Mars Center for Cocoa Science

Ribeirão Preto, Brazil

Mars Center for Cocoa Science

Ribeirão Preto, Brazil
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Kuhn D.N.,U.S. Department of Agriculture | Figueira A.,University of Sao Paulo | Lopes U.,Mars Center for Cocoa Science | Motamayor J.C.,Mars Inc. | And 5 more authors.
Tree Genetics and Genomes | Year: 2010

The seeds of Theobroma cacao (cacao) are the source of cocoa, the raw material for the multi-billion dollar chocolate industry. Cacao's two most important traits are its unique seed storage triglyceride (cocoa butter) and the flavor of its fermented beans (chocolate). The genome of T. cacao is being sequenced, and to expand the utility of the genome sequence to the improvement of cacao, we are evaluating Theobroma grandiflorum, the closest economically important species of Theobroma for its potential use in a comparative genomic study. T. grandiflorum differs from cacao in important agronomic traits such as flavor of the fermented beans, disease resistance to witches' broom and abscission of mature fruits. By comparing genomic sequences and analyzing viable inter-specific hybrids, we hope to identify the key genes that regulate cacao's most important traits. We have investigated the utility in T. grandiflorum of three types of markers (microsatellite markers, single-strand conformational polymorphism markers and single nucleotide polymorphism (SNP) markers) developed in cacao. Through sequencing of amplicons of 12 diverse individuals of both cacao and T. grandiflorum, we have identified new intra- and inter-specific SNPs. Two markers which had no overlap of alleles between the species were used to genotype putative inter-specific hybrid seedlings. Sequence conservation was significant and species-specific differences numerous enough to suggest that comparative genomics of T. grandiflorum and T. cacao will be useful in elucidating the genetic differences that lead to a variety of important agronomic trait differences. © 2010 US Government.


Stack J.C.,Mars Incorporated | Royaert S.,Mars Center for Cocoa Science | Gutierrez O.,U.S. Department of Agriculture | Nagai C.,Hawaii Agriculture Research Center | And 3 more authors.
Tree Genetics and Genomes | Year: 2015

Linkage disequilibrium (LD) measured over the genomes of a species can provide important indications for how future association analyses should proceed. This information can be advantageous especially for slow-growing, perennial crops such as Theobroma cacao, where experimental crosses are inherently time-consuming and logistically expensive. While LD has been evaluated in cacao, previous work has been focused on relatively narrow genetic bases. We use microsatellite marker data collected from a uniquely diverse sample of individuals broadly covering both wild and cultivated varieties to gauge the LD present in the different cacao diversity groups and populations. We find that genome-wide LD decays far more rapidly in the wild and primitive diversity groups of cacao as compared to those representing cultivated varieties. The impact that such differences can have on association analyses is demonstrated using phenotypic data on pod color and genotypic data from two cacao populations with contrasting patterns of LD decay. Our results indicate that the more rapid LD decay in wild and primitive germplasm can lead to higher-resolution mapping intervals when compared to results from cultivated germplasm. Through simulations, we demonstrate how future association mapping analyses, comprising of cacao samples with a wild or primitive background, will likely exhibit lower LD and would be more suitable for fine-scale association mapping analyses. As many traits targeted by cacao breeders are found exclusively in wild and primitive germplasm, association mapping in wild cacao populations holds significant promise for cacao improvement through marker-assisted breeding and emphasize the need to further explore the natural diversity of Amazonian cacao. © 2015, The Author(s).


de Oliveira G.A.P.,Federal University of Rio de Janeiro | Pereira E.G.,Federal University of Rio de Janeiro | Dias C.V.,University Estadual Of Santa Cruz | Dias C.V.,Mars Center For Cocoa Science | And 8 more authors.
PLoS ONE | Year: 2012

Understanding how Nep-like proteins (NLPs) behave during the cell cycle and disease progression of plant pathogenic oomycetes, fungi and bacteria is crucial in light of compelling evidence that these proteins play a role in Witches' Broom Disease (WBD) of Theobroma cacao, one of the most important phytopathological problems to afflict the Southern Hemisphere. The crystal structure of MpNep2, a member of the NLP family and the causal agent of WBD, revealed the key elements for its activity. This protein has the ability to refold after heating and was believed to act as a monomer in solution, in contrast to the related homologs MpNep1 and NPP from the oomyceteous fungus Phytophthora parasitica. Here, we identify and characterize a metastable MpNep2 dimer upon over-expression in Escherichia coli using different biochemical and structural approaches. We found using ultra-fast liquid chromatography that the MpNep2 dimer can be dissociated by heating but not by dilution, oxidation or high ionic strength. Small-angle X-ray scattering revealed a possible tail-to-tail interaction between monomers, and nuclear magnetic resonance measurements identified perturbed residues involved in the putative interface of interaction. We also explored the ability of the MpNep2 monomer to refold after heating or chemical denaturation. We observed that MpNep2 has a low stability and cooperative fold that could be an explanation for its structure and activity recovery after stress. These results can provide new insights into the mechanism for MpNep2′s action in dicot plants during the progression of WBD and may open new avenues for the involvement of NLP- oligomeric species in phytopathological disorders. © 2012 de Oliveira et al.


