Development of a Diet Production System for Conopomorpha cramerella (Lepidoptera: Gracillariidae), a Major Cocoa Production Pest in Southeast Asia and the Pacific Islands.

The development of artificial diets for the cocoa pod borer Conopomorpha cramerella, a major pest of cocoa plants, has undergone significant advancements. In this study, we present the success rates of two diet formulations, MM1 and MM4, which have been progressively improved. Nutritional composition analysis revealed that the MM1 diet differed from the natural host, cocoa pods, in several aspects, including protein, carbohydrate, and vitamin C content. To address these differences, modifications were made to the diet compositions, leading to the MM4 diet version. These modifications resulted in improved diet quality and reduced contamination, leading to enhanced success rates in all stages of C. cramerella development. Larval development, pupation success rates, and adult emergence rates were significantly higher in the MM4 diet compared with the MM1 diet. Moreover, the duration of larval development and pupal stage decreased, while adult longevity increased with the MM4 diet. The overall development success of diet-reared insects from egg to adult was comparable with that of insects reared on cocoa pods. However, the cocoon formation, body length and fresh weight of the adults reared on the artificial diets were lower than those reared on cocoa pods. This diet formulation provides a promising approach for laboratory rearing of C. cramerella and opens avenues for further research and mass-rearing initiatives to mitigate the impact of this pest on cocoa production.

Clonal reproduction of Moniliophthora roreri and the emergence of unique lineages with distinct genomes during range expansion.

The basidiomycete Moniliophthora roreri causes frosty pod rot of cacao (Theobroma cacao) in the western hemisphere. Moniliophthora roreri is considered asexual and haploid throughout its hemibiotrophic life cycle. To understand the processes driving genome modification, using long-read sequencing technology, we sequenced and assembled 5 high-quality M. roreri genomes out of a collection of 99 isolates collected throughout the pathogen's range. We obtained chromosome-scale assemblies composed of 11 scaffolds. We used short-read technology to sequence the genomes of 22 similarly chosen isolates. Alignments among the 5 reference assemblies revealed inversions, translocations, and duplications between and within scaffolds. Isolates at the front of the pathogens' expanding range tend to share lineage-specific structural variants, as confirmed by short-read sequencing. We identified, for the first time, 3 new mating type A locus alleles (5 in total) and 1 new potential mating type B locus allele (3 in total). Currently, only 2 mating type combinations, A1B1 and A2B2, are known to exist outside of Colombia. A systematic survey of the M. roreri transcriptome across 2 isolates identified an expanded candidate effector pool and provided evidence that effector candidate genes unique to the Moniliophthoras are preferentially expressed during the biotrophic phase of disease. Notably, M. roreri isolates in Costa Rica carry a chromosome segment duplication that has doubled the associated gene complement and includes secreted proteins and candidate effectors. Clonal reproduction of the haploid M. roreri genome has allowed lineages with unique genome structures and compositions to dominate as it expands its range, displaying a significant founder effect.

Single-base resolution methylomes of somatic embryogenesis in Theobroma cacao L. reveal epigenome modifications associated with somatic embryo abnormalities.

Propagation by somatic embryogenesis in Theobroma cacao has some issues to be solved, as many morphologically abnormal somatic embryos that do not germinate into plants are frequently observed, thus hampering plant production on a commercial scale. For the first time the methylome landscape of T. cacao somatic embryogenesis was examined, using whole-genome bisulfite sequencing technique, with the aim to understand the epigenetic basis of somatic embryo abnormalities. We identified 873 differentially methylated genes (DMGs) in the CpG context between zygotic embryos, normal and abnormal somatic embryos, with important roles in development, programmed cell death, oxidative stress, and hypoxia induction, which can help to explain the morphological abnormalities of somatic embryos. We also identified the role of ethylene and its precursor 1-aminocyclopropane-1-carboxylate in several biological processes, such as hypoxia induction, cell differentiation and cell polarity, that could be associated to the development of abnormal somatic embryos. The biological processes and the hypothesis of ethylene and its precursor involvement in the somatic embryo abnormalities in cacao are discussed.

Development of a Kairomone-Based Attractant as a Monitoring Tool for the Cocoa Pod Borer, Conopomorpha cramerella (Snellen) (Lepidoptera: Gracillariidae).

