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.

The American Cherimoya Genome Reveals Insights into the Intra-Specific Divergence, the Evolution of Magnoliales, and a Putative Gene Cluster for Acetogenin Biosynthesis.

Annona cherimola (cherimoya) is a species renowned for its delectable fruit and medicinal properties. In this study, we developed a chromosome-level genome assembly for the cherimoya 'Booth' cultivar from the United States. The genome assembly has a size of 794 Mb with a N50 = 97.59 Mb. The seven longest scaffolds account for 87.6% of the total genome length, which corresponds to the seven pseudo-chromosomes. A total of 45,272 protein-coding genes (≥30 aa) were predicted with 92.9% gene content completeness. No recent whole genome duplications were identified by an intra-genome collinearity analysis. Phylogenetic analysis supports that eudicots and magnoliids are more closely related to each other than to monocots. Moreover, the Magnoliales was found to be more closely related to the Laurales than the Piperales. Genome comparison revealed that the 'Booth' cultivar has 200 Mb less repeats than the Spanish cultivar 'Fino de Jete', despite their highly similar (>99%) genome sequence identity and collinearity. These two cultivars were diverged during the early Pleistocene (1.93 Mya), which suggests a different origin and domestication of the cherimoya. Terpene/terpenoid metabolism functions were found to be enriched in Magnoliales, while TNL (Toll/Interleukin-1-NBS-LRR) disease resistance gene has been lost in Magnoliales during evolution. We have also identified a gene cluster that is potentially responsible for the biosynthesis of acetogenins, a class of natural products found exclusively in Annonaceae. The cherimoya genome provides an invaluable resource for supporting characterization, conservation, and utilization of Annona genetic resources.

Cacao pod transcriptome profiling of seven genotypes identifies features associated with post-penetration resistance to Phytophthora palmivora.

The oomycete Phytophthora palmivora infects the fruit of cacao trees (Theobroma cacao) causing black pod rot and reducing yields. Cacao genotypes vary in their resistance levels to P. palmivora, yet our understanding of how cacao fruit respond to the pathogen at the molecular level during disease establishment is limited. To address this issue, disease development and RNA-Seq studies were conducted on pods of seven cacao genotypes (ICS1, WFT, Gu133, Spa9, CCN51, Sca6 and Pound7) to better understand their reactions to the post-penetration stage of P. palmivora infection. The pod tissue-P. palmivora pathogen assay resulted in the genotypes being classified as susceptible (ICS1, WFT, Gu133 and Spa9) or resistant (CCN51, Sca6 and Pound7). The number of differentially expressed genes (DEGs) ranged from 1625 to 6957 depending on genotype. A custom gene correlation approach identified 34 correlation groups. De novo motif analysis was conducted on upstream promoter sequences of differentially expressed genes, identifying 76 novel motifs, 31 of which were over-represented in the upstream sequences of correlation groups and associated with gene ontology terms related to oxidative stress response, defense against fungal pathogens, general metabolism and cell function. Genes in one correlation group (Group 6) were strongly induced in all genotypes and enriched in genes annotated with defense-responsive terms. Expression pattern profiling revealed that genes in Group 6 were induced to higher levels in the resistant genotypes. An additional analysis allowed the identification of 17 candidate cis-regulatory modules likely to be involved in cacao defense against P. palmivora. This study is a comprehensive exploration of the cacao pod transcriptional response to P. palmivora spread after infection. We identified cacao genes, promoter motifs, and promoter motif combinations associated with post-penetration resistance to P. palmivora in cacao pods and provide this information as a resource to support future and ongoing efforts to breed P. palmivora-resistant cacao.

Genome-wide characterization and evolutionary analysis of the AP2/ERF gene family in lettuce (Lactuca sativa).

