Search

The glutathione peroxidases (GPXs) are enzymes which are part of the cell antioxidant system inhibiting the ROS-induced damages of membranes and proteins. In cacao (Theobroma cacao L.) genome, five GPX genes were identified. Cysteine insertion codons (UGU) were found in TcPHGPX, TcGPX2, TcGPX4, TcGPX6 and tryptophan insertion codon (UGG) in TcGPX8. Multiple alignments revealed conserved domains between TcGPXs and other plants and human GPXs. Homology modeling was performed using the Populus trichocarpa GPX5 structure as template, and the molecular modeling showed that TcGPXs have affinity with selenometionine in their active site. In silico analysis of the TcGPXs promoter region revealed the presence of conserved cis-elements related to biotic stresses and hormone responsiveness. The expression analysis of TcGPXs in cacao plantlet meristems infected by M. perniciosa showed that TcGPXs are most expressed in susceptible variety than in resistant one, mainly in disease stages in which oxidative stress and programmed cell death occurred. This data, associated with phylogenetic and location analysis suggested that TcGPXs may play a role in protecting cells from oxidative stress as a try of disease progression reduction. To our knowledge, this is the first study of the overall GPX family from T. cacao.

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.

O gênero Theobroma abrange 22 espécies nativas da região amazônica, dentre elas, duas merecem destaque de produção no Brasil: T. cacao e T. grandiflorum. Devido à importância dessas culturas e a possibilidade de obter informações através de transferibilidade de marcadores moleculares específicos de T. grandiflorum em T. cacao; ii) construir um mapa genético a partir de um cruzamento de T. grandiflorum; iii) posicionar no genoma do cacaueiro os microssatélites de T. grandiflorum. A partir de trabalhos anteriores, foram selecionados e testados 181 marcadores específicos do cacaueiro nos genitores e em cinco indivíduos da população de cupuaçuzeiro, e 44 marcadores específicos do cupuaçuzeiro em cinco genótipos de cacaueiro representativos da diversidade da espécie. As análises de transferibilidade de microssatélites entre T. cacao e T. grandiflorum mostraram uma baixa taxa de transferibilidade dos marcadores de cacaueiro quando amplificados em cupuaçuzeiro (43,09%), e uma alta taxa de transferibilidade dos marcadores de cupuaçuzeiro quando amplificados em cacaueiro (93,3%). Foi possível obter um mapeamento genético parcial de T. grandiflorum, permitindo flanquear microssatélites, das duas espécies, em sete grupos de ligação do mapa de referência do cacaueiro em um mapa genético consenso da espécie. Os resultados do presente trabalho permitiram obter mais informações sobre a transferibilidade e mapeamento de microssatélites entre T. cacao e T. grandiflorum. Tais conhecimentos possibilitam estudos posteriores sobre a diversidade genética, caracterização de germoplasma, construção de mapas genéticos e localização de QTLs.

The selenium-binding proteins are known to be inducers of apoptosis in human and animals, and have been studied as target for the treatment of various types of cancer. In plants, SBP expression has been related to abiotic and biotic stress resistance. The SBP from Theobroma cacao (TcSBP) was first identified from a cocoa-Moniliophthora perniciosa cDNA library. The present study provides details on the TcSBP gene and protein structure. Multiple alignments revealed conserved domains between SBP from plants, human and archea. Homology modeling and molecular docking were performed and showed that the TcSBP has affinity to selenite in the active CSSC site. This result was confirmed by circular dichroism of the recombinant TcSBP, which also presented thermostable behavior. RT-qPCR analysis showed that TcSBP was differentially expressed in resistant vs susceptible cacao varieties inoculated by M. perniciosa and its expression was probably due to hormone induction via cis-regulating elements present in its promotor. The presence of the CSSC domain suggested that TcSBP acted by altering oxidation/reduction of proteins during H2O2 production and programmed cell death in the final stages of the witches' broom disease. To our knowledge, this is the first in silico and in vitro analysis of the SBP from cacao.

Cocoa self-compatibility is an important yield factor and has been described as being controlled by a late gameto-sporophytic system expressed only at the level of the embryo sac. It results in gametic non-fusion and involves several loci. In this work, we identified two loci, located on chromosomes 1 and 4 (CH1 and CH4), involved in cocoa self-incompatibility by two different processes. 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 the gamete fusion step and independently of the CH4 locus. Using fine-mapping and genome-wide association studies, we focused analyses on restricted regions and identified candidate genes. Some of them showed a differential expression between incompatible and compatible reactions. Immunolocalization experiments provided evidence of CH1 candidate genes expressed in ovule and style tissues. Highly polymorphic simple sequence repeat (SSR) diagnostic markers were designed in the CH4 region that had been identified by fine-mapping. They are characterized by a strong linkage disequilibrium with incompatibility alleles, thus allowing the development of efficient diagnostic markers predicting self-compatibility and fruit setting according to the presence of specific alleles or genotypes. SSR alleles specific to self-compatible Amelonado and Criollo varieties were also identified, thus allowing screening for self-compatible plants in cocoa populations.

Transcriptional regulation, led by transcription factors (TFs) such as those of the WRKY family, is a mechanism used by the organism to enhance or repress gene expression in response to stimuli. Here, we report on the genome-wide analysis of the Theobroma cacao WRKY TF family and also investigate the expression of WRKY genes in cacao infected by the fungus Moniliophthora perniciosa. In the cacao genome, 61 non-redundant WRKY sequences were found and classified in three groups (I to III) according to the WRKY and zinc-finger motif types. The 61 putative WRKY sequences were distributed on the 10 cacao chromosomes and 24 of them came from duplication events. The sequences were phylogenetically organized according to the general WRKY groups. The phylogenetic analysis revealed that subgroups IIa and IIb are sister groups and share a common ancestor, as well as subgroups IId and IIe. The most divergent groups according to the plant origin were IIc and III. According to the phylogenetic analysis, 7 TcWRKY genes were selected and analyzed by RT-qPCR in susceptible and resistant cacao plants infected (or not) with M. perniciosa. Some TcWRKY genes presented interesting responses to M. perniciosa such as Tc01_p014750/Tc06_p013130/AtWRKY28, Tc09_p001530/Tc06_p004420/AtWRKY40, Tc04_p016130/AtWRKY54 and Tc10_p016570/ AtWRKY70. Our results can help to select appropriate candidate genes for further characterization in cacao or in other Theobroma species.

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.

In Theobroma cacao the main biotic stresses are cause by fungi, i.e Moniliophthora perniciosa (Mp), causal agent of witches' broom disease of cacao (WBD). Breeding of Mp-resistant varieties is confronted with two major difficulties at present. First, cacao resistant sources have been identified, but most of them are Scavina 6 descendants. Second, resistance from Scavina sources has shown to be unstable. The OMICS with the classical phytopatological and breeding approaches have allowed identifying genotypes with distinction in relation to WBD resistance, thus, carrying different resistance genes. New microsatellites and SNPs markers, and new QTLs (under natural and artificial inoculations) linked to WBD disease resistance have been identified. In parallel, histopatological studies of the cacao-Mp interaction revealed multiples modes of penetration of the fungus into cacao plants as well different mechanisms of resistance. The adaptability of Mp has also been considered, and advances in the understanding the breakdown of witches´ broom resistance, in Bahia, Brazil, have been achieved. Partial results of these projects and the overall strategy will be presented.

Advances in DNA technology, such as marker assisted selection, detection of quantitative trait loci and genomic selection also require the isolation of DNA from a large number of samples and the preservation of tissue samples for future use in cacao genome studies. The present study proposes a method for the preservation of sample tissues for DNA extraction and for manual extraction of large number of samples using spheres. The integrity and concentration of the DNA by these methods were assessed and compared with conventional method using mortar. The best parameters in order to obtain a fine powder using spheres was the use of 4 lyophilized leaf disks (50 mg), a single steel ball of 6 mm in diameter, followed by 30 s of manual maceration. The quantity of DNA obtained was four times higher than the conventional method. The purity of the DNA obtained was satisfactory and proved to be amplifiable by PCR using SSR primers. The present approach is a reliable, rapid, simple and consistent DNA isolation method for cacao, compared to the conventional methods. The protocol greatly increases the efficiency of extraction and suggests an inexpensive and practical way of DNA isolation of cacao for large scale.

Studies to determine the genetic bases to witches' broom disease (WBD) resistance in Theobroma cacao L. were carried out in order to identify different genetic sources of resistance and to improve the efficiency of selection using molecular markers. A major QTL for WBD resistance and linked to Scavina 6 - the mains source of resistance, was detected in chromosome 9 from a F2 Scavina 6 x ICS 1 progeny. In this paper, phonotypical quantification of vegetative and flower cushions brooms was done in the same progeny growing in two fields: F2-1M and F2-1C; respectively with 62 individuals and 142 individuals, observed over a period from 2003 to 2008. QTLs analyses were carried out with MapQTL 5.0 software to confirm the presence in chromosome 9 of the previously detected QTL. A significant instability of this QTL was observed with a LOD and the percentage of variation explained decreasing and moving in the confidence interval of mTcCIR157. Others QTLs of resistance were also detected such as one in Ch2, which was found in an opposite direction. These new QTLs analyzes, under natural infections of M. perniciosa, suggest a change in the host and pathogen relationships.

Cacao was introduced in Bahia in 1756, becoming later the largest producer state in the country. In order to support the planting of cacao in the region, a breeding program was established by CEPEC at the beginning of the 1970s. For a long time, the program consisted in testing new hybrids (full-sibs) and releasing a mixture of the best ones to farmers. Lately, particularly after the witches´ broom arrival in the region, in 1989, recurrent breeding strategies were implemented, aiming mainly the development of clones. From 1993 to 2010, more than 500 progenies, accumulating 30 thousand trees, were developed by crossing many parents with resistance to witches´ broom, high yield and other traits. In this period, more than 500 clones were put in trials and 39 clones and 3 hybrids were released to farmers. In this paper the strategies and results achieved by the program are reviewed. Overall the program has good interface with pathology and genomic programs.