and known inhibitor of Phytophthora spp., Conclusions: Our results indicate most expression variation associated with resistance is unique to populations. Moreover, our findings demonstrate the value of using a broad sample of evolutionarily diverged populations for revealing the genetic bases of cacao resistance to P. palmivora. This approach has promise for further revealing and harnessing valuable genetic resources in this and other long-generation plants., (© 2024. The Author(s).)

Cacao (Theobroma cacao) is a highly valuable crop with growing demand in the global market. However, cacao farmers often face challenges posed by black pod disease caused by Phytophthora spp., with P. palmivora being the most dominant. Regulations of various gene expressions influence plant resistance to pathogens. One mechanism involves targeting the mRNA of virulence genes in the invading pathogens, suppressing their infection. However, resistance also could be suppressed by plant-derived miRNAs that target their own defence genes. The objective of this study is to identify differentially expressed miRNAs in black pod-resistant and susceptible cacao varieties and to predict their targets in T. cacao and P. palmivora transcripts. Extracted miRNA from resistant and susceptible varieties of T. Cacao was sequenced, identified, and matched to host and pathogen mRNA. In total, 54 known miRNAs from 40 miRNA families and 67 novel miRNAs were identified. Seventeen miRNAs were differentially expressed in susceptible variety compared to resistant one, with 9 miRNAs upregulated and 8 miRNAs downregulated. In T. cacao transcripts, the upregulated miRNAs were predicted to target several genes, including defence genes. The suppression of these defense genes can lead to a reduction in plant resistance against pathogen infection. In P. palmivora transcripts, the upregulated miRNAs were predicted to target several genes, including P. palmivora effector genes. In the future, limiting expression of miRNAs that target T. cacao's defence genes and applying miRNAs that target P. palmivora effector genes hold promise for enhancing cacao plant resistance against P. palmivora infection., (© 2024. The Author(s).)

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., Competing Interests: No competing interests were disclosed., (Copyright: © 2024 Huamán-Pilco AF et al.)

Transient in planta transformation is a fast and cost-effective alternative for plant genetic transformation. Most protocols for in planta transformation rely on the use of Agrobacterium-mediated transformation. However, the protocols currently in use are standardized for small-sized plants due to the physical and economic constraints of submitting large-sized plants to a vacuum treatment. This work presents an effective protocol for localized vacuum-based agroinfiltration customized for large-sized plants. To assess the efficacy of the proposed method, we tested its use in cacao plants, a tropical plant species recalcitrant to genetic transformation. Our protocol allowed applying up to 0.07 MPa vacuum, with repetitions, to a localized aerial part of cacao leaves, making it possible to force the infiltration of Agrobacterium into the intercellular spaces of attached leaves. As a result, we achieved the Agrobacterium-mediated transient in planta transformation of attached cacao leaves expressing for the RUBY reporter system. This is also the first Agrobacterium-mediated in planta transient transformation of cacao. This protocol would allow the application of the vacuum-based agroinfiltration method to other plant species with similar size constraints and open the door for the in planta characterization of genes in recalcitrant woody, large-size species.

The oomycete pathogen Phytophthora palmivora , which causes black pod rot (BPR) on cacao ( Theobroma cacao L.), is responsible for devastating yield losses worldwide. Genetic variation in resistance to Phytophthora spp. is well documented among cacao cultivars, but variation has also been observed in the incidence of BPR even among trees of the same cultivar. In light of evidence that the naturally occurring phyllosphere microbiome can influence foliar disease resistance in other host-pathogen systems, it was hypothesized that differences in the phyllosphere microbiome between two field accessions of the cultivar Gainesville II 164 could be responsible for their contrasting resistance to P. palmivora . Bacterial alpha diversity was higher but fungal alpha diversity was lower in the more resistant accession MITC-331, and the accessions harbored phyllosphere microbiomes with distinct community compositions. Six bacterial and 82 fungal amplicon sequence variants (ASVs) differed in relative abundance between MITC-333 and MITC-331, including bacterial putative biocontrol agents and a high proportion of fungal pathogens, and nine fungal ASVs were correlated with increased lesion development. The roles of contrasting light availability and host mineral nutrition, particularly potassium, are also discussed. Results of this preliminary study can be used to guide research into microbiome-informed integrated pest management strategies effective against Phytophthora spp. in cacao. IMPORTANCE Up to 40% of the world's cacao is lost each year to diseases, the most devastating of which is black pod rot, caused by Phytophthora palmivora . Though disease resistance is often attributed to cacao genotypes (i.e., disease-resistant rootstocks), this study highlights the role of the microbiome in contributing to differences in resistance even among accessions of the same cacao cultivar. Future studies of plant-pathogen interactions may need to account for variation in the host microbiome, and optimizing the cacao phyllosphere microbiome could be a promising new direction for P. palmivora resistance research., Competing Interests: J.E.S. and A.E.D. are employed by Mars Inc.

