Rafael Moysés Alves

Mating system in a natural population of Theobroma grandiflorum (Willd. ex Spreng.) Schum., by microsatellite markers

The aim of this research was to study the mating system of a natural population of Theobroma grandiflorum (cupuassu) from Nova Ipixuna, Para state, using microsatellite markers. Eight polymorphic microsatellite loci were analyzed in eight families, each represented by 10 six-month old seedlings derived from open-pollinated pods. The estimation for the multilocus outcrossing rate (m = 1.0) and individual outcrossing rate ( = 1.0) for this population suggests that T. grandiflorum may be a perfect outbreeding (allogamous) species. Likewise, for the studied population the estimate for single locus outcrossing rate (S) was elevated (0.946), but lower than m, confirming the likely outcrossing character of the species and suggesting the occurrence of 5.4% biparental inbreeding rate (m - S). The estimation of genetic divergence (st) between allelic frequencies in ovules and pollen revealed a deviation from random mating in 75% of the evaluated loci. Likewise, the estimate of correlation of paternity (P = 0.930) and the mean coefficient of co-ancestrality within families (XY = 0.501) indicated that the outcrossings were predominantly correlated, and the offspring were full-sibs. These results suggested that for this particular population of T. grandiflorum, the sampling strategy for genetic conservation and breeding should adopt specific models for families derived from correlated outcrossing (full-sibs) and not the ones usually adopted in classic outcrossing species breeding programs (half-sibs).

High levels of genetic divergence and inbreeding in populations of cupuassu (Theobroma grandiflorum)

Theobroma grandiflorum (cupuassu) is an important fruit tree native to the Brazilian Amazon. Establishing the genetic diversity and structure of populations is critical to define long-term strategies for cupuassu conservation presently threatened by rapid deforestation. Three natural populations collected at the putative center of diversity, three groups of accessions established at a germplasm collection, and one derived from commercial plantings were analyzed. The genetic diversity was assessed using 21 polymorphic microsatellite loci originally developed for Theobroma cacao, disclosing a total of 113 alleles. The estimated genetic diversity parameters averaged over cupuassu populations (A = 3.53 alleles per locus; He = 0.426; Ho = 0.346) were lower than the values reported for other Neotropical tree species. The three natural populations presented a positive and significant fixation index (f), ranging from 0.133 to 0.234. Cupuassu apparently adhered to a general pattern of genetic diversity structure of some Neotropical tree species occurring at low densities, with a low intrapopulation genetic diversity and important levels of endogamy, possibly due to biparental inbreeding derived from the presence of spatial genetic structure in the populations. A high level of genetic divergence was detected among the natural populations (θp = 0.301), a strong differentiation caused by limited gene flow, and suggesting that human interference in spreading and/or stimulating plantings might have had a smaller effect than expected. The approximate location of the T. grandiflorum center of diversity could not be confirmed by analyzing natural populations from the putative region.

Diversidade genética em coleções amazônicas de germoplasma de cupuaçuzeiro [Theobroma grandiflorum (Willd. ex Spreng.) Schum.]

This study aimed to evaluate the genetic diversity and relatedness of 24 accessions of Theobroma grandiflorum, originating from three units of Embrapa, aiming their use as parents in hybridization specie programs. The genetic markers used were heterologous microsatellite loci developed for cocoa. In the population studied 45 alleles were found. The effective average number of alleles per locus (2.33) was less than the average number of alleles per locus (3.21), indicating that many alleles have low frequency. The observed heterozygosity at polymorphic loci ranged from 0.33 to 1.00 with a mean of 0.54 and expected heterozygosity ranged from 0.48 to 0.76 with a mean of 0.54. The fixation index medium between loci (0.003) was not significantly different from zero. The estimate of relatedness between pairs of individuals indicates that some may be relatives, including half-brothers and clones. The results suggest that the accesses of T. grandiflorum analyzed contain a moderate level of genetic diversity and absence of inbreeding and therefore great potential for use in breeding programs.