The cocoa pod borer (CPB), Conopomorpha cramerella, is a major economic pest of cocoa, Theobroma cacao, in Southeast Asia. CPB monitoring programs currently use a costly synthetic pheromone lure attractive to males. Field trapping experiments demonstrating an effective plant-based alternative are presented in this study. Five lychee-based products were compared for their attractiveness to CPB males. The organic lychee flavor extract (OLFE), the most attractive product, captured significantly more CPB as a 1 mL vial formulation than unbaited traps, while being competitive with the commercial pheromone lures. Additional experiments show that a 20 mL membrane OLFE lure was most effective, attracting significantly more CPB than the pheromone. When the kairomone and pheromone lures were combined, no additive or synergistic effects were observed. Concentrating the OLFE product (OLFEc) using a rotary evaporator increased the lure attractiveness to field longevity for up to 28 weeks; in contrast, pheromone lures were effective for approximately 4 weeks. The 20 mL concentrated OLFE membrane lures should provide a cheaper and more efficient monitoring tool for CPB than the current commercial pheromone lures.

Mealybug (Hemiptera: Pseudococcidae) Species Associated with Cacao Mild Mosaic Virus and Evidence of Virus Acquisition.

Theobroma cacao is affected by viruses on every continent where the crop is cultivated, with the most well-known ones belonging to the Badnavirus genus. One of these, cacao mild mosaic virus (CaMMV), is present in the Americas, and is transmitted by several species of Pseudococcidae (mealybugs). To determine which species are associated with virus-affected cacao plants in North America, and to assess their potential as vectors, mealybugs (n = 166) were collected from infected trees in Florida, and identified using COI, ITS2, and 28S markers. The species present were Pseudococcus jackbeardsleyi (38%; n = 63), Maconellicoccus hirsutus (34.3%; n = 57), Pseudococcus comstocki (15.7%; n = 26), and Ferrisia virgata (12%; n = 20). Virus acquisition was assessed by testing mealybug DNA (0.8 ng) using a nested PCR that amplified a 500 bp fragment of the movement protein-coat protein region of CaMMV. Virus sequences were obtained from 34.6 to 43.1% of the insects tested; however, acquisition did not differ among species, X2 (3, N = 166) = 0.56, p < 0.91. This study identified two new mealybug species, P. jackbeardsleyi and M. hirsutus, as potential vectors of CaMMV. This information is essential for understanding the infection cycle of CaMMV and developing effective management strategies.

Complete genome sequences of three newly discovered cacao mild mosaic virus isolates from Theobroma cacao L. in Brazil and Puerto Rico and evidence for recombination.

To analyze the DNA virome associated with cacao (Theobroma cacao L.) trees showing virus-like symptoms in Brazil (BR) and Puerto Rico (PR) during 2018-2019, total DNA was isolated from symptomatic leaves and subjected to high-throughput Illumina sequencing. The assembled complete badnaviral genome sequences were verified by PCR amplification, cloning, and DNA sequencing. Based on pairwise distances and phylogenetic analysis, three badnaviral genomes were identified, and these viruses were found to be isolates of the previously described cacao mild mosaic virus (CaMMV). The three genomes were 7,520, 7,524, and 7,514 bp in size for the isolates CaMMV-BR321, CaMMV-BR322, and CaMMV-PR3, respectively. Each genome contained four predicted open reading frames: ORFs 1-3 and ORFY. The CaMMV-PR3 isolate was identified as a probable recombinant, with a CaMMV-BR-like virus as the major parent.

Phytophthora theobromicola sp. nov.: A New Species Causing Black Pod Disease on Cacao in Brazil.

