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Cocoa is an important crop for South American countries such as Ecuador, Peru, and Colombia, providing a source of income to nearly 300,000 households, mainly small farm holders, who have, until the recent past, been able to improve their revenue thanks to the high quality of their cocoa. Unfortunately, a large part of these fine cocoa producers is no longer allowed to export their cocoa to E.U., because of its level of cadmium, in many cases higher than the value permitted by an E.U regulation effective since January 2019. The ClimaLoca Project, initiated in 2021 aims at 1) precisely assessing the geographical extent of the cadmium in soil and cocoa beans, 2) assessing the impact of the new regulation on the income of the farmers of the three countries, 3) assessing the impact of climatic change on cadmium uptake, 4) assess the efficiency and cost-effectiveness of soil amendments to reduce cadmium uptake and 5) identify cocoa genotypes with low cadmium uptake and identify the genomic regions involved in this attribute. A participatory approach has been adopted, including on-farm experiments and the establishment of stakeholders' platforms. In addition, a strong collaboration has been maintained between participating research institutions from the three LATAM countries and from the European countries participating (France, Belgium, Netherlands). This collaboration is ensured through seminars and workshops and through the project website (climaloca. org). The strong link between the research team and the cocoa producers' organization has already permitted a successful transfer of a new quick and low-cost methodology of cadmium measurement in cocoa beans to a Peruvian cocoa cooperative, allowing the selection of the cocoa batches complying with the regulation of cadmium, that can be exported to E.U.

Theobroma cacao is native to the Upper Amazon. The species has undergone centuries of environmental and human selection and has spread throughout Latin America. The T. cacao germplasm is characterized by a high genetic diversity with at least ten major genetically differentiated groups. Little is known about the environmental and climatic drivers, as well as the genomic basis of adaptation in the species. Yet, a better understanding of how plants adapt to their environment is crucial to improve predictions of species' adaptive capacities and to provide accurate conservation strategies of genetic resources in the context of increasing environmental pressure. Environmental changes along altitudinal gradients allow the identification of functionally important genes contributing to the adaptive potential of species at a restricted geographic scale. In this study, using landscape genomics approaches, we deciphered the genomic basis of cocoa tree adaptation to temperature and precipitation from accessions sampled along an altitudinal gradient. A total of 102 geolocalised accessions of cacao trees collected in Ecuador along an altitudinal gradient ranging from 200 to 1000 m above sea level have been re-sequenced to identify genome-wide biallelic single nucleotide polymorphism (SNP) markers and presence/absence gene variation (PAV). The climate variables associated with sampled locations were obtained from the public database WorldClim. Using genome-scan, we detected genes under selection along the altitudinal gradient and potentially involved in trees adaptation to climate. Then, we searched for significant associations between genomic variation and the climate variables by conducting genome-environment association studies. Functional categories of genes associated with adaptation to elevation were investigated. This work will enhance our understanding of T. cacao response to continued climatic change. It allows to better characterize the available adaptive genetic diversi

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.

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.

Societal Impact Statement: “Fine flavor” cocoa, known for its superior flavor and aroma, commands a higher price for farmers than “bulk” cocoa produced for market. These fine flavor cocoa varieties make an important contribution to the agricultural sector in Ecuador. However, cocoa diversity is threatened by deforestation. The effective preservation, characterization, and use of cocoa tree diversity are therefore essential to the future development of this market. We carried out participatory collection surveys with local communities in the Ecuadorian Amazon Forest, to evaluate the genetic diversity of native cocoa trees and protect trees as a resource for local communities. Accessing this wealth of diversity will aid farmers to safeguard cocoa against climate change and develop new varieties for market. Summary: The aromatic Nacional variety of Theobroma cacao, emblematic of Ecuador, is highly sought after by the chocolate industry. The modern Nacional is a hybrid population resulting from genetic admixture that has lost the specificity of the ancestral variety. In the context of progressive forest disappearance, the objective of our study was to collect, safeguard, and evaluate native aromatic cocoa trees from Ecuadorian Amazon areas previously identified as areas of origin of the Nacional variety, as well as those that extend toward the northern Amazon. Four collection expeditions were organized in the Ecuadorian Amazon provinces of Zamora-Chinchipe, Morona-Santiago, and Pastaza in close collaboration with local communities. A total of 283 native accessions were collected and safeguarded at experimental stations and in local communities. The genetic diversity of the cocoa trees was analyzed by comparison to known genetic groups with a set of 48 simple sequence repeat (SSR) markers. This new collection clearly enriches the currently known diversity and improves knowledge of the global genetic structure of cocoa trees. Our results clarify the geographic origin of t