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.

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Masson SAS. All rights reserved.)

Cocoa (Theobroma cacao L.) is an international commodity used as an ingredient in the manufacturing of chocolate making its authentication a key issue in the cocoa chain. Various molecular techniques have been increasingly applied for quality requirements. These issues highlight the need for techniques that allow the extraction and detection of cocoa DNA from highly processed cocoa products and chocolate. The applicability of real-time PCR to highly processed cocoa-derived products for authentication purposes depends on the possibility of extracting high-quality and amplifiable DNA and further developing efficient PCR tests. This methodology herein describes the use of a classical CTAB method providing DNA suitable for TaqMan real-time PCR amplification. Real-time PCR is a simple and fast method, with a high potential application in a wide range of food products. The main features of this technique are focused on two DNA targets, one located in the nuclear genome (vicilin-li PCR test) and a second one based on chloroplast DNA (lipids PCR test), which successfully passed the performance criteria considering the specificity, sensitivity, efficiency of amplification, robustness, and applicability in processed cocoa-derived products and chocolate., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

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.

GRAS genes exist widely and play vital roles in various physiological processes in plants. In this study, to identify Theobroma cacao (T. cacao) GRAS genes involved in environmental stress and phytohormones, we conducted a genome-wide analysis of the GRAS gene family in T. cacao. A total of 46 GRAS genes of T. cacao were identified. Chromosomal distribution analysis showed that all the TcGRAS genes were evenly distributed on ten chromosomes. Phylogenetic relationships revealed that GRAS proteins could be divided into twelve subfamilies (HAM: 6, LISCL: 10, LAS: 1, SCL4/7: 1, SCR: 4, DLT: 1, SCL3: 3, DELLA: 4, SHR: 5, PAT1: 6, UN1: 1, UN2: 4). Of the T. cacao GRAS genes, all contained the GRAS domain or GRAS superfamily domain. Subcellular localization analysis predicted that TcGRAS proteins were located in the nucleus, chloroplast, and endomembrane system. Gene duplication analysis showed that there were two pairs of tandem repeats and six pairs of fragment duplications, which may account for the rapid expansion in T. cacao. In addition, we also predicted the physicochemical properties and cis-acting elements. The analysis of GO annotation predicted that the TcGRAS genes were involved in many biological processes. This study highlights the evolution, diversity, and characterization of the GRAS genes in T. cacao and provides the first comprehensive analysis of this gene family in the cacao genome.

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., (© 2022 The Authors. The Plant Genome published by Wiley Periodicals LLC on behalf of Crop Science Society of America.)

Theobroma grandiflorum, T. bicolor and T. subincanum represent underutilized genetic resources for cocoa quality. The bioactive components and flavor volatiles of different bean genotypes of these species were profiled and compared with those of T. cacao. T. bicolor and T. grandiflorum had different profiles of methylxanthines and polyphenols from T. bicolor and T. cacao. T. subincanum and T. grandiflorum were rich in theacrine and flavones. T. grandiflorum, T. bicolor and T. subincanum beans generally had less phenolics than T. cacao. Roasting decreased the concentrations of polyphenols and methylxanthines in the beans. Roasted T. grandiflorum and T. subincanum beans had higher concentrations of pyrazines and esters than T. cacao. T. grandiflorum and T. subincanum beans had more odor-active volatiles than T. cacao. Overall, the underutilized Theobroma species have potential to be exploited to improve the flavor and nutritional quality of cocoa products., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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., Competing Interests: The authors have declared that no competing interests exist.

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., Competing Interests: The authors declare that they have no competing interests.

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., (© 2022. The Author(s).)

The MYB gene family is involved in the regulation of plant growth, development and stress responses. In this paper, to identify Theobroma cacao R2R3-MYB (TcMYB) genes involved in environmental stress and phytohormones, we conducted a genome-wide analysis of the R2R3-MYB gene family in Theobroma cacao (cacao). A total of 116 TcMYB genes were identified, and they were divided into 23 subgroups according to the phylogenetic analysis. Meanwhile, the conserved motifs, gene structures and cis-acting elements of promoters were analyzed. Moreover, these TcMYB genes were distributed on 10 chromosomes. We conducted a synteny analysis to understand the evolution of the cacao R2R3-MYB gene family. A total of 37 gene pairs of TcMYB genes were identified through tandem or segmental duplication events. Additionally, we also predicted the subcellular localization and physicochemical properties. All the studies showed that TcMYB genes have multiple functions, including responding to environmental stresses. The results provide an understanding of R2R3-MYB in Theobroma cacao and lay the foundation for a further functional analysis of TcMYB genes in the growth of cacao.

Cocoa cultivation is dominated by the clone "Colleción Castro Naranjal 51" (CCN-51). In contrast, CCN-51 is the expensive and aromatic fine cocoa "Arriba Nacional" from Ecuador. The differences in the overall quality of the beans and in the prices show that it is necessary to develop a rapid and accurate method to distinguish these varieties and prevent food fraud. To this end, we used a CRISPR-Cpf1 assay suitable for AT-rich targets such as the chloroplast genome (cpGenome). SNPs in cocoa plastid genomes were selected to replace the canonical PAM sequence of Cpf1 (5'-TTTV-3'). We developed two assay systems to digest both Arriba and CCN-51. The results were tested qualitatively by agarose gel electrophoresis and quantitatively by capillary gel electrophoresis. Using the assay described here, we were able to reliably detect admixtures of 5% CCN-51 ( P < 0.01) and 10% Arriba ( P < 0.05). The application to processed cocoa products was also successful.

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., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

&lt;b&gt;Background and Objective:&lt;/b&gt; In plants, the 70 kDa heat shock proteins (Hsp70-s) play important roles during growth and development and in response to stresses. This study aimed to provide comprehensive information on the &lt;i&gt;Hsp70 &lt;/i&gt;family in cocoa (&lt;i&gt;Theobroma cacao&lt;/i&gt;), one of the most important tropical perennial cash crops worldwide. &lt;b&gt;Materials and Methods:&lt;/b&gt; A basic local alignment search tool (TBLASTN) against the cocoa genome was performed using &lt;i&gt;Arabidopsis&lt;/i&gt; Hsp70-s as queries to detect TcHsp70-s. Sequence analyses were carried out by various bioinformatics tools. &lt;b&gt;Results:&lt;/b&gt; A total of 18 members of the &lt;i&gt;Hsp70&lt;/i&gt; family has been detected and characterized in the cocoa genome. All general properties, such as physic-chemical parameters, gene structure, phylogenetic tree and sub-cellular localization, were determined using a variety of bioinformatics tools. The expression patterns of the &lt;i&gt;TcHsp70&lt;/i&gt; genes in different stages of the zygotic and somatic embryos were investigated. In addition, expression profiles of the &lt;i&gt;TcHsp70&lt;/i&gt; genes under &lt;i&gt;Phytophthora megakarya&lt;/i&gt; inoculation were analyzed. &lt;b&gt;Conclusion:&lt;/b&gt; The results of this study revealed the features and expression analyses of &lt;i&gt;Hsp70&lt;/i&gt; genes in cocoa. These findings could provide a strong foundation for further research of the &lt;i&gt;TcHsp70&lt;/i&gt; family, which could aid in the development and stress tolerance of cocoa species.