The APETALA2/ETHYLENE RESPONSIVE FACTOR (AP2/ERF) gene family plays vital roles in plants, serving as a key regulator in responses to abiotic stresses. Despite its significance, a comprehensive understanding of this family in lettuce remains incomplete. In this study, we performed a genome-wide search for the AP2/ERF family in lettuce and identified a total of 224 members. The duplication patterns provided evidence that both tandem and segmental duplications contributed to the expansion of this family. Ka/Ks ratio analysis demonstrated that, following duplication events, the genes have been subjected to purifying selection pressure, leading to selective constraints on their protein sequence. This selective pressure provides a dosage benefit against stresses in plants. Additionally, a transcriptome analysis indicated that some duplicated genes gained novel functions, emphasizing the contribution of both dosage effect and functional divergence to the family functionalities. Furthermore, an orthologous relationship study showed that 60% of genes descended from a common ancestor of Rosid and Asterid lineages, 28% from the Asterid ancestor, and 12% evolved in the lettuce lineage, suggesting lineage-specific roles in adaptive evolution. These results provide valuable insights into the evolutionary mechanisms of the AP2/ERF gene family in lettuce, with implications for enhancing abiotic stress tolerance, ultimately contributing to the genetic improvement of lettuce crop production.

Genotyping of Jujube (Ziziphus spp.) Germplasm in New Mexico and Southwestern Texas.

Since the early 19th century, a substantial amount of jujube (Ziziphus spp.) germplasm has been introduced from China and Europe into the United States. However, due to a lack of passport data, cultivar mislabeling is common and the genetic background of the introduced germplasm remains unknown. In the present study, a low-density SNP array was employed to genotype 204 jujube trees sampled from multiple locations in New Mexico, Texas, Missouri, and Kentucky. Multilocus matching of SNP profiles revealed a significant rate of genetic redundancy among these jujube samples. A total of 14 synonymous groups were detected, comprising 48 accessions. Bayesian clustering analysis and neighbor-joining tree partitioned the US jujube germplasm into two major clusters. The first cluster included cultivated genotypes (Ziziphus jujuba Mill.), whereas the other major cluster comprised the wild/sour jujube (Ziziphus spinosa Hu.). The results also revealed a unique jujube population at Fabens/Tornillo, Texas, and a semi-naturalized population at Tucumcari, NM. These findings will provide valuable guidance to jujube growers and researchers on the effective utilization of jujube germplasm in the horticultural industry.

The complete genome sequences of Erythroxylum coca and Erythroxylum novogranatense.

The flowering plant genus Erythroxylum contains approximately 300 species, including the economically and socially consequential crops called coca. We present the genome sequences of Erythroxylum coca and E. novogranatense, two cultigens produced for medicinal and quotidian use in the Andes and Amazon regions of South America, as well as the international cocaine industry. Sequencing was performed on an Illumina X-Ten platform, and reads were assembled by a de novo method followed by finishing via comparison with several species from the same genus. The BioProject, raw and assembled data can be accessed in GenBank for E. coca (PRJNA676123; JAJMLV000000000) and E. novogranatense (PRJNA675212; JAJKBF000000000).

Elucidation of tropane alkaloid biosynthesis in Erythroxylum coca using a microbial pathway discovery platform.

Tropane alkaloids (TAs) are heterocyclic nitrogenous metabolites found across seven orders of angiosperms, including Malpighiales (Erythroxylaceae) and Solanales (Solanaceae). Despite the well-established euphorigenic properties of Erythroxylaceae TAs like cocaine, their biosynthetic pathway remains incomplete. Using yeast as a screening platform, we identified and characterized the missing steps of TA biosynthesis in Erythroxylum coca. We first characterize putative E. coca polyamine synthase- and amine oxidase-like enzymes in vitro, in yeast, and in planta to show that the first tropane ring closure in Erythroxylaceae occurs via bifunctional spermidine synthase/N-methyltransferases and both flavin- and copper-dependent amine oxidases. We next identify a SABATH family methyltransferase responsible for the 2-carbomethoxy moiety characteristic of Erythroxylaceae TAs and demonstrate that its coexpression with methylecgonone reductase in yeast engineered to express the Solanaceae TA pathway enables the production of a hybrid TA with structural features of both lineages. Finally, we use clustering analysis of Erythroxylum transcriptome datasets to discover a cytochrome P450 of the CYP81A family responsible for the second tropane ring closure in Erythroxylaceae, and demonstrate the function of the core coca TA pathway in vivo via reconstruction and de novo biosynthesis of methylecgonine in yeast. Collectively, our results provide strong evidence that TA biosynthesis in Erythroxylaceae and Solanaceae is polyphyletic and that independent recruitment of unique biosynthetic mechanisms and enzyme classes occurred at nearly every step in the evolution of this pathway.

