Molecular Characterization of Cacao (<Emphasis Type="Italic">Theobroma cacao</Emphasis>) Germplasm from Jamaica Using Single Nucleotide Polymorphism (SNP) Markers

Cacao is an economically important commodity in Jamaica. Knowledge of the genetic diversity of Jamaican cacao germplasm is essential for their conservation and management. In spite of cacao’s economic importance in Jamaica, the crop is under studied, therefore limiting sound decisions toward improving productivity. Assessment of germplasm and on-farm genetic diversity is required to assist selecting superior genotypes to propagate and distribute across the island, as well as to use them as parental clones in breeding programs. Using 94 single nucleotide polymorphism (SNP) markers, 140 Jamaican cacao samples from two germplasm collections and a farmer’s estate along with 150 reference samples were analyzed. The principal coordinate analysis demonstrated that the majority of the Jamaican cacao selections were hybrids derived from five original germplasm groups, including Criollo, Amelonado and three Upper Amazon Forastero groups. Among the Upper Amazon groups, the Bayesian clustering analysis revealed that the Parinari (PA) ancestral lineage contributed the most (29.9%) to the Jamaican cacao germplasm. The germplasm collections showed greater diversity in terms of ancestral contributions compared to the farmer’s estate. However, the genetic differentiation between the three collecting sites was small (Fst?=?0.036), indicating that samples collected from the three sites were derived from a common pool of germplasm. The current study supports the historical records and clarified the ancestry of Jamaican cacao. Although the majority of the cacao genetic groups were observed in the Jamaican cacao collections, several diversity gaps were found in both germplasm collections and in the farmer’s estate, especially germplasm with disease resistance to cacao frosty pod rot that was recently found in Jamaica.     

The globalization of a ceremonial tree: The case of cacao (Theobroma cacao) among the Mopan Maya

The uses, perceptions, and economic significance of cacao have radically changed in the past 25 years among the Mopan Maya in southern Belize. Cacao was once perceived as a ceremonial crop with little cash value. Over the past 25 years though, cacao has become the most important cash crop grown by the Mopan Maya. The Mopan Maya grow organic cacao that has allowed them to tap into a specialized, high-end chocolate market. However, the emergence of cacao as an important cash crop has altered traditional uses and created conflicts in villages where increasing acreage of reservation lands are planted with cacao, thereby assigning a commercial value to previously communal lands.     

Genetic diversity of naturalized cacao (<Emphasis Type="Italic">Theobroma cacao</Emphasis> L.) in Puerto Rico

Identification of genetically diverse cacao with disease resistance, high productivity, and desirable organoleptic traits is vitally important to the agricultural crop’s long-term sustainability. Environmental changes, pests, and diseases as well as nation’s sovereign property rights have led to a decrease in accessibility and exchange of germplasm of interest. Having been introduced during colonial times, naturalized cacao in Puerto Rico could serve as an unexplored source of genetic diversity in improvement programs. An island-wide survey was carried out to identify naturalized trees and to determine their genetic associations to reference cacao accessions. Samples were genotyped with Expressed Sequence Tag-derived single nucleotide polymorphism (SNP) markers. Principal coordinate, cluster, and population structure analysis using the genotype data for both local and reference samples assigned individuals into five distinct genetic backgrounds: Criollo, Trinitario, Amelonado, Upper Amazon Forastero (UAF), and Nacional. Puerto Rican cacao fit into four (Criollo, Trinitario, Amelonado and UAF) of the five genetic backgrounds, being mainly composed of individuals of Criollo ancestry. Based on historical evidence, cacao of Criollo background was probably brought to Puerto Rico from Venezuela and/or Central America during colonial times. Trinitario, Amelonado, and UAF genetic backgrounds are most likely products of more modern introductions. Genotyping cacao in Puerto Rico provides information on the history and possible origin of the naturalized trees on the island. In addition, the assessment has allowed the targeting of material for incorporation and long-term conservation filling gaps in the existing collection and providing new germplasm to be evaluated for agronomic performance.     

