Three de novo assembled wild cacao genomes from the Upper Amazon.

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.

Development and characterization of microsatellite markers for population genetics of the cocoa pod borer Conopomorpha cramerella (Snellen) (Lepidoptera: Gracillaridae).

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.

A revisited history of cacao domestication in pre-Columbian times revealed by archaeogenomic approaches.

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.

Multi-trait selection for nutritional and physiological quality of cacao genotypes in irrigated and non-irrigated environments.

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.

Identification of self- and pathogen-targeted miRNAs from resistant and susceptible Theobroma cacao variety to black pod disease.

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.

The surface of leaves and fruits of Peruvian cacao is home for several Hannaella yeast species, including the new species Hannaella theobromatis sp. nov.

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.

A combination of conserved and diverged responses underlies Theobroma cacao's defense response to Phytophthora palmivora.

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.

A scarab beetle (Coleoptera: Scarabaeidae) causes tree mortality, delayed growth, and yield reduction in cacao with genotype-specific susceptibility to herbivory.

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.

Vacuum-Forced Agroinfiltration for In planta Transformation of Recalcitrant Plants: Cacao as a Case Study.

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.

Phyllosphere microbial diversity and specific taxa mediate within-cultivar resistance to Phytophthora palmivora in cacao.

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.

DNA Isolation from Cocoa-Derived Products and Cocoa Authentication by TaqMan Real-Time PCR.

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.

State of the Art of the Molecular Biology of the Interaction between Cocoa and Witches' Broom Disease: A Systematic Review.

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.

Molecular, biochemical and micromorphological responses of cacao seedlings of the Parinari series, carrying the lethal gene Luteus-Pa, in the presence and absence of cotyledons.

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.

Chemical Characterization of Quality-Related Compounds in Cocoa Matrices: An Overview of Analytical Methods Applied for Their Analysis.

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.

Comparative analyses of Theobroma cacao and T. grandiflorum mitogenomes reveal conserved gene content embedded within complex and plastic structures.

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.

Morphological, phylogenetic, and genomic evidence reveals the causal agent of thread blight disease of cacao in Peru is a new species of Marasmius in the section Neosessiles, Marasmius infestans sp. nov.

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.

Comparison of bioactive components and flavor volatiles of diverse cocoa genotypes of Theobroma grandiflorum, Theobroma bicolor, Theobroma subincanum and Theobroma cacao.

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.

Genome-wide association studies and genomic selection assays made in a large sample of cacao (Theobroma cacao L.) germplasm reveal significant marker-trait associations and good predictive value for improving yield potential.

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.

Genome-wide SNP genotyping as a simple and practical tool to accelerate the development of inbred lines in outbred tree species: An example in cacao (Theobroma cacao L.).

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.

Single-base resolution methylomes of somatic embryogenesis in Theobroma cacao L. reveal epigenome modifications associated with somatic embryo abnormalities.

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.