Royaert S.,Mars Center for Cocoa Science | Jansen J.,Wageningen University | da Silva D.V.,University Estadual Of Santa Cruz | de Jesus Branco S.M.,University Estadual Of Santa Cruz | And 6 more authors.
BMC Genomics | Year: 2016

Background: Witches' broom disease (WBD) caused by the fungus Moniliophthora perniciosa is responsible for considerable economic losses for cacao producers. One of the ways to combat WBD is to plant resistant cultivars. Resistance may be governed by a few genetic factors, mainly found in wild germplasm. Results: We developed a dense genetic linkage map with a length of 852.8 cM that contains 3,526 SNPs and is based on the MP01 mapping population, which counts 459 trees from a cross between the resistant 'TSH 1188' and the tolerant 'CCN 51' at the Mars Center for Cocoa Science in Barro Preto, Bahia, Brazil. Seven quantitative trait loci (QTL) that are associated with WBD were identified on five different chromosomes using a multi-trait QTL analysis for outbreeders. Phasing of the haplotypes at the major QTL region on chromosome IX on a diversity panel of genotypes clearly indicates that the major resistance locus comes from a well-known source of WBD resistance, the clone 'SCAVINA 6'. Various potential candidate genes identified within all QTL may be involved in different steps leading to disease resistance. Preliminary expression data indicate that at least three of these candidate genes may play a role during the first 12 h after infection, with clear differences between 'CCN 51' and 'TSH 1188'. Conclusions: We combined the information from a large mapping population with very distinct parents that segregate for WBD, a dense set of mapped markers, rigorous phenotyping capabilities and the availability of a sequenced genome to identify several genomic regions that are involved in WBD resistance. We also identified a novel source of resistance that most likely comes from the 'CCN 51' parent. Thanks to the large population size of the MP01 population, we were able to pick up QTL and markers with relatively small effects that can contribute to the creation and selection of more tolerant/resistant plant material. © 2016 Royaert et al.


Villela-Dias C.,University Estadual Of Santa Cruz | Villela-Dias C.,Mars Center for Cocoa Science | Camillo L.R.,University Estadual Of Santa Cruz | de Oliveira G.A.P.,Federal University of Rio de Janeiro | And 7 more authors.
Physiologia Plantarum | Year: 2014

NEP1 (necrosis- and ethylene-inducing peptide 1)-like proteins (NLPs) have been identified in a variety of taxonomically unrelated plant pathogens and share a common characteristic of inducing responses of plant defense and cell death in dicotyledonous plants. Even though some aspects of NLP action have been well characterized, nothing is known about the global range of modifications in proteome and metabolome of NLP-treated plant cells. Here, using both proteomic and metabolomic approaches we were able to identify the global molecular and biochemical changes in cells of Nicotiana benthamiana elicited by short-term treatment with MpNEP2, a NLP of Moniliophthora perniciosa, the basidiomycete responsible for the witches' broom disease on cocoa (Theobroma cacao L.). Approximately 100 protein spots were collected from 2-DE gels in each proteome, with one-third showing more than twofold differences in the expression values. Fifty-three such proteins were identified by mass spectrometry (MS)/MS and mapped into specific metabolic pathways and cellular processes. Most MpNEP2 upregulated proteins are involved in nucleotide-binding function and oxidoreductase activity, whereas the downregulated proteins are mostly involved in glycolysis, response to stress and protein folding. Thirty metabolites were detected by gas spectrometry (GC)/MS and semi-quantified, of which eleven showed significant differences between the treatments, including proline, alanine, myo-inositol, ethylene, threonine and hydroxylamine. The global changes described affect the reduction-oxidation reactions, ATP biosynthesis and key signaling molecules as calcium and hydrogen peroxide. These findings will help creating a broader understanding of NLP-mediated cell death signaling in plants. © 2013 Scandinavian Plant Physiology Society.


PubMed | University Estadual Of Santa Cruz, Mars Center for Cocoa Science, Rural University and U.S. Department of Agriculture
Type: Journal Article | Journal: Genetics and molecular research : GMR | Year: 2016

Theobroma cacao is a species of great economic importance with its beans used for chocolate production. The tree has been a target of various molecular studies. It contains many polyphenols, which complicate the extraction of nucleic acids with the extraction protocols requiring a large amount of plant material. These issues, therefore, necessitate the optimization of the protocols. The aim of the present study was to evaluate different methods for extraction of total RNA from shoot apical meristems of T. cacao CCN 51 and to assess the influence of storage conditions for the meristems on the extraction. The study also aimed to identify the most efficient protocol for RNA extraction using a small amount of plant material. Four different protocols were evaluated for RNA extraction using one shoot apical meristem per sample. Among these protocols, one that was more efficient was then tested to extract RNA using four different numbers of shoot apical meristems, subjected to three different storage conditions. The best protocol was tested for cDNA amplification using reverse transcription-polymerase chain reaction; the cDNA quality was determined to be satisfactory for molecular analyses. The study revealed that with the best RNA extraction protocol, one shoot apical meristem was sufficient for extraction of high-quality total RNA. The results obtained might enable advances in genetic analyses and molecular studies using reduced amount of plant material.


PubMed | Wageningen University, Mars Center for Cocoa Science, Mars Incorporated, Rural University and University Estadual Of Santa Cruz
Type: | Journal: BMC genomics | Year: 2016

Witches broom disease (WBD) caused by the fungus Moniliophthora perniciosa is responsible for considerable economic losses for cacao producers. One of the ways to combat WBD is to plant resistant cultivars. Resistance may be governed by a few genetic factors, mainly found in wild germplasm.We developed a dense genetic linkage map with a length of 852.8 cM that contains 3,526 SNPs and is based on the MP01 mapping population, which counts 459 trees from a cross between the resistant TSH 1188 and the tolerant CCN 51 at the Mars Center for Cocoa Science in Barro Preto, Bahia, Brazil. Seven quantitative trait loci (QTL) that are associated with WBD were identified on five different chromosomes using a multi-trait QTL analysis for outbreeders. Phasing of the haplotypes at the major QTL region on chromosome IX on a diversity panel of genotypes clearly indicates that the major resistance locus comes from a well-known source of WBD resistance, the clone SCAVINA 6. Various potential candidate genes identified within all QTL may be involved in different steps leading to disease resistance. Preliminary expression data indicate that at least three of these candidate genes may play a role during the first 12 h after infection, with clear differences between CCN 51 and TSH 1188.We combined the information from a large mapping population with very distinct parents that segregate for WBD, a dense set of mapped markers, rigorous phenotyping capabilities and the availability of a sequenced genome to identify several genomic regions that are involved in WBD resistance. We also identified a novel source of resistance that most likely comes from the CCN 51 parent. Thanks to the large population size of the MP01 population, we were able to pick up QTL and markers with relatively small effects that can contribute to the creation and selection of more tolerant/resistant plant material.


Dias C.V.,State University of Santa Cruz | Dias C.V.,Mars Center for Cocoa Science | Mendes J.S.,State University of Santa Cruz | dos Santos A.C.,State University of Santa Cruz | And 9 more authors.
Plant Physiology and Biochemistry | Year: 2011

In plant-pathogen interaction, the hydrogen peroxide (H 2O 2) may play a dual role: its accumulation inhibits the growth of biotrophic pathogens, while it could help the infection/colonization process of plant by necrotrophic pathogens. One of the possible pathways of H 2O 2 production involves oxalic acid (Oxa) degradation by apoplastic oxalate oxidase. Here, we analyzed the production of H 2O 2, the presence of calcium oxalate (CaOx) crystals and the content of Oxa and ascorbic acid (Asa) - the main precursor of Oxa in plants - in susceptible and resistant cacao (Theobroma cacao L.) infected by the hemibiotrophic fungus Moniliophthora perniciosa. We also quantified the transcript level of ascorbate peroxidase (Apx), germin-like oxalate oxidase (Glp) and dehydroascorbate reductase (Dhar) by RT-qPCR. We report that the CaOx crystal amount and the H 2O 2 levels in the two varieties present distinct temporal and genotype-dependent patterns. Susceptible variety accumulated more CaOx crystals than the resistant one, and the dissolution of these crystals occurred in the early infection steps and in the final stage of the disease in the resistant and the susceptible variety, respectively. High expression of the Glp and accumulation of Oxa were observed in the resistant variety. The content of Asa increased in the inoculated susceptible variety, but remained constant in the resistant one. The susceptible variety presented reduced Dhar expression. The role of H 2O 2 and its formation from Oxa via Apx and Glp in resistant and susceptible variety infected by M. perniciosa were discussed. © 2011 Elsevier Masson SAS.


Motamayor J.C.,Mars Incorporated | Mockaitis K.,Indiana University | Schmutz J.,Mars Incorporated | Schmutz J.,HudsonAlpha Institute for Biotechnology | And 30 more authors.
Genome Biology | Year: 2013

Background: Theobroma cacao L. cultivar Matina 1-6 belongs to the most cultivated cacao type. The availability of its genome sequence and methods for identifying genes responsible for important cacao traits will aid cacao researchers and breeders.Results: We describe the sequencing and assembly of the genome of Theobroma cacao L. cultivar Matina. 1-6. The genome of the Matina 1-6 cultivar is 445 Mbp, which is significantly larger than a sequenced Criollo cultivar, and more typical of other cultivars. The chromosome-scale assembly, version 1.1, contains 711 scaffolds covering 346.0 Mbp, with a contig N50 of 84.4 kbp, a scaffold N50 of 34.4 Mbp, and an evidence-based gene set of 29,408 loci. Version 1.1 has 10x the scaffold N50 and 4x the contig N50 as Criollo, and includes 111 Mb more anchored sequence. The version 1.1 assembly has 4.4% gap sequence, while Criollo has 10.9%. Through a combination of haplotype, association mapping and gene expression analyses, we leverage this robust reference genome to identify a promising candidate gene responsible for pod color variation. We demonstrate that green/red pod color in cacao is likely regulated by the R2R3 MYB transcription factor TcMYB113, homologs of which determine pigmentation in Rosaceae, Solanaceae, and Brassicaceae. One SNP within the target site for a highly conserved trans-acting siRNA in dicots, found within TcMYB113, seems to affect transcript levels of this gene and therefore pod color variation.Conclusions: We report a high-quality sequence and annotation of Theobroma cacao L. and demonstrate its utility in identifying candidate genes regulating traits. © 2013 Motamayor et al.; licensee BioMed Central Ltd.


Melnick R.L.,U.S. Department of Agriculture | Marelli J.-P.,Mars Center for Cocoa Science | Sicher R.C.,U.S. Department of Agriculture | Strem M.D.,U.S. Department of Agriculture | Bailey B.A.,U.S. Department of Agriculture
Tree Genetics and Genomes | Year: 2012

Witches' broom disease of Theobroma cacao L. is caused by the hemibiotrophic basidiomycete Moniliophthora perniciosa. Infection of flower cushions by M. perniciosa results in parthenocarpy. Healthy and parthenocarpic immature cacao pods were obtained from seven cacao clones. Microscopic observations of parthenocarpic pods from two clones confirmed that fruits lack viable seed. Septate mycelia colonized parthenocarpic pods, but were absent from healthy pods. Parthenocarpic pods had increased concentrations of leucine, methionine, serine, phenylalanine, and valine. Major transport metabolites sucrose and asparagine were decreased by 63 and 40 %, respectively, during parthenocarpy. M. perniciosa expressed sequence tags (ESTs) related to detoxification (MpSOD2 and MpCTA1) and nutrient acquisition (MpAS, MpAK, MpATG8, MpPLY, and MpPME) were induced in parthenocarpic pods. Most M. perniciosa ESTs related to plant hormone biosynthesis were repressed (MpGAox, MpCPS, MpDES, MpGGPPS, and MpCAO) in parthenocarpic pods. RT-qPCR analysis was conducted for 54 defense-related cacao ESTs and 93 hormone-related cacao ESTs. Specific cacao ESTs related to plant defense were induced (TcPR5, TcChi4, TcThau-ICS) while others were repressed (TcPR1, TcPR6, TcP12, and TcChiB). Cacao ESTs related to GA biosynthesis (TcGA20OX1B) were repressed in parthenocarpic pods. Cacao ESTs putatively related to maintaining cytokinin (TcCKX3 and TcCKX5) and IAA (TcGH3. 17a, TcGH3. 1, TcARF18) homeostasis were induced in parthenocarpic pods, suggesting an attempt to regulate cytokinin and auxin concentrations. In conclusion, M. perniciosa expresses specific sets of transcripts targeting nutrient acquisition and survival while altering the host physiology without causing significant necrosis resulting in parthenocarpy. Only a general host defense response is elicited. © 2012 Springer-Verlag (outside the USA).

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