Genetic identity and diversity of Nigerian cacao genebank collections verified by single nucleotide polymorphisms (SNPs): a guide to field genebank management and utilization

Nigeria is the sixth largest cacao producer in the world. Field performance and quality of cacao hybrid families is largely dependent on the genetic integrity of parental clones obtained in field genebank collections. However, information on the impact of mislabeling on seed garden output in Nigeria is lacking. Using 63 single nucleotide polymorphism (SNP) markers, we analyzed 1457 cacao trees sampled from seven major field genebank plots in Nigeria to assess the genetic integrity in Nigerian cacao germplasm. The procedure of multilocus matching with known reference clones revealed up to 78% mislabeling in recently introduced international germplasm. A high rate of mislabeling was also revealed in the West African local selections and breeding lines, using Bayesian assignment test. The problem of mislabeling has been attributed to errors from the sources of introduction, pre-planting labeling errors, and rootstocks overtaking budded scions due to poor field management. The analysis of genetic diversity revealed a good representation of the available cacao germplasm groups in Nigerian field genebanks, indicating that the genetic base of Nigeria cacao germplasm has been significantly widened through germplasm introductions. However, only a small proportion of the available germplasm in the genebank have been utilized for variety development. This study proved the utility of SNP markers for cleaning up the genebanks and reducing offtypes; thereby providing a strong basis for improving the accuracy and efficiency in cacao genebank management and breeding, as well as for mobilizing improved varieties to cacao farmers in Nigeria.     

Cacao domestication I: the origin of the cacao cultivated by the Mayas

Criollo cacao (Theobroma cacao ssp. cacao) was cultivated by the Mayas over 1500 years ago. It has been suggested that Criollo cacao originated in Central America and that it evolved independently from the cacao populations in the Amazon basin. Cacao populations from the Amazon basin are included in the second morphogeographic group: Forastero, and assigned to T. cacao ssp. sphaerocarpum. To gain further insight into the origin and genetic basis of Criollo cacao from Central America, RFLP and microsatellite analyses were performed on a sample that avoided mixing pure Criollo individuals with individuals classified as Criollo but which might have been introgressed with Forastero genes. We distinguished these two types of individuals as Ancient and Modern Criollo. In contrast to previous studies, Ancient Criollo individuals formerly classified as 'wild', were found to form a closely related group together with Ancient Criollo individuals from South America. The Ancient Criollo trees were also closer to Colombian-Ecuadorian Forastero individuals than these Colombian-Ecuadorian trees were to other South American Forastero individuals. RFLP and microsatellite analyses revealed a high level of homozygosity and significantly low genetic diversity within the Ancient Criollo group. The results suggest that the Ancient Criollo individuals represent the original Criollo group. The results also implies that this group does not represent a separate subspecies and that it probably originated from a few individuals in South America that may have been spread by man within Central America.     

Microsatellite variation and population structure in the “Refractario” cacao of Ecuador

Utilization of germplasm for crop improvement is often hampered by absence of information regarding origin, genetic identity and genealogical relationships of germplasm groups or populations. Molecular marker technology offers an efficient tool to verify or reconstruct passport data. Using a high-throughput genotyping system with 15 microsatellite loci, we fingerprinted 482 accessions in 48 putative half-sib families of Refractario cacao (a group of germplasm collected from nine farms in Ecuador). Based on the multilocus profiles, a Bayesian method for individual assignment was applied to verify membership in each half-sib family. Multivariate statistical analysis showed that the Refractario genetic profile was different from other groups tested, except for the “Nacional” cacao from the coastal valley of Ecuador. Hierarchical partitioning of genetic variance in the Refractario cacao showed that 76% of the variation was contributed by intra-family difference, whereas the inter-family and inter-farm difference accounted for 15 and 9% of total variance, respectively. All three sources of variation were highly significant (P < 0.01). Cluster and Principal Coordinates Analyses revealed a population sub-structure in Refractario, which was also highly heterozygous, suggesting hybridization derived from Nacional cacao and multiple other parental varieties, which all shared a similar genetic background. The improved understanding of identities and structure in Refractario cacao will contribute to more efficient conservation and use of this germplasm group in cacao breeding.     