United States tea: A synopsis of ongoing tea research and solutions to United States tea production issues.

Tea is a steeped beverage made from the leaves of Camellia sinensis. Globally, this healthy, caffeine-containing drink is one of the most widely consumed beverages. At least 50 countries produce tea and most of the production information and tea research is derived from international sources. Here, we discuss information related to tea production, genetics, and chemistry as well as production issues that affect or are likely to affect emerging tea production and research in the United States. With this review, we relay current knowledge on tea production, threats to tea production, and solutions to production problems to inform this emerging market in the United States.

Mitochondrial Genomics of Six Cacao Pathogens From the Basidiomycete Family Marasmiaceae.

Thread blight disease has recently been described as an emerging disease on cacao (Theobroma cacao) in Ghana. In Ghana, thread blight disease is caused by multiple species of the Marasmiaceae family: Marasmius tenuissimus, M. crinis-equi, M. palmivorus, and Marasmiellus scandens. Interestingly, two additional members of the Marasmiaceae; Moniliophthora roreri (frosty pod rot) and Moniliophthora perniciosa (witches' broom disease), are major pathogens of cacao in the Western hemisphere. It is important to accurately characterize the genetic relationships among these economically important species in support of their disease management. We used data from Illumina NGS-based genome sequencing efforts to study the mitochondrial genomes (mitogenomes) of the four cacao thread blight associated pathogens from Ghana and compared them with published mitogenomes of Mon. roreri and Mon. perniciosa. There is a remarkable interspecies variation in mitogenome size within the six cacao-associated Marasmiaceae species, ranging from 43,121 to 109,103 bp. The differences in genome lengths are primarily due to the number and lengths of introns, differences in intergenic space, and differences in the size and numbers of unidentified ORFs (uORF). Among seven M. tenuissimus mitogenomes sequenced, there is variation in size and sequence pointing to divergent evolution patterns within the species. The intronic regions show a high degree of sequence variation compared to the conserved sequences of the 14 core genes. The intronic ORFs identified, regardless of species, encode GIY-YIG or LAGLIDADG domain-containing homing endonuclease genes. Phylogenetic relationships using the 14 core proteins largely mimic the phylogenetic relationships observed in gene order patterns, grouping M. tenuissimus with M. crinis-equi, and M. palmivorus with Mon. roreri and Mon. perniciosa, leaving Mar. scandens as an outlier. The results from this study provide evidence of independent expansion/contraction events and sequence diversification in each species and establish a foundation for further exploration of the evolutionary trajectory of the fungi in Marasmiaceae family.

The chromosome-level genome of dragon fruit reveals whole-genome duplication and chromosomal co-localization of betacyanin biosynthetic genes.

Dragon fruits are tropical fruits economically important for agricultural industries. As members of the family of Cactaceae, they have evolved to adapt to the arid environment. Here we report the draft genome of Hylocereus undatus, commercially known as the white-fleshed dragon fruit. The chromosomal level genome assembly contains 11 longest scaffolds corresponding to the 11 chromosomes of H. undatus. Genome annotation of H. undatus found ~29,000 protein-coding genes, similar to Carnegiea gigantea (saguaro). Whole-genome duplication (WGD) analysis revealed a WGD event in the last common ancestor of Cactaceae followed by extensive genome rearrangements. The divergence time between H. undatus and C. gigantea was estimated to be 9.18 MYA. Functional enrichment analysis of orthologous gene clusters (OGCs) in six Cactaceae plants found significantly enriched OGCs in drought resistance. Fruit flavor-related functions were overrepresented in OGCs that are significantly expanded in H. undatus. The H. undatus draft genome also enabled the discovery of carbohydrate and plant cell wall-related functional enrichment in dragon fruits treated with trypsin for a longer storage time. Lastly, genes of the betacyanin (a red-violet pigment and antioxidant with a very high concentration in dragon fruits) biosynthetic pathway were found to be co-localized on a 12 Mb region of one chromosome. The consequence may be a higher efficiency of betacyanin biosynthesis, which will need experimental validation in the future. The H. undatus draft genome will be a great resource to study various cactus plants.