Societal Impact Statement: Recent surveys conducted on Amazonian cocoa trees in their home range are a unique opportunity to assess the aromas, diversity and potential of the Ecuadorian Amazon to create new aromatic cocoa varieties. Our results reveal informations about the diversity and genesis of aromas in Ecuadorian fine cocoa. The great aromatic diversity could enrich cocoa flavour selection programmes and provide Amazonian populations with new income linked to aromatic varieties, which could lead to a 'grand cru' chocolate. Until now, breeding programmes have been mainly focused on improving characteristics for production, but in recent years cocoa quality has been increasingly examined to meet market expectations. Summary: Ecuador is known worldwide for its fine or flavour cocoa from the Nacional variety. Currently, farmers mainly grow the modern Nacional variety of cocoa trees (hybrids between the ancestral Nacional and Trinitario) while the ancestral Nacional variety tends to be neglected. In order to enlarge the genetic resources related to this ancestral variety, several surveys were carried out in its area of origin located in the South Ecuadorian Amazonia. The 202 trees resulting from these surveys were genotyped using GBS (Genotyping By Sequencing) markers and their dried-fermented beans were characterised for both their aromatic volatile compounds and sensorial quality. A genome wide association study (GWAS) was carried out with the aim to study the genetic and biochemical bases of the aroma traits of this population and to better exploit them in breeding programmes. Some association areas and candidate genes related to spicy and woody aromas were found for the first time in Theobroma cacao. Some association areas and candidate genes related to floral and fruity aromas common to other studies were identified. Our results support the hypothesis that aroma formation may be related to a defence reaction to biotic and abiotic stresses generated by the ferm

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 rat

Cacao (Theobroma cacao L.) is considered a key crop in Colombian social programs aiming at alleviating rural poverty, promoting peace in post-conflict regions and, replacing crops used for illicit purposes. Colombia is thought to be part of the center of origin of cacao; several germplasm collecting expeditions have been implemented, dating back to the 1940s. Despite that history, the first breeding program based on creating, selecting, and releasing full-sib progenies made extensive use of accessions introduced from other countries as parents. A new breeding strategy was adopted in the 1990s, based on mass selection of promising trees (high-yield and disease-resistant) in farmers' fields, resulting in the selection of clones released to farmers as planting material. In 2012, a new strategy, Recurrent Selection, was adopted by the Colombian Corporation for Agricultural Research, Agrosavia, based on the development of improved populations and allowing the selection of clones at the end of each cycle of recombination. The use of molecular markers is being integrated into this program in order to assist breeders in selecting material. This review provides details about the history and perspectives of the cacao breeding program in Colombia.

The archaeological site of Santa Ana-La Florida (SALF), located in the Ecuadorian upper Amazon, is in the region of Theobroma spp. greatest genetic diversity, thus making it ideal to investigate the origins of domestication of this enigmatic tree. We present research showing that the residents of SALF were involved in the domestication of cacao, traditionally thought to have been first domesticated in Mesoamerica and/or Central America. We used three independent lines of evidence—starch grains, theobromine residues and ancient DNA—dating from approximately 5,300 years ago, to establish the earliest evidence of T. cacao use in the Americas, the first unequivocal archaeological example of its pre-Columbian use in South America and reveal the upper Amazon region as the oldest centre of cacao domestication yet identified. We suggest that new paleoethnobotanical research will expand our knowledge of this process, including the timing, locations, and uses of cacao by Indigenous South Americans.

Genomic selection (GS) is a statistical approach that utilizes all available genome-wide markers simultaneously and phenotypic traits of a “training population” to estimate breeding values or total genetic values. For breeding programs, GS is a promising alternative to the traditional marker-assisted selection for manipulating complex polygenic traits often controlled by many small effect genes. A major interest of this method is also to be able to make predictions of trait values, from a training population, on trees only genotyped by molecular markers The use of the appropriate statistical model remains one of the critical issues of the GS. The relative performance of biometrical models is expected to depend on the genetic background of the traits under assessment. The objective of this study was to estimate the reliability of different models of genomic selection to predict two agronomic traits of cacao - yield and resistance to Phytophthora pod rot. The study was performed on 287 trees present in a cacao farm plot in Cameroon, belonging to several full-sib progenies released to farmers as commercial varieties. Each tree was genotyped, using more than 5,000 GBS (genotyping by sequencing) based on SNP markers, and assessed for the mean average of one bean, a trait contributing to cocoa yield, and the % of rotten, as a measure of resistance to Phytophthora megakarya. Two models were used: Best linear unbiased prediction model and Bayesian lasso model. Cross-validation was used to test their predictive ability. It is an assumption-free method using an estimation set for model training and an independent test set for prediction. Predictive ability of models was good for both traits indicating that GS is a promising method to improve these cocoa traits. However, it was slightly higher for average weight of a bean (R= 0.59) than for % of rotten pods (R= 0.42).