Evaluation and selection of Theobroma grandiflorum parents, in Belem, Para

Este trabalho teve por objetivo a selecao entre e dentro de progenies de irmaos completos de cupuacuzeiro. Foram avaliadas 21 progenies de irmaos completos, em dois experimentos, com cinco plantas por parcela. Os experimentos foram avaliados ao nivel de individuos, em cada safra, para os caracteres: producao de frutos, polpa e semente, bem como resistencia a vassoura-de-bruxa. Para efeito de selecao, tambem foi considerado o grau de parentesco das matrizes. As analises foram conduzidas via metodologia de modelos lineares mistos, como delineamento em blocos incompletos, desbalanceados com tratamentos comuns. Foram estimados os parâmetros geneticos e os valores genotipicos ajustados de progenies, bem como os valores geneticos aditivos e genotipicos individuais. Os resultados demonstraram que as progenies 12; 13; 18; 20; 21 e 24 tiveram os melhores desempenhos para as variaveis de producao. Entre elas, as progenies 12; 13 e 18 foram as que mais se destacaram. Foram selecionadas tres matrizes da progenie 18, duas matrizes da progenie 12 e uma matriz de cada uma das progenies 13; 20 e 21. E possivel concluir que essas matrizes, juntamente com materiais selecionados em outras areas experimentais, apresentam potencial para compor um pomar de sementes clonais, estabelecido em lote isolado de outros plantios, onde sera produzida uma populacao melhorada de primeiro ciclo que se constituira na nova cultivar de cupuacuzeiro.

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.

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.

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.

MODELO DE EFEITOS FIXOS COM MEDIDA REPETIDA APLICADO EM EXPERIMENTOS DE MELHORAMENTO GENÉTICO DO CUPUAÇUZEIRO

This study was conducted to evaluate vegetative performance of cupuassu progenies, in the immaturity phase, through analysis of repeated measures. The experimental area was installed in the municipality of Tome Acu, Northeast of the state of Para. We employed 25 full-sib progeny of cupuassu, who had vegetative growth (height and diameter of the plant), monitored for three years. Because of the nature of longitudinal observations it was assessed primarily through Mauchly sphericity test, which type of statistical analysis should be applied. For the variable diameter, sphericity conditionwas not violated, therefore, proceeded to review the design scheme of split plot. For the height variable, the test was significant, indicating necessarily evaluate the best structure to explain the correlation of errors, being chosen the covariance structure Heterogeneous Composed Symmetry. Differences were found between the progenies only in the third year of evaluation, and the variable diameter of the plant allowed better discriminate the progenies.

Phenotypic and genotypic characterization and compatibility among genotypes to select elite clones of cupuassu.

A ampliacao da base genetica dos materiais de cultivo e trabalho continuo do programa de melhoramento genetico do cupuacuzeiro (Theobroma grandiflorum). Ha necessidade, entretanto, que os parentais envolvidos estejam caracterizados fenotipica e genotipicamente, para auxiliar na escolha dos individuos que serao hibridizados, bem como para garantir os mecanismos de rastreabilidade e protecao das cultivares. Este estudo teve como objetivo identificar e selecionar clones de cupuacuzeiro inter-compativeis e geneticamente divergentes, utilizando como ferramentas as distâncias genotipica, fenotipica e combinada mistas, assim como as taxas de compatibilidade entre os clones. Estimativas das distâncias geneticas entre os clones foram realizadas com base em 14 iniciadores microssatelites heterologos de cacaueiro (Theobroma cacao) que amplificam o DNA do cupuacuzeiro. Para a caracterizacao fenotipica foram empregadas 14 variaveis relacionadas a producao de frutos. A matriz de dissimilaridade conjunta foi obtida por meio da soma das matrizes de dissimilaridade fenotipica e molecular. A compatibilidade intra e inter-clonal foi estimada atraves de cruzamentos controlados. Houve uma baixa correlacao entre as matrizes de dissimilaridade com base nos dados moleculares e agronomicos. Quanto a compatibilidade, todos os clones foram auto-incompativeis, contudo, compativeis entre si, com diferentes taxas. O indice de compatibilidade foi fortemente influenciado pelo grau de relacionamento dos clones. Foi possivel identificar e selecionar os conjuntos de clones de cupuacuzeiro mais promissores para ser usados em melhoramento genetico, apesar da ocorrencia de relacao genetica entre eles.