Cocoa swollen shoot virus disease (CSSVD) is a major disease of cacao (Theobroma cacao L.) in Ghana and other West African countries that grow the crop. Attempts to develop resistant varieties since the discovery of the disease in 1936 have yielded little success. Recently, planting materials that are tolerant to the disease have been recommended for planting in areas with high CSSVD prevalence. However, the effect of CSSVD on yield component traits of most cacao clones including the tolerant varieties has not been well studied. To investigate the impact of CSSVD on these traits, reduction in bean weight (BW), number of beans per pod (NoBP) and dry bean yield (DBY) between symptomless and symptomatic trees, and disease incidence (DI) among 210 different cacao clones were evaluated. The clones were transplanted in June 2010 following a randomised complete block design with four replications consisting of three trees per clone per block. Response of the clones to CSSVD incidence had 180 of the genotypes having < 50% symptoms. Clones of Contanama, Iquitos, Marañon, Nanay and unknown derived from Upper Amazon parentage had less DI at the end of the study. The CSSVD effect (symptomless vs. symptomatic) was significant (p < 0.05) for DBY and NoBP, and the symptomless trees surpassed on average their symptomatic trees by 21.17% for DBY. Some of the best-performing clones identified under natural CSSVD infection were; COCA 3348/52 and GU 219/V among the underrepresented clones, B 36, ICS 40, NA 33 x IMC 67, T30/628, T60/887, T63/971, T 81/1879 and T 82/503 among those that combine high DBY with low yield reduction, and NA 124, T17/358, T35/78, T57/305, T63/971 x SCA 6, T65/239, T76/1835 and T82/2294 among those that combine high DBY with no disease incidence. Their inclusion in breeding programs that seek to develop resistant CSSVD varieties or deployment as planting materials in endemic areas to improve yield production in Ghana is recommended on the basis of the present observations., Competing Interests: The authors have declared that no competing interests exist.

Nacional is a variety of cocoa tree known for its "Arriba" aroma characterised mainly by fruity, floral, and spicy aromatic notes. In this study, the genetic basis of the fruity aroma of modern Nacional cocoa was investigated. GWAS studies have been conducted on biochemical and sensorial fruity traits and allowed to identify a large number of association zones. These areas are linked to both the volatile compounds known to provide fruity flavours and present in the beans before and after roasting, and to the fruity notes detected by sensorial analysis. Five main metabolic pathways were identified as involved in the fruity traits of the Nacional population: the protein degradation pathway, the sugar degradation pathway, the fatty acid degradation pathway, the monoterpene pathway, and the L-phenylalanine pathway. Candidate genes involved in the biosynthetic pathways of volatile compounds identified in association areas were detected for a large number of associations., (Copyright © 2021 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

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., Competing Interests: Luigy Barragan is employed by Mars la Chola C.L. (MLCH, Ecuador)., (©2022 Jaimez et al.)

Cutinases are enzymes produced by phytopathogenic fungi like Moniliophthora roreri. The three genome-located cutinase genes of M. roreri were amplified from cDNA of fungi growing in different induction culture media for cutinase production. The mrcut1 gene was expressed in the presence of a cacao cuticle, while the mrcut2 and mrcut3 genes were expressed when an apple cuticle was used as the inducer. The sequences of all genes were obtained and analyzed by bioinformatics tools to determine the presence of signal peptides, introns, glycosylation, and regulatory sequences. Also, the theoretical molecular weight and pI were obtained and experimentally confirmed. Finally, cutinase 1 from M. roreri (MRCUT1) was selected for heterologous expression in Escherichia coli. Successful overexpression of MRCUT1 was observed with the highest enzyme activity of 34,036 U/mg under the assay conditions at 40°C and pH 8. Furthermore, the degradation of different synthetic polyesters was evaluated; after 21 days, 59% of polyethylene succinate (PES), 43% of polycaprolactone (PCL), and 31% of polyethylene terephthalate (PET) from plastic residues were degraded. IMPORTANCE Plastic pollution is exponentially increasing; even the G20 has recognized an urgent need to implement actions to reduce it. In recent years, searching for enzymes that can degrade plastics, especially those based on polyesters such as PET, has been increasing as they can be a green alternative to the actual plastic degradation process. A promising option in recent years refers to biological tools such as enzymes involved in stages of partial and even total degradation of some plastics. In this context, the MRCUT1 enzyme can degrade polyesters contained in plastic residues in a short time. Besides, there is limited knowledge about the biochemical properties of cutinases from M. roreri. Commonly, fungal enzymes are expressed as inclusion bodies in E. coli with reduced activity. Interestingly, the successful expression of one cutinase of M. roreri in E. coli with enhanced activity is described.

The MADS-box family gene is a class of transcription factors that have been extensively studied and involved in several plant growth and development processes, especially in floral organ specificity, flowering time and initiation and fruit development. In this study, we identified 69 candidate MADS-box genes and clustered these genes into five subgroups (Mα: 11; Mβ: 2; Mγ: 14; Mδ: 9; MIKC: 32) based on their phylogenetical relationships with Arabidopsis . Most TcMADS genes within the same subgroup showed a similar gene structure and highly conserved motifs. Chromosomal distribution analysis revealed that all the TcMADS genes were evenly distributed in 10 chromosomes. Additionally, the cis-acting elements of promoter, physicochemical properties and subcellular localization were also analyzed. This study provides a comprehensive analysis of MADS-box genes in Theobroma cacao and lays the foundation for further functional research.