Accurate Differentiation of Green Beans of Arabica and Robusta Coffee Using Nanofluidic Array of Single Nucleotide Polymorphism (SNP) Markers.

Green (unroasted) coffee is one of the most traded agricultural commodities in the world. The Arabica (Coffea arabica L.) and Robusta (Coffea canephora Pierre ex A. Froehner) species are the two main types of coffees for commercial production. In general, Arabica coffee is known to have better quality in terms of sensory characteristics; thus, it has a higher market value than Robusta coffee. Accurate differentiation of green beans of the two species is, therefore, of commercial interest in the coffee industry. Using the newly developed single nucleotide polymorphism (SNP) markers, we analyzed a total of 80 single green bean samples, representing 20 Arabica cultivars and four Robusta accessions. Reliable SNP fingerprints were generated for all tested samples. Unambiguous differentiation between Robusta and Arabica coffees was achieved using multivariate analysis and assignment test. The SNP marker panel and the genotyping protocol are sufficiently robust to detect admixture of green coffee in a high-throughput fashion. Moreover, the multilocus SNP approach can differentiate every single bean within Robusta and 55% of Arabica samples. This advantage, together with the single-bean sensitivity, suggests a significant potential for practical application of this technology in the coffee industry.

Elevated temperature and drought stress significantly affect fruit quality and activity of anthocyanin-related enzymes in jujube (Ziziphus jujuba Mill. cv. 'Lingwuchangzao').

The quality attributes of jujube fruit can be directly and indirectly affected by abiotic stresses associated with climate change. Increased temperature and drought are among the most important factors challenging sustainable jujube production in the temperate semi-arid region in northwest China. The main objective of the present study was to understand the effects of elevated air temperature and drought stress on sugar and acid accumulation and coloration of jujube fruits. The content of soluble sugar, organic acid and pigments of traditional jujube "Linwuchangzao" under different atmospheric temperatures and drought stresses were analyzed during three different fruit ripening stages. The elevated temperature (1.5-2.5° C than normal temperature) significantly increased the fruit sugar content, sugar-acid ratio, anthocyanins, flavonoids and carotenoids content. Under the drought stress where the soil moisture was 30% -50% of the field capacity, sugar content, anthocyanin, flavonoid and carotenoid content of the fruit were significantly reduced at the same temperature, but the chlorophyll and organic acid content increased. No significant interaction of Temperature x Drought was observed for all the analyzed quality parameters. The current results showed that the fruit quality of jujube variety "Lingwuchangzao" could be improved when the atmospheric temperature increases by 2° C in this region. However, drought stress had a negative impact on the fruit's sugar-acid ratio and pigment content. The present results also showed that the synthesis and accumulation of anthocyanins in jujube fruit were positively correlated with sugar content and related enzyme activities, especially Phenylalanine Ammonia-lyase (PAL) activity. This study, therefore, provides novel information for understanding the influence of growth environment on the quality properties of jujube fruits. This knowledge will help develop appropriate crop management practices for jujube production in arid and semi-arid areas in northwest China.

Effectiveness of Single Nucleotide Polymorphism Markers in Genotyping Germplasm Collections of Coffea canephora Using KASP Assay.

Accurate genotype identification is imperative for effective use of Coffea canephora L. germplasm to breed new varieties with tolerance or resistance to biotic and abiotic stresses (including moisture stress and pest and disease stresses such as coffee berry borer and rust) and for high yield and improved cup quality. The present study validated 192 published single nucleotide polymorphism (SNP) markers and selected a panel of 120 loci to examine parentage and labeling errors, genetic diversity, and population structure in 400 C. canephora accessions assembled from different coffee-producing countries and planted in a field gene bank in Ghana. Of the 400 genotypes analyzed, both synonymous (trees with same SNP profiles but different names, 12.8%) and homonymous (trees with same name but different SNP profiles, 5.8%) mislabeling were identified. Parentage analysis showed that 33.3% of the progenies derived from controlled crossing and 0% of the progenies derived from an open pollinated biclonal seed garden had parentage (both parents) corresponding to breeder records. The results suggest mislabeling of the mother trees used in seed gardens and pollen contamination from unwanted paternal parents. After removing the duplicated accessions, Bayesian clustering analysis partitioned the 270 unique genotypes into two main populations. Analysis of molecular variance (AMOVA) showed that the between-population variation accounts for 41% of the total molecular variation and the genetic divergence was highly significant (Fst = 0.256; P < 0.001). Taken together, our results demonstrate the effectiveness of using the selected SNP panel in gene bank management, varietal identification, seed garden management, nursery verification, and coffee bean authentication for C. canephora breeding programs.