Black pod disease, caused by Phytophthora species, is among the main limiting factors of cacao (Theobroma cacao L.) production. High incidence levels of black pod disease have been reported in Brazil, being induced by Phytophthora capsici, Phytophthora citrophthora, Phytophthora heveae, and Phytophthora palmivora. To assess the diversity of Phytophthora species affecting cacao in Brazil, 40 new isolates were obtained from cacao pods exhibiting symptoms of black pod disease collected in different smallholder farms in 2017. Further, ten cacao-infecting isolates morphologically identified as P. citrophthora and P. palmivora were molecularly characterized. The genomic regions beta-tubulin, elongation factor 1 alpha, heat shock protein 90, and internal transcribed spacer, and the mitochondrially encoded cytochrome c oxidase I and II genes were PCR-amplified and Sanger-sequenced from the cacao-infecting Phytophthora isolates. The morphological characterization and evaluation of the mycelial growth rates for the Phytophthora isolates were performed in vitro. Based on the molecular analysis and morphological comparisons, 19 isolates were identified as P. palmivora (clade 4). Interestingly, 31 isolates grouped together in the phylogenetic tree and were placed apart from previously known species in Phytophthora clade 2. Therefore, these isolates are considered as a new species herein referred to as Phytophthora theobromicola sp. nov., which produced papillate, semipapillate, and persistent sporangia on simple sporangiophores. The P. palmivora isolates were identified as A1 mating type by pairing each isolate with known A1 and A2 tester strains of P. capsici, but no oogonia/antheridia were observed when P. theobromicola was paired with the different tester strains. The P. theobromicola and P. citrophthora isolates showed higher mycelial growth rates, when compared to P. palmivora, on different media at 10, 15, and 20°C, but similar values were observed when grown on clarified CA media at 25 and 30°C. The pathogenicity tests carried out on pods of four cacao clones (CCN51, PS1319, Cepec2004, and CP49) showed significant variability among the isolates of both Phytophthora species, with P. theobromicola inducing higher rates of necrotic lesion expansion, when compared to P. palmivora. Here, two Phytophthora species were found associated with black pod disease in the state of Bahia, Brazil, and the previously undescribed P. theobromicola seems to be prevalent in field conditions. This is the first report of P. theobromicola on T. cacao. Also, these findings are crucial to improve the disease control strategies, and for the development of cacao materials genetically resistant to Phytophthora.

Independent Whole-Genome Duplications Define the Architecture of the Genomes of the Devastating West African Cacao Black Pod Pathogen Phytophthora megakarya and Its Close Relative Phytophthora palmivora.

Phytophthora megakarya and P. palmivora are oomycete pathogens that cause black pod rot of cacao (Theobroma cacao), the most economically important disease on cacao globally. While P. palmivora is a cosmopolitan pathogen, P. megakarya, which is more aggressive on cacao than P. palmivora, has been reported only in West and Central Africa where it has been spreading and devastating cacao farms since the 1950s. In this study, we reconstructed the complete diploid genomes of multiple isolates of both species using single-molecule real-time sequencing. Thirty-one additional genotypes were sequenced to analyze inter- and intra-species genomic diversity. The P. megakarya genome is exceptionally large (222 Mbp) and nearly twice the size of P. palmivora (135 Mbp) and most known Phytophthora species (∼100 Mbp on average). Previous reports pointed toward a whole-genome duplication (WGD) in P. palmivora In this study, we demonstrate that both species underwent independent and relatively recent WGD events. In P. megakarya we identified a unique combination of WGD and large-scale transposable element driven genome expansion, which places this genome in the upper range of Phytophthora genome sizes, as well as effector pools with 1,382 predicted RxLR effectors. Finally, this study provides evidence of adaptive evolution of effectors like RxLRs and Crinklers, and discusses the implications of effector expansion and diversification.

A Complex of Badnavirus Species Infecting Cacao Reveals Mixed Infections, Extensive Genomic Variability, and Interspecific Recombination.

The incidence of cacao swollen shoot disease (CSSD) in cacao (Theobroma cacao L.) has increased in West Africa since ~2000. To investigate the genomic and species diversity of the CSSD-badnaviruses infecting cacao in Côte d'Ivoire and Ghana, symptomatic leaves were subjected to high-throughput sequencing. Among the 30 newly determined genomes, three badnaviruses were identified, Cacao swollen shoot Togo B virus (CSSTBV), Cacao swollen shoot CD virus, and Cacao swollen shoot CE virus (CSSCEV). The phylogenetic trees reconstructed for the reverse transcriptase (RT) and ribonuclease H (RNase H) sequences were incongruent with the complete viral genomes, which had the most robust statistical support. Recombination seems to be involved in the CSSD-badnavirus diversification. The genomic diversity varied among different CSSD-badnaviruses, with CSSTBV showing the lowest nucleotide diversity (π = 0.06236), and CSSCEV exhibiting the greatest variability (π = 0.21911). Evidence of strong purifying selection was found in the coding regions of the CSSTBV isolates.

Identification of Climate and Genetic Factors That Control Fat Content and Fatty Acid Composition of Theobroma cacao L. Beans.

The main ingredients of chocolate are usually cocoa powder, cocoa butter, and sugar. Both the powder and the butter are extracted from the beans of the cacao tree (Theobroma cacao L.). The cocoa butter represents the fat in the beans and possesses a unique fatty acid profile that results in chocolate's characteristic texture and mouthfeel. Here, we used a linkage mapping population and phenotypic data of 3,292 samples from 420 progeny which led to the identification of 27 quantitative trait loci (QTLs) for fatty acid composition and six QTLs for fat content. Progeny showed extensive variation in fat levels and composition, with the level of palmitic acid negatively correlated to the sum of stearic acid, oleic acid, and linoleic acid. A major QTL explaining 24% of the relative level of palmitic acid was mapped to the distal end of chromosome 4, and those higher levels of palmitic acid were associated with the presence of a haplotype from the "TSH 1188" parent in the progeny. Within this region of chromosome 4 is the Thecc1EG017405 gene, an orthologue and isoform of the stearoyl-acyl carrier protein (ACP) desaturase (SAD) gene in plants, which is involved in fatty acid biosynthesis. Besides allelic differences, we also show that climate factors can change the fatty acid composition in the beans, including a significant positive correlation between higher temperatures and the higher level of palmitic acid. Moreover, we found a significant pollen donor effect from the variety "SIAL 70" which was associated with decreased palmitic acid levels.

Rapid Molecular Identification of Scolytinae (Coleoptera: Curculionidae).

Routine identification of bark and ambrosia beetles is done using morphology. For people lacking the necessary taxonomic knowledge, proper identification of a novel specimen can be challenging and time consuming. This study compares the usefulness of four genetic markers (28S, EF-1a, ITS2, and COI) and five primer pairs (D2F1/D3R2, eflafor1/eflarev1, ets149/efa754, ITS2F/ITS2R, and LCO1490/HCO2198) to identify Scolytinae beetles, and outlines a molecular identification strategy, with results possible in two days. Markers COI and EF-1a were selected based on the ability of the respective primers to amplify DNA from multiple genera (Coptoborus, Xyleborus, Hypothenemus, Theoborus, and Araptus) and the ability of the resulting sequences to provide accurate and unambiguous matches in GenBank. BLASTn analysis of EF-1a sequences (both primer pairs) correctly identified four out of the five genera and COI sequences identified at least one sample of every genus tested and was the only primer pair to correctly identify Araptus specimens. Further, 28S sequences successfully identified Coptoborus, Xyleborus, and Theoborus but not Hypothenemus or Araptus. The low number of EF-1a (1), 28S (7), and ITS2 (0) sequences from Araptus individuals present in GenBank compared with COI (137) is likely the reason that only the latter marker was capable of identifying members of this genus. ITS2 sequences were insufficient to identify any of the samples tested. This study also determined the minimum quantity of DNA that could be used for molecular identification. Primers D2F1 and D3R2, which had the highest rate of amplification in all genera tested, could yield an informative sequence with as little as 0.00048 ng of DNA, however, at least 0.0024 ng was needed for reliable amplification.

Chocolate Under Threat from Old and New Cacao Diseases.

Theobroma cacao, the source of chocolate, is affected by destructive diseases wherever it is grown. Some diseases are endemic; however, as cacao was disseminated from the Amazon rain forest to new cultivation sites it encountered new pathogens. Two well-established diseases cause the greatest losses: black pod rot, caused by several species of Phytophthora, and witches' broom of cacao, caused by Moniliophthora perniciosa. Phytophthora megakarya causes the severest damage in the main cacao producing countries in West Africa, while P. palmivora causes significant losses globally. M. perniciosa is related to a sister basidiomycete species, M. roreri which causes frosty pod rot. These Moniliophthora species only occur in South and Central America, where they have significantly limited production since the beginnings of cacao cultivation. The basidiomycete Ceratobasidium theobromae causing vascular-streak dieback occurs only in South-East Asia and remains poorly understood. Cacao swollen shoot disease caused by Cacao swollen shoot virus is rapidly spreading in West Africa. This review presents contemporary research on the biology, taxonomy and genomics of what are often new-encounter pathogens, as well as the management of the diseases they cause.

Somatic Embryogenesis in Theobroma cacao L.

Theobroma cacao L. is a tropical tree originating in the Amazon, where it grows naturally in the shade of tropical rainforests. Cacao sub-products, such as butter and powder, are produced as principal components of chocolate and contain important nutritional compounds such as polyphenols and flavonoids. However, bean production is decreasing because plantations are antiquated and unproductive. Cacao propagation has been traditionally performed through classical propagation methods, such as grafting or rooted cuttings, but those methods are not sufficient to obtain large quantities of planting material with the desired genetic quality and optimal plant health. In the search for solutions to this problem, somatic embryogenesis (SE) is a vegetative method used for cacao propagation that has the potential to be explored. SE is a type of clonal propagation by which totipotent cells in the somatic tissue can develop into embryos and subsequently convert into plants.This method offers significant technological advantages because it is possible to obtain a large quantity of disease-free planting material with good agronomic characteristics and genetic stability. In T. cacao, tow techniques of in vitro micropropagation have been reported as direct and indirect SE. Indirect SE requires the additional step of cell dedifferentiation, unlike direct SE, which does not require this step. Here, we report a protocol using direct and indirect SE techniques using two types of culture methodologies-solid and liquid culture media.

Transcription Factors: Their Role in the Regulation of Somatic Embryogenesis in Theobroma cacao L. and Other Species.

Transcription factors are proteins that help with the control and regulation in the transcription of the DNA to mRNA by binding to special DNA sequences. With the aim to understand more about gene transcription regulation in Theobroma cacao L., this review outlines the principal transcription factors that were reported in other plants especially Arabidopsis thaliana and attempts at looking for the homologies with transcription factors in T. cacao. The information cited in this work is about the initiation, development, and maturation of the cacao somatic embryos and other crops. It is important to underline that there are very few publications in T. cacao discussing transcription factors that control the somatic embryogenesis process, but there is some information about transcription factors in other crops that we have used as a guide to try to understand this process.

Climate change could threaten cocoa production: Effects of 2015-16 El Niño-related drought on cocoa agroforests in Bahia, Brazil.

Climate models predict a possible increase in the frequency of strong climate events such as El Niño-Southern Oscillation (ENSO), which in parts of the tropics are the cause of exceptional droughts, these threaten global food production. Agroforestry systems are often suggested as promising diversification options to increase farmers' resilience to extreme climatic events. In the Northeastern state of Bahia, where most Brazilian cocoa is grown in wildlife-friendly agroforests, ENSOs cause severe droughts which negatively affect forest and agriculture. Cocoa (Theobroma cacao) is described as being sensitive to drought but there are no field-studies of the effect of ENSO-related drought on adult cocoa trees in the America's; there is one study of an experimentally-imposed drought in Indonesia which resulted in 10 to 46% yield loss. In our study, in randomly chosen farms in Bahia, Brazil, we measured the effect of the 2015-16 severe ENSO, which caused an unprecedented drought in cocoa agroforests. We show that drought caused high cocoa tree mortality (15%) and severely decreased cocoa yield (89%); the drought also increased infection rate of the chronic fungal disease witches' broom (Moniliophthora perniciosa). Ours findings showed that Brazilian cocoa agroforests are at risk and that increasing frequency of strong droughts are likely to cause decreased cocoa yields in the coming decades. Furthermore, because cocoa, like many crops, is grown somewhat beyond its climatic limits, it and other crops could be the 'canaries in the coalmine' warning of forthcoming major drought effects on semi-natural and natural vegetation.

Mapping of a Major QTL for Ceratocystis Wilt Disease in an F1 Population of Theobroma cacao.

Cacao is an important crop, its beans are key raw materials for the chocolate and cosmetic industries. Ceratocystis wilt of cacao (CWC) caused by Ceratocystis cacaofunesta is a lethal disease for the crop. Therefore, the selection of resistant cacao varieties is one of the viable ways to minimize losses in cacao production. In this paper, we described the identification of a major QTL associated with CWC in an F1 mapping population from a cross between a resistant, "TSH 1188," and a susceptible genotype, "CCN 51." A set of 266 trees were genotyped using 3,526 single nucleotide polymorphic markers and then multiple QTL mapping analyses were performed. Two QTLs were identified on chromosomes IV and VI. The major QTL was located at 20 cM from the top position of chromosome VI, accounting for more than 60% of the phenotypic variation. The favorable allele T1, with haplotype GTT, came from the "TSH 1188" parent. It was evident that the haplotype combination T1C2 on chromosome VI was the most significant for resistance, since 93% of resistant trees had this haplotype. The major QTL converged to a genomic region of 739.4 kb that harbored nine candidate genes, including two major classes of resistance genes, which would make them the primary candidates involved in the resistance to CWC. The haplotypes detected are now used to improve the efficiency and precision of the selection of resistant trees in cacao breeding.

Genetic Parameters and the Impact of Off-Types for Theobroma cacao L. in a Breeding Program in Brazil.

Breeding programs of cacao (Theobroma cacao L.) trees share the many challenges of breeding long-living perennial crops, and genetic progress is further constrained by both the limited understanding of the inheritance of complex traits and the prevalence of technical issues, such as mislabeled individuals (off-types). To better understand the genetic architecture of cacao, in this study, 13 years of phenotypic data collected from four progeny trials in Bahia, Brazil were analyzed jointly in a multisite analysis. Three separate analyses (multisite, single site with and without off-types) were performed to estimate genetic parameters from statistical models fitted on nine important agronomic traits (yield, seed index, pod index, % healthy pods, % pods infected with witches broom, % of pods other loss, vegetative brooms, diameter, and tree height). Genetic parameters were estimated along with variance components and heritabilities from the multisite analysis, and a trial was fingerprinted with low-density SNP markers to determine the impact of off-types on estimations. Heritabilities ranged from 0.37 to 0.64 for yield and its components and from 0.03 to 0.16 for disease resistance traits. A weighted index was used to make selections for clonal evaluation, and breeding values estimated for the parental selection and estimation of genetic gain. The impact of off-types to breeding progress in cacao was assessed for the first time. Even when present at <5% of the total population, off-types altered selections by 48%, and impacted heritability estimations for all nine of the traits analyzed, including a 41% difference in estimated heritability for yield. These results show that in a mixed model analysis, even a low level of pedigree error can significantly alter estimations of genetic parameters and selections in a breeding program.

Identification of candidate genes involved in Witches' broom disease resistance in a segregating mapping population of Theobroma cacao L. in Brazil.

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.

The genome sequence of the most widely cultivated cacao type and its use to identify candidate genes regulating pod color.

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.

Over-expression of a cacao class I chitinase gene in Theobroma cacao L. enhances resistance against the pathogen, Colletotrichum gloeosporioides.

Theobroma cacao L. plants over-expressing a cacao class I chitinase gene (TcChi1) under the control of a modified CaMV-35S promoter were obtained by Agrobacterium-mediated transformation of somatic embryo cotyledons. Southern blot analysis confirmed insertion of the transgene in eight independent lines. High levels of TcChi1 transgene expression in the transgenic lines were confirmed by northern blot analysis. Chitinase activity levels were measured using an in vitro fluorometric assay. The transgene was expressed at varying levels in the different transgenic lines with up to a sixfold increase of endochitinase activity compared to non-transgenic and transgenic control plants. The in vivo antifungal activity of the transgene against the foliar pathogen Colletotrichum gloeosporioides was evaluated using a cacao leaf disk bioassay. The assay demonstrated that the TcChi1 transgenic cacao leaves significantly inhibited the growth of the fungus and the development of leaf necrosis compared to controls when leaves were wound inoculated with 5,000 spores. These results demonstrate for the first time the utility of the cacao transformation system as a tool for gene functional analysis and the potential utility of the cacao chitinase gene for increasing fungal pathogen resistance in cacao.