The Theobroma cacao presents a wide diversity in pod color among different cultivars. Although flavonoid biosynthesis has been studied in many plants, molecular mechanisms governing the diversity of coloration in cacao pods are largely unknown. The flavonoid metabolite profiles and flavonoid biosynthetic gene expression in the pod exocarps of light green pod 'TAS 410' (GW), green pod 'TAS 166' (GF), and mauve pod 'TAS 168' (PF) were determined. Changes in flavonoid metabolites, particularly the anthocyanins (cyanidin 3-O-galactoside, cyanidin 3-O-glucoside, and cyanidin O-syringic acid) were significantly up-accumulated in the mauve phenotype (PF) compared to the light green or green phenotypes, endowing the pod color change from light green or green to mauve. Consistently, the PF phenotype showed different expression patterns of flavonoid biosynthetic structural genes in comparison with GW/GF phenotypes. The expression level of LAR and ANR in GW/GF was significantly higher than PF, while the expression level of UFGT in GW/GF was lower than PF. These genes likely generated more anthocyanins in the exocarps samples of PF than that of GW/GF. Simultaneously, colorless flavan-3-ols (catechin, epicatechin and proanthocyanidin) content in the exocarp samples of PF was lower than GW/GF. Additionally, MYB (gene18079) and bHLH (gene5045 and gene21575) may participate in the regulation of the pod color. This study sheds light on the molecular basis of cacao pod color variation, which will contribute to breeding cacao varieties with enhanced flavonoid profiles for nutritional applications., (© 2021. The Botanical Society of Japan.)

High salt environments can induce stress in different plants. The genes containing the ZAT domain constitute a family that belongs to a branch of the C 2 H 2 family, which plays a vital role in responding to abiotic stresses. In this study, we identified 169 ZAT genes from seven plant species, including 44 ZAT genes from G. hirsutum. Phylogenetic tree analysis divided ZAT genes in six groups with conserved gene structure, protein motifs. Two C 2 H 2 domains and an EAR domain and even chromosomal distribution on At and Dt sub-genome chromosomes of G. hirsutum was observed. GhZAT6 was primarily expressed in the root tissue and responded to NaCl and ABA treatments. Subcellular localization found that GhZAT6 was located in the nucleus and demonstrated transactivation activity during a transactivation activity assay. Arabidopsis transgenic lines overexpressing the GhZAT6 gene showed salt tolerance and grew more vigorously than WT on MS medium supplemented with 100 mmol NaCl. Additionally, the silencing of the GhZAT6 gene in cotton plants showed more obvious leaf wilting than the control plants, which were subjected to 400 mmol NaCl treatment. Next, the expressions of GhAPX1, GhFSD1, GhFSD2, and GhSOS3 were significantly lower in the GhZAT6-silenced plants treated with NaCl than the control. Based on these findings, GhZAT6 may be involved in the ABA pathway and mediate salt stress tolerance by regulating ROS-related gene expression., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Genomic structural variants (SVs) can play important roles in adaptation and speciation. Yet the overall fitness effects of SVs are poorly understood, partly because accurate population-level identification of SVs requires multiple high-quality genome assemblies. Here, we use 31 chromosome-scale, haplotype-resolved genome assemblies of Theobroma cacao- an outcrossing, long-lived tree species that is the source of chocolate-to investigate the fitness consequences of SVs in natural populations. Among the 31 accessions, we find over 160,000 SVs, which together cover eight times more of the genome than single-nucleotide polymorphisms and short indels (125 versus 15 Mb). Our results indicate that a vast majority of these SVs are deleterious: they segregate at low frequencies and are depleted from functional regions of the genome. We show that SVs influence gene expression, which likely impairs gene function and contributes to the detrimental effects of SVs. We also provide empirical support for a theoretical prediction that SVs, particularly inversions, increase genetic load through the accumulation of deleterious nucleotide variants as a result of suppressed recombination. Despite the overall detrimental effects, we identify individual SVs bearing signatures of local adaptation, several of which are associated with genes differentially expressed between populations. Genes involved in pathogen resistance are strongly enriched among these candidates, highlighting the contribution of SVs to this important local adaptation trait. Beyond revealing empirical evidence for the evolutionary importance of SVs, these 31 de novo assemblies provide a valuable resource for genetic and breeding studies in T cacao ., Competing Interests: The authors declare no competing interest.