The first draft genome of the species, from the Belizian Criollo B97-61/B2 cultivar, was published in 2011. Although a useful resource, some improvements were possible, including to identify misassemblies, to reduce the number of scaffolds and gaps, and to anchor un-anchored sequences to the 10 chromosomes. In 2017, we used a combination Next Generation Sequencing data to produce the version 2 of the assembly. We corrected misassembled regions and reduced the number of scaffolds from 4,792 in assembly V1 to 554 in V2 with a N50 increased from 0.47 Mb in V1 to 6.5 Mb in V2. A total of 96.7% of the assembly was anchored to the 10 chromosomes compared to 66.8% in the previous version. Unknown sites (Ns) were reduced from 10.8% to 5.7%. In addition, we updated the functional annotations and performed a new RefSeq structural annotation based on RNAseq evidence. In that context and to support post-genomics efforts, we developed the Cocoa Genome Hub (http://cocoagenome- hub.southgreen.fr/), an integrated web-based database providing centralized access to T. cacao genome and analysis tools to facilitate basic, translational and applied research in cocoa. We provide access to the complete criollo genome sequence V2 along with gene structure, gene product information, metabolism, gene families, transcriptomics (ESTs, RNA-Seq), genetic markers and genetic maps. The hub relies on generic software (e.g. GMOD tools) for easy querying, visualizing and downloading research data. It includes a Genome Browser enhanced by a Community Annotation System, enabling the improvement of automatic gene annotation through an annotation editor.

We explored the Theobroma cacao genome sequence to progress in the knowledge of the T. cacao incompatibility system. Cocoa self-compatibility is an important yield factor and has been described as controlled by a late gameto-sporophytic system involving several locus, and resulting in gametic non-fusion. In this work, we identified two different mechanisms controlling the T. cacao self-incompatibility system at two separate loci, located on chromosome one and four (CH1 and CH4). Both loci are responsible for gametic selection, but only one (the CH4 locus) is involved in the main fruit drop. The CH1 locus acts prior to gamete fusion and independently of CH4 locus. Fine mapping and genome wide association studies focused analyses of restricted regions without recombinant plants where several candidate genes were identified. Their expression analysis showed differential expression during incompatible or compatible reactions for some of them. Highly polymorphic SSR diagnostic markers, designed in the CH4 region identified by fine mapping, allowed the development of efficient diagnostic markers predicting selfcompatibility and fruit setting according to allele or genotype presence. SSR alleles specific to self-compatible Amelonado and Criollo varieties were also identified allowing screening for self-compatible plants in cocoa populations.

Background: There is great potential for the genetic improvement of oil palm yield. Traditional progeny tests allow accurate selection but limit the number of individuals evaluated. Genomic selection (GS) could overcome this constraint. We estimated the accuracy of GS prediction of seven oil yield components using A × B hybrid progeny tests with almost 500 crosses for training and 200 crosses for independent validation. Genotyping-by-sequencing (GBS) yielded +5000 single nucleotide polymorphisms (SNPs) on the parents of the crosses. The genomic best linear unbiased prediction method gave genomic predictions using the SNPs of the training and validation sets and the phenotypes of the training crosses. The practical impact was illustrated by quantifying the additional bunch production of the crosses selected in the validation experiment if genomic preselection had been applied in the parental populations before progeny tests. Results: We found that prediction accuracies for cross values plateaued at 500 to 2000 SNPs, with high (0.73) or low (0.28) values depending on traits. Similar results were obtained when parental breeding values were predicted. GS was able to capture genetic differences within parental families, requiring at least 2000 SNPs with less than 5% missing data, imputed using pedigrees. Genomic preselection could have increased the selected hybrids bunch production by more than 10%. Conclusions: Finally, preselection for yield components using GBS is the first possible application of GS in oil palm. This will increase selection intensity, thus improving the performance of commercial hybrids. Further research is required to increase the benefits from GS, which should revolutionize oil palm breeding.

Theobroma cacao L., native from the Amazonian basin of South America is an economically important fruit tree crop for tropical countries, source of chocolate. The first draft genome of the species, from a Criollo cultivar was published In 2011. Although a useful resource, some improvements can be made, including efforts to identify misassemblies, and reduction of the number of scaffolds, gaps, and un-anchored sequences to the ten chromosomes. In this work, we used a NGS-based approach to significantly improve the assembly of the Belizian Criollo B97-61/B2 genome. We combined 4 Illumina large insert size mate paired libraries with 52x of Pacific Biosciences long reads to correct misassembled regions, reduce the number of scaffolds to 554 (4,792 in assembly V1) with a N50 increased from 0.47 Mb to 6.5 Mb. 96.7% of the assembly was anchored to the 10 chromosomes compared to the previous 66.8%. Unknown sites (Ns) were reduced from 10.8% to 5.7%. Moreover, the NCBI Eukaryotic Genome Annotation Pipeline carried out a new RefSeq structural annotation based on RNAseq evidences and functional annotations have been updated. The release of the Theobroma cacao Criollo genome version 2 will be a valuable resource for investigating complex traits at the genomic level and is an important step for future comparative genomics and genetics studied on cocoa. New functional tools and annotations are available through the cacao genome hub (http://cocoa-genome-hub.southgreen.fr). (Texte integral)

Background: Theobroma cacao L., native to the Amazonian basin of South America, is an economically important fruit tree crop for tropical countries as a source of chocolate. The first draft genome of the species, from a Criollo cultivar, was published in 2011. Although a useful resource, some improvements are possible, including identifying misassemblies, reducing the number of scaffolds and gaps, and anchoring un-anchored sequences to the 10 chromosomes. Methods: We used a NGS-based approach to significantly improve the assembly of the Belizian Criollo B97-61/B2 genome. We combined four Illumina large insert size mate paired libraries with 52x of Pacific Biosciences long reads to correct misassembled regions and reduced the number of scaffolds. We then used genotyping by sequencing (GBS) methods to increase the proportion of the assembly anchored to chromosomes. Results: The scaffold number decreased from 4,792 in assembly V1 to 554 in V2 while the scaffold N50 size has increased from 0.47 Mb in V1 to 6.5 Mb in V2. A total of 96.7% of the assembly was anchored to the 10 chromosomes compared to 66.8% in the previous version. Unknown sites (Ns) were reduced from 10.8% to 5.7%. In addition, we updated the functional annotations and performed a new RefSeq structural annotation based on RNAseq evidence. Conclusion: Theobroma cacao Criollo genome version 2 will be a valuable resource for the investigation of complex traits at the genomic level and for future comparative genomics and genetics studies in cacao tree. New functional tools and annotations are available on the Cocoa Genome Hub (http://cocoa-genome-hub.southgreen.fr).

Uno de los pilares fundamentales en los que se basa hoy en día el desarrollo de la cacaocultura ecuatoriana, es el aprovechamiento de sus recursos genéticos disponibles. Con esta visión, desde el año 1995, se diseñaron nuevas estrategias de mejoramiento genético, con el objetivo de: a) obtener nuevos materiales genéticos con elevada producción y calidad organoléptica; y b) Potenciar el desarrollo de nuevos nichos de producción en áreas no tradicionales del cultivo. Para esto, un equipo multidisciplinario de trabajo ha venido combinando esfuerzos de investigación en las áreas de: mejoramiento convencional (cruzamientos), biología molecular, calidad organoléptica del grano (físico, químico y sensorial) y más recientemente también de tipo arqueológico, con el propósito de alcanzar los objetivos propuestos. El primer objetivo se empezó a cumplir desde el año 2009, con la liberación comercial de 4 clones altamente productivos: 2 para la Provincia de Manabí y 2 para la Provincia de Santa Elena, convirtiéndose esta última provincia a la época actual, en la región del país donde se evidencian los niveles más altos de producción de cacao fino por hectárea, en base al material genético liberado por INIAP. El resultado anterior, se consolidó en el año 2016, con la entrega para otra zona agroecológica de dos nuevos clones que inclusive superan en producción a nuestro referente territorial (el clon CCN-51). Al momento, se han obtenido nuevos resultados, con base en los cuales se planifica la entrega de dos nuevos clones de alta producción (2019) y que serán específicos para otra región cacaotera del Ecuador. El segundo objetivo también se está materializando con la identificación de zonas con potencial para la producción de nuevos cacaos especiales y de manera particular en la amazonia ecuatoriana, en donde se han realizado hasta el momento tres prospecciones en la región sur, con el propósito de aprovechar al máximo la diversidad natural presente en la zona. Este accionar ha pe