Genetic diversity and relationships of cacao (Theobroma cacao L.) in southern Mexico

 Neotropical tree crops are affected by a combination of biological and human factors that complicate the study of genetic diversity and crop evolution. Genetic diversity and relationships among southern Mexican populations and horticultural collections of Theobroma cacao (chocolate, cocoa, cacao) are examined in light of the agricultural practices of the Maya. Collections of cacao were obtained from the extremes of its geographic range including archeological sites in southern Mexico where cacao was first domesticated. Genetic diversity was assayed by 57 informative random amplified polymorphic DNA (RAPD) marker loci. A unique sample of the total diversity found in this study exists in the southern Mexican populations. These populations are significantly different from all other cacao with regards to their profile of RAPD bands, including the ‘criollo’ variety, their morphological and geographical group. A population of cacao found in a sinkhole (cenote) in northern Yucatan with genetic affinities to populations in Chiapas suggests the Maya maintained plants far away from their native habitat. This finding concurs with known agroforestry practices of the Maya. Modern efforts to increase germplasm of tropical tree crops such as cacao should carefully examine archeological sites where genetic diversity, either deliberately or by chance, was collected and maintained by ancient cultures. Received: 21 May 1997 / Accepted: 9 October 1997     

Geographic and genetic population differentiation of the Amazonian chocolate tree (Theobroma cacao L)

Numerous collecting expeditions of Theobroma cacao L. germplasm have been undertaken in Latin-America. However, most of this germplasm has not contributed to cacao improvement because its relationship to cultivated selections was poorly understood. Germplasm labeling errors have impeded breeding and confounded the interpretation of diversity analyses. To improve the understanding of the origin, classification, and population differentiation within the species, 1241 accessions covering a large geographic sampling were genotyped with 106 microsatellite markers. After discarding mislabeled samples, 10 genetic clusters, as opposed to the two genetic groups traditionally recognized within T. cacao, were found by applying Bayesian statistics. This leads us to propose a new classification of the cacao germplasm that will enhance its management. The results also provide new insights into the diversification of Amazon species in general, with the pattern of differentiation of the populations studied supporting the palaeoarches hypothesis of species diversification. The origin of the traditional cacao cultivars is also enlightened in this study.     

Tracing the native ancestors of the modern Theobroma cacao L. population in Ecuador

The native Theobroma cacao L. population from Ecuador, known as Nacional, is famous for its fine cocoa flavour. From the beginning of the twentieth century, however, it has been subjected to genetic erosion due principally to successive introductions of foreign germplasm whose hybrid descendants gradually replaced the native plantations, implying a decrease in cocoa quality. We attempted to trace this native cacao within a wide pool of modern Ecuadorian cacao population. Three hundred and twenty-two cacao accessions collected from different geographical areas along the pacific coast of Ecuador and maintained in two living collections were analysed using 40 simple-sequence repeat markers. Most of Ecuadorian cacao accessions displayed a high diversity and heterozygosity level. A factorial analysis of correspondence (FAC) showed a continuous variation among them, with a few ones, grouped at an extreme side of the FAC cloud, showing higher levels of homozygosity and lower introgression level by foreign cacaos. A paternity analysis revealed that these highly homozygous individuals are the most probable ancestors of the modern Nacional hybrid pool. These particular accessions studied could represent the native Nacional cacao present in Ecuador before the foreign introductions. Their identification will help to conserve valuable genetic material and to improve cocoa quality in new cacao varieties.     

Genetic Structure and Molecular Diversity of Cacao Plants Established as Local Varieties for More than Two Centuries: The Genetic History of Cacao Plantations in Bahia,Brazil

Bahia is the most important cacao-producing state in Brazil, which is currently the sixth-largest country worldwide to produce cacao seeds. In the eighteenth century, the Comum, Pará and Maranhão varieties of cacao were introduced into southern Bahia, and their descendants, which are called ‘Bahian cacao’ or local Bahian varieties, have been cultivated for over 200 years. Comum plants have been used to start plantations in African countries and extended as far as countries in South Asia and Oceania. In Brazil, two sets of clones selected from Bahian varieties and their mutants, the Agronomic Institute of East (SIAL) and Bahian Cacao Institute (SIC) series, represent the diversity of Bahian cacao in germplasm banks. Because the genetic diversity of Bahian varieties, which is essential for breeding programs, remains unknown, the objective of this work was to assess the genetic structure and diversity of local Bahian varieties collected from farms and germplasm banks. To this end, 30 simple sequence repeat (SSR) markers were used to genotype 279 cacao plants from germplasm and local farms. The results facilitated the identification of 219 cacao plants of Bahian origin, and 51 of these were SIAL or SIC clones. Bahian cacao showed low genetic diversity. It could be verified that SIC and SIAL clones do not represent the true diversity of Bahian cