Genomic decoding of Theobroma grandiflorum (cupuassu) at chromosomal scale: evolutionary insights for horticultural innovation.

BACKGROUND: Theobroma grandiflorum (Malvaceae), known as cupuassu, is a tree indigenous to the Amazon basin, valued for its large fruits and seed pulp, contributing notably to the Amazonian bioeconomy. The seed pulp is utilized in desserts and beverages, and its seed butter is used in cosmetics. Here, we present the sequenced telomere-to-telomere genome of cupuassu, disclosing its genomic structure, evolutionary features, and phylogenetic relationships within the Malvaceae family. FINDINGS: The cupuassu genome spans 423 Mb, encodes 31,381 genes distributed in 10 chromosomes, and exhibits approximately 65% gene synteny with the Theobroma cacao genome, reflecting a conserved evolutionary history, albeit punctuated with unique genomic variations. The main changes are pronounced by bursts of long-terminal repeat retrotransposons at postspecies divergence, retrocopied and singleton genes, and gene families displaying distinctive patterns of expansion and contraction. Furthermore, positively selected genes are evident, particularly among retained and dispersed tandem and proximal duplicated genes associated with general fruit and seed traits and defense mechanisms, supporting the hypothesis of potential episodes of subfunctionalization and neofunctionalization following duplication, as well as impact from distinct domestication process. These genomic variations may underpin the differences observed in fruit and seed morphology, ripening, and disease resistance between cupuassu and the other Malvaceae species. CONCLUSIONS: The cupuassu genome offers a foundational resource for both breeding improvement and conservation biology, yielding insights into the evolution and diversity within the genus Theobroma.

Finding the origin of domestication of cupuaçu requires more than genomics

Colli-Silva et al.1 recently proposed that cupuaçu (Theobroma grandiflorum) was domesticated in northwestern Amazonia as a selection from its sister species, cupuí (T. subincanum). This proposal ignores long-term research in taxonomy, botany, biogeography, and genetics about Theobroma, including cupuaçu. Our review of the research that was ignored and of Colli-Silva et al.'s results demonstrates that cupuaçu is a valid species, as they now agree2, but cupuí may be paraphyletic, the distribution of wild cupuaçu was not included in their samples so the origin of domestication continues unknown, precolonial archaeology lacks remains that can be attributed to either species, historical linguistics indicates that the term cupuaçu references the species' wild distribution and is a recent introduction in northwestern Amazonia, history suggests that cupuaçu started to be domesticated during the last 100-200 years, and the genomics results are not about the domestication of cupuaçu because it is a valid species and its wild distribution was not sampled.

Simultaneous selection of cupuassu tree and Brazilian mahogany genotypes in an agroforestry system in Pará state, Brazil

This study aimed to evaluate and select progenies and matrices of cupuassu-tree siblings, and to select Brazilian mahogany matrices with superior characteristics to be used in agroforestry systems (AFS). Twenty-five full cupuassu tree sibling progenies and one Brazilian mahogany half-sibling progeny were evaluated. The study was conducted for 14 years in a commercial property in Tomé Açu, Pará State, Brazil. The number of fruits and fruit yield per plant, as well as the rate of plants with symptoms of witches' broom disease, were used as response variables for cupuassu. For mahogany, the following response variables were used: total height, commercial height, crown height, diameter at breast height and commercial wood volume. There were five cupuassu tree progenies with interesting characteristics to be used in AFS. Based on the high values observed in the selection accuracy and heritabilities, a good potential for the selection of promising individuals in the cupuassu tree population is inferred. Based on the ranking of genotypic values and their agronomic performance, 10 matrices of cupuassu tree were selected. For Brazilian mahogany, three matrices showed excellent silvicultural performance. The matrices of these two species should be propagated vegetatively in order to evaluate clonal tests. (AU)

Involvement of structurally distinct cupuassu chitinases and osmotin in plant resistance to the fungus Moniliophthora perniciosa.

The cupuassu tree (Theobroma grandiflorum) is a crop of great economic importance to Brazil, mainly for its pulp and seeds, which are used in food industry. However, cupuassu fruit production is threatened by witches' broom disease caused by the fungus Moniliophthora perniciosa. As elements of its defense mechanisms, the plant can produce and accumulate pathogenesis-related (PR) proteins such as chitinases and osmotins. Here, we identified three cupuassu PR proteins (TgPR3, TgPR5 and TgPR8) from cupuassu-M. perniciosa interaction RNA-seq data. TgPR3 and TgPR8 corresponded to chitinases, and TgPR5 to osmotin; they are phylogenetically related to cacao and to Arabidopsis PR sequences involved in biotic and abiotic stress. The TgPR proteins' tridimensional structure was obtained through homology modeling, and molecular docking with chitin and chitosan showed that the TgPR proteins can interact with both cell wall molecules and presented a higher affinity for chitosan. TgPR gene expression was analyzed by RT-qPCR on resistant and susceptible cupuassu genotypes infected by M. perniciosa at 8, 24, 48 and 72 h after infection (hai). The TgPR genes showed higher expression in resistant plants compared to the susceptible ones, mainly for TgPR5 at 8 and 24 hai, while the expression was lower in the susceptible cupuassu plants. To our knowledge, this is the first in silico and in vitro reports of cupuassu PR protein. The data suggested that TgPRs could be involved in recognizing mechanisms of the plant's innate immune system through chitin receptors. Our results also suggest a putative role of chitinase/chitosanase for the TgPR5/osmotin.

Phenotypic and genotypic characterization and compatibility among genotypes to select elite clones of cupuassu / Caracterização fenotípica, genotípica e compatibilidade entre genótipos para seleção de clones elite de cupuaçuzeiro

ABSTRACT The expansion of the genetic base of cultivated materials is an ongoing activity of the cupuassu (Theobroma grandiflorum) breeding program. However, the parents involved need to be genotypically and phenotypically characterized to ensure compatibility of crossings, as well as to assist in the selection of more promising individuals for hybridization. This study aimed to identify and select T. grandiflorum clones that are compatible and genetically divergent using tools such as the estimates of genotypic, phenotypic, and combined distances, as well as the compatibility rates among clones. The genetic distance analysis of the clones was performed with 14 heterologous microsatellite primers of cocoa (Theobroma cacao) that amplify the DNA of cupuassu. Phenotypic characterization was based on 14 variables related to fruit production. The joint dissimilarity matrix was obtained by means of the sum of the phenotypic and molecular dissimilarity matrices. The intra- and inter-clonal compatibility was estimated through controlled crossings. A low correlation was noted between the dissimilarity matrices based on the molecular and agronomic data. As for compatibility, all clones were self-incompatible, with different compatibility rates when crossed. The compatibility index was strongly influenced by the degree of relationship of the clones. It was possible to identify and select the most promising sets of cupuassu clones to be used in breeding programs, despite their genetic relationship.

RESUMO A ampliação da base genética dos materiais de cultivo é trabalho contínuo do programa de melhoramento genético do cupuaçuzeiro (Theobroma grandiflorum). Há necessidade, entretanto, que os parentais envolvidos estejam caracterizados fenotípica e genotipicamente, para auxiliar na escolha dos indivíduos que serão hibridizados, bem como para garantir os mecanismos de rastreabilidade e proteção das cultivares. Este estudo teve como objetivo identificar e selecionar clones de cupuaçuzeiro inter-compatíveis e geneticamente divergentes, utilizando como ferramentas as distâncias genotípica, fenotípica e combinada mistas, assim como as taxas de compatibilidade entre os clones. Estimativas das distâncias genéticas entre os clones foram realizadas com base em 14 iniciadores microssatélites heterólogos de cacaueiro (Theobroma cacao) que amplificam o DNA do cupuaçuzeiro. Para a caracterização fenotípica foram empregadas 14 variáveis ​​relacionadas à produção de frutos. A matriz de dissimilaridade conjunta foi obtida por meio da soma das matrizes de dissimilaridade fenotípica e molecular. A compatibilidade intra e inter-clonal foi estimada através de cruzamentos controlados. Houve uma baixa correlação entre as matrizes de dissimilaridade com base nos dados moleculares e agronômicos. Quanto à compatibilidade, todos os clones foram auto-incompatíveis, contudo, compatíveis entre si, com diferentes taxas. O índice de compatibilidade foi fortemente influenciado pelo grau de relacionamento dos clones. Foi possível identificar e selecionar os conjuntos de clones de cupuaçuzeiro mais promissores para ser usados em melhoramento genético, apesar da ocorrência de relação genética entre eles.

Genome size, cytogenetic data and transferability of EST-SSRs markers in wild and cultivated species of the genus Theobroma L. (Byttnerioideae, Malvaceae).

The genus Theobroma comprises several trees species native to the Amazon. Theobroma cacao L. plays a key economic role mainly in the chocolate industry. Both cultivated and wild forms are described within the genus. Variations in genome size and chromosome number have been used for prediction purposes including the frequency of interspecific hybridization or inference about evolutionary relationships. In this study, the nuclear DNA content, karyotype and genetic diversity using functional microsatellites (EST-SSR) of seven Theobroma species were characterized. The nuclear content of DNA for all analyzed Theobroma species was 1C = ~ 0.46 pg. These species presented 2n = 20 with small chromosomes and only one pair of terminal heterochromatic bands positively stained (CMA+/DAPI- bands). The small size of Theobroma ssp. genomes was equivalent to other Byttnerioideae species, suggesting that the basal lineage of Malvaceae have smaller genomes and that there was an expansion of 2C values in the more specialized family clades. A set of 20 EST-SSR primers were characterized for related species of Theobroma, in which 12 loci were polymorphic. The polymorphism information content (PIC) ranged from 0.23 to 0.65, indicating a high level of information per locus. Combined results of flow cytometry, cytogenetic data and EST-SSRs markers will contribute to better describe the species and infer about the evolutionary relationships among Theobroma species. In addition, the importance of a core collection for conservation purposes is highlighted.

Selection of Reference Genes for Expression Study in Pulp and Seeds of Theobroma grandiflorum (Willd. ex Spreng.) Schum.

Cupuassu (Theobroma grandiflorum [Willd. ex Spreng.] Schum) is a species of high economic importance in Brazil with great potential at international level due to the multiple uses of both its seeds and pulp in the industry of sweets and cosmetics. For this reason, the cupuassu breeding program focused on the selection of genotypes with high pulp and seed quality-selection associated with the understanding of the mechanisms involved in fruit formation. Gene expression is one of the most used approaches related to such understanding. In this sense, quantitative real-time PCR (qPCR) is a powerful tool, since it rapidly and reliably quantifies gene expression levels across different experimental conditions. The analysis by qPCR and the correct interpretation of data depend on signal normalization using reference genes, i.e. genes presenting a uniform pattern of expression in the analyzed samples. Here, we selected and analyzed the expression of five genes from cupuassu (ACP, ACT, GAPDH, MDH, TUB) to be used as candidates for reference genes on pulp and seed of young, maturing and mature cupuassu fruits. The evaluation of the gene expression stability was obtained using the NormFinder, geNorm and BestKeeper programs. In general, our results indicated that the GAPDH and MDH genes constituted the best combination as reference genes to analyze the expression of cupuassu samples. To our knowledge, this is the first report of reference gene definition in cupuassu, and these results will support subsequent analysis related to gene expression studies in cupuassu plants subjected to different biotic or abiotic conditions as well as serve as a tool for diversity analysis based on pulp and seed quality.

First Microsatellite Markers Developed from Cupuassu ESTs: Application in Diversity Analysis and Cross-Species Transferability to Cacao.

The cupuassu tree (Theobroma grandiflorum) (Willd. ex Spreng.) Schum. is a fruitful species from the Amazon with great economical potential, due to the multiple uses of its fruit´s pulp and seeds in the food and cosmetic industries, including the production of cupulate, an alternative to chocolate. In order to support the cupuassu breeding program and to select plants presenting both pulp/seed quality and fungal disease resistance, SSRs from Next Generation Sequencing ESTs were obtained and used in diversity analysis. From 8,330 ESTs, 1,517 contained one or more SSRs (1,899 SSRs identified). The most abundant motifs identified in the EST-SSRs were hepta- and trinucleotides, and they were found with a minimum and maximum of 2 and 19 repeats, respectively. From the 1,517 ESTs containing SSRs, 70 ESTs were selected based on their functional annotation, focusing on pulp and seed quality, as well as resistance to pathogens. The 70 ESTs selected contained 77 SSRs, and among which, 11 were polymorphic in cupuassu genotypes. These EST-SSRs were able to discriminate the cupuassu genotype in relation to resistance/susceptibility to witches' broom disease, as well as to pulp quality (SST/ATT values). Finally, we showed that these markers were transferable to cacao genotypes, and that genome availability might be used as a predictive tool for polymorphism detection and primer design useful for both Theobroma species. To our knowledge, this is the first report involving EST-SSRs from cupuassu and is also a pioneer in the analysis of marker transferability from cupuassu to cacao. Moreover, these markers might contribute to develop or saturate the cupuassu and cacao genetic maps, respectively.