Genome and transcriptome analysis of the latent pathogen Lasiodiplodia theobromae, an emerging threat to the cacao industry.

Lasiodiplodia theobromae (Pat.) Griffon & Maubl., a member of the family Botryosphaeriaceae, is becoming a significant threat to crops and woody plants in many parts of the world, including the major cacao growing areas. While attempting to isolate Ceratobasidium theobromae, a causal agent of vascular streak dieback (VSD), from symptomatic cacao stems, 74% of isolated fungi were Lasiodiplodia spp. Sequence-based identification of 52 putative isolates of L. theobromae indicated that diverse species of Lasiodiplodia were associated with cacao in the studied areas, and the isolates showed variation in aggressiveness when assayed using cacao leaf discs. The present study reports a 43.75 Mb de novo assembled genome of an isolate of L. theobromae from cacao. Ab initio gene prediction generated 13 061 protein-coding genes, of which 2862 are unique to L. theobromae, when compared with other closely related Botryosphaeriaceae. Transcriptome analysis revealed that 11 860 predicted genes were transcriptionally active and 1255 were more highly expressed in planta compared with cultured mycelia. The predicted genes differentially expressed during infection were mainly those involved in carbohydrate, pectin, and lignin catabolism, cytochrome P450, necrosis-inducing proteins, and putative effectors. These findings significantly expand our knowledge of the genome of L. theobromae and the genes involved in virulence and pathogenicity.

Draft genome sequence of fastidious pathogen Ceratobasidium theobromae, which causes vascular-streak dieback in Theobroma cacao.

BACKGROUND: Ceratobasidium theobromae, a member of the Ceratobasidiaceae family, is the causal agent of vascular-streak dieback (VSD) of cacao, a major threat to the chocolate industry in the South-East Asia. The fastidious pathogen is very hard to isolate and maintain in pure culture, which is a major bottleneck in the study of its genetic diversity and genome. RESULT: This study describes for the first time, a 33.90 Mbp de novo assembled genome of a putative C. theobromae isolate from cacao. Ab initio gene prediction identified 9264 protein-coding genes, of which 800 are unique to C. theobromae when compared to Rhizoctonia spp., a closely related group. Transcriptome analysis using RNA isolated from 4 independent VSD symptomatic cacao stems identified 3550 transcriptionally active genes when compared to the assembled C. theobromae genome while transcripts for only 4 C. theobromae genes were detected in 2 asymptomatic stems. De novo assembly of the non-cacao associated reads from the VSD symptomatic stems uniformly produced genes with high identity to predicted genes in the C. theobromae genome as compared to Rhizoctonia spp. or genes found in Genbank. Further analysis of the predicted C. theobromae transcriptome was carried out identifying CAZy gene classes, KEGG-pathway associated genes, and 138 putative effector proteins. CONCLUSION: These findings put forth, for the first time, a predicted genome for the fastidious basidiomycete C. theobromae causing VSD on cacao providing a model for testing and comparison in the future. The C. theobromae genome predicts a pathogenesis model involving secreted effector proteins to suppress plant defense mechanisms and plant cell wall degrading enzymes.

Morphological variants of Moniliophthora roreri on artificial media and the biotroph/necrotroph shift.

Moniliophthora roreri (Mr) causes frosty pod rot of Theobroma cacao in a hemibiotrophic association. The Mr biotroph-like phase has not been studied in culture. Mr spores (isolates Co12, Co52, and B3) were germinated on high (V8) and low (BPMM) nutrients with different media hardness (0.5% to 3% agarose). Germination was high on V8 media. Hardness affected germination on BPMM. Most colonies on V8 were slow-growing, failing to sporulate. Colony morphology depended on the isolate. On BPMM, exaggerated mycelia formed of limited length with enlarged cells. On agarose, rapidly expanding sporulating necrotrophic colonies formed rarely. Co12 and B3 spores were germinated on V8 and BPMM with low melting point (LMP) agarose. Slow-growing colonies of B3 on BPMM were unstable on LMP agarose, often forming slow-growing/rapidly expanding hybrids. Slow-growing colonies are hypothesized to represent the biotrophic phase. One nucleus was common in Mr cells, other than spores. Binucleate cells were occasionally observed in aged cells of slow-growing mycelia. Co52 cells often had more than two nuclei per cell after germination. Mr mycelia cells typically carry a single nucleus, being considered haploid. Biotroph- and necrotroph-like mycelia displayed differential gene expression but results were inconsistent with published in vivo results and require further study.

Moniliophthora roreri, causal agent of cacao frosty pod rot.

Taxonomy: Moniliophthora roreri (Cif.) H.C. Evans et al. ; Phylum Basidiomycota; Class Agaricomycetes; Order Agaricales; Family Marasmiaceae; Genus Moniliophthora. Biology: Moniliophthora roreri attacks Theobroma and Herrania species causing frosty pod rot. Theobroma cacao (cacao) is the host of major economic concern. Moniliophthora roreri is a hemibiotroph with a long biotrophic phase (45-90 days). Spore masses, of apparent asexual origin, are produced on the pod surface after initiation of the necrotrophic phase. Spores are spread by wind, rain and human activity. Symptoms of the biotrophic phase can include necrotic flecks and, in some cases, pod malformation, but pods otherwise remain asymptomatic. Relationship to Moniliophthora perniciosa: Moniliophthora roreri and Moniliophthora perniciosa, causal agent of witches' broom disease of cacao, are closely related. Their genomes are similar, including many of the genes they carry which are considered to be important in the disease process. Moniliophthora perniciosa, also a hemibiotroph, has a typical basidiomycete lifestyle and morphology, forming clamp connections and producing mushrooms. Basidiospores infect meristematic tissues including flower cushions, stem tips and pods. Moniliophthora roreri does not form clamp connections or mushrooms and infects pods only. Both pathogens are limited to the Western Hemisphere and are a threat to cacao production around the world. Agronomic importance: Disease losses caused by frosty pod rot can reach 90% and result in field abandonment. Moniliophthora roreri remains in the invasive phase in the Western Hemisphere, not having reached Brazil, some islands within the Caribbean and a few specific regions within otherwise invaded countries. DISEASE MANAGEMENT: The disease can be managed by a combination of cultural (for example, maintenance of tree height and removal of infected pods) and chemical methods. These methods benefit from regional application, but can be cost prohibitive. Breeding for disease resistance offers the greatest potential for frosty pod rot management and new tolerant materials are becoming available.

Assessing hidden parentage and genetic integrity of the "United Fruit Clones" of cacao (Theobroma cacao) from Costa Rica using SNP markers.

The international cacao collection in CATIE, Costa Rica contains nearly 1200 accessions of cacao, mainly from the center of genetic diversity of this species. Among these accessions, the United Fruit clones (UF clones) were developed by the United Fruit Company in Costa Rica, and they represent one of the earliest groups of improved cacao germplasm in the world. Some of these UF clones have been used as key progenitors for breeding resistance/tolerance to Frosty Pod and Black Pod diseases in the Americas. Accurate information on the identity and background of these clones is important for their effective use in breeding. Using Single Nucleotide Polymorphism (SNP) markers, we genotyped 273 cacao germplasm accessions including 44 UF clones and 229 reference accessions. We verified the true-to-type identity of UF clones in the CATIE cacao collection and analyzed their population memberships using maximum-likelihood-based approaches. Three duplicate groups, representing approximately 30% of the UF clones, were identified. Both distance- and model-based clustering methods showed that the UF clones were mainly composed of Trinitario, ancient Nacional and hybrids between ancient Nacional and Amelonado. This result filled the information gap about the UF clones thus will improve their utilization for cacao breeding.