Geographical indications may stimulate collective actions of governance for quality control, trade and marketing as well as innovation based on the use of local resources and regional biodiversity. Cocoa production, however, dominated by small family agriculture in tropical regions, has rarely made use of such strategies. This review is aimed at understanding major research interests and emerging technologies helpful for the origin differentiation of cocoa quality. Results from literature search and cited references of publications on cocoa research were imported into VOSviewer for data analysis, which aided in visualizing major research hotpots. Co-occurrence analysis yielded major research clusters which guided the discussion of this review. Observed was a consensus recognizing cocoa quality resulting from the interaction of genotype, fermentation variables and geographical origin. A classic view of cocoa genetics based on the dichotomy of 'fine versus bulk' has been reexamined by a broader perspective of human selection and cocoa genotype evolution. This new approach to cocoa genetic diversity, together with the understanding of complex microbiome interactions through fermentation, as well as quality reproducibility challenged by geographical conditions, have demonstrated the importance of terroir in the production of special attributes. Cocoa growing communities around the tropics have been clearly enabled by new omics and chemometrics to systematize producing conditions and practices in the designation of specifications for the differentiation of origin quality. © 2021 Society of Chemical Industry., (© 2021 Society of Chemical Industry.)

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., (© 2021. The Author(s).)

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., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Convergent evolution is widespread but the extent to which common ancestral conditions are necessary to facilitate the independent acquisition of similar traits remains unclear. In order to better understand how ancestral biosynthetic catalytic capabilities might lead to convergent evolution of similar modern-day biochemical pathways, we resurrected ancient enzymes of the caffeine synthase (CS) methyltransferases that are responsible for theobromine and caffeine production in flowering plants. Ancestral CS enzymes of Theobroma, Paullinia, and Camellia exhibited similar substrate preferences but these resulted in the formation of different sets of products. From these ancestral enzymes, descendants with similar substrate preference and product formation independently evolved after gene duplication events in Theobroma and Paullinia. Thus, it appears that the convergent modern-day pathways likely originated from ancestral pathways with different inferred flux. Subsequently, the modern-day enzymes originated independently via gene duplication and their convergent catalytic characteristics evolved to partition the multiple ancestral activities by different mutations that occurred in homologous regions of the ancestral proteins. These results show that even when modern-day pathways and recruited genes are similar, the antecedent conditions may be distinctive such that different evolutionary steps are required to generate convergence., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Background: In angiosperms the transition to flowering is controlled by a complex set of interacting networks integrating a range of developmental, physiological, and environmental factors optimizing transition time for maximal reproductive efficiency. The molecular mechanisms comprising these networks have been partially characterized and include both transcriptional and post-transcriptional regulatory pathways. Florigen, encoded by FLOWERING LOCUS T (FT) orthologs, is a conserved central integrator of several flowering time regulatory pathways. To characterize the molecular mechanisms involved in controlling cacao flowering time, we have characterized a cacao candidate florigen gene, TcFLOWERING LOCUS T (TcFT). Understanding how this conserved flowering time regulator affects cacao plant's transition to flowering could lead to strategies to accelerate cacao breeding., Results: BLAST searches of cacao genome reference assemblies identified seven candidate members of the CENTRORADIALIS/TERMINAL FLOWER1/SELF PRUNING gene family including a single florigen candidate. cDNA encoding the predicted cacao florigen was cloned and functionally tested by transgenic genetic complementation in the Arabidopsis ft-10 mutant. Transgenic expression of the candidate TcFT cDNA in late flowering Arabidopsis ft-10 partially rescues the mutant to wild-type flowering time. Gene expression studies reveal that TcFT is spatially and temporally expressed in a manner similar to that found in Arabidopsis, specifically, TcFT mRNA is shown to be both developmentally and diurnally regulated in leaves and is most abundant in floral tissues. Finally, to test interspecies compatibility of florigens, we transformed cacao tissues with AtFT resulting in the remarkable formation of flowers in tissue culture. The morphology of these in vitro flowers is normal, and they produce pollen that germinates in vitro with high rates., Conclusion: We have identified the cacao CETS gene family, central to developmental regulation in angiosperms. The role of the cacao's single FT-like gene (TcFT) as a general regulator of determinate growth in cacao was demonstrated by functional complementation of Arabidopsis ft-10 late-flowering mutant and through gene expression analysis. In addition, overexpression of AtFT in cacao resulted in precocious flowering in cacao tissue culture demonstrating the highly conserved function of FT and the mechanisms controlling flowering in cacao.

Theobroma cacao is one of the most economically important tropical trees, being the source of chocolate. As part of an ongoing study to understand the diversity of the badnavirus complex, responsible for the cacao swollen shoot virus disease in West Africa, evidence was found recently of virus-like sequences in asymptomatic cacao plants. The present study exploited the wealth of genomic resources in this crop, and combined bioinformatic, molecular, and genetic approaches to report for the first time the presence of integrated badnaviral sequences in most of the cacao genetic groups. These sequences, which we propose to name eTcBV for endogenous T. cacao bacilliform virus, varied in type with each predominating in a specific genetic group. A diagnostic multiplex PCR method was developed to identify the homozygous or hemizygous condition of one specific insert, which was inherited as a single Mendelian trait. These data suggest that these integration events occurred before or during the species diversification in Central and South America, and prior to its cultivation in other regions. Such evidence of integrated sequences is relevant to the management of cacao quarantine facilities and may also aid novel methods to reduce the impact of such viruses in this crop.

Predicting the potential fate of a species in the face of climate change requires knowing the distribution of molecular adaptations across the geographic range of the species. In this work, we analysed 79 genomes of Theobroma cacao, an Amazonian tree known for the fruit from which chocolate is produced, to evaluate how local and regional molecular signatures of adaptation are distributed across the natural range of the species. We implemented novel techniques that incorporate summary statistics from multiple selection scans to infer selective sweeps. The majority of the molecular adaptations in the genome are not shared among populations. We show that ~71.5% of genes under selection also show significant associations with changes in environmental variables. Our results support the interpretation that these genes contribute to local adaptation of the populations in response to abiotic factors. We also found strong patterns of molecular adaptation in a diverse array of disease resistance genes (6.5% of selective sweeps), suggesting that differential adaptation to pathogens also contributes significantly to local adaptations. Our results are consistent with the interpretation that local selective pressures are more important than regional selective pressures in explaining adaptation across the range of a species., (© 2020 John Wiley & Sons Ltd.)

The glutathione peroxidases (GPXs) are enzymes which are part of the cell antioxidant system inhibiting the ROS-induced damages of membranes and proteins. In cacao (Theobroma cacao L.) genome, five GPX genes were identified. Cysteine insertion codons (UGU) were found in TcPHGPX, TcGPX2, TcGPX4, TcGPX6 and tryptophan insertion codon (UGG) in TcGPX8. Multiple alignments revealed conserved domains between TcGPXs and other plants and human GPXs. Homology modeling was performed using the Populus trichocarpa GPX5 structure as template, and the molecular modeling showed that TcGPXs have affinity with selenometionine in their active site. In silico analysis of the TcGPXs promoter region revealed the presence of conserved cis-elements related to biotic stresses and hormone responsiveness. The expression analysis of TcGPXs in cacao plantlet meristems infected by M. perniciosa showed that TcGPXs are most expressed in susceptible variety than in resistant one, mainly in disease stages in which oxidative stress and programmed cell death occurred. This data, associated with phylogenetic and location analysis suggested that TcGPXs may play a role in protecting cells from oxidative stress as a try of disease progression reduction. To our knowledge, this is the first study of the overall GPX family from T. cacao., Competing Interests: Declaration of competing interest No conflicts of interest to declare., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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 ( H e = 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.

The Sul 1 cacao ( Theobroma cacao ) genotype has become more popular among cocoa producers due to its resistance toward vascular streak dieback, a devastating dieback disease in cacao plants. Sul 1 cocoa beans were subjected to prolonged fermentation of 10 days (240 h). Changes in the composition of methylxanthines, polyphenols, and volatiles and sensory profiles of the cocoa beans as affected by fermentation were analyzed. The first 48 h of fermentation significantly determined the composition of the methylxanthines and polyphenols in the cocoa beans. A prolonged fermentation (>96 h) period did not further reduce the contents of methylxanthines and polyphenols in the cocoa beans. Important volatiles characteristic of the cocoa/chocolate flavor were mostly developed after 48 h of fermentation. The fermentation for 72 h retained considerable amounts of methylxanthines and polyphenols in the beans while producing cocoa mass with good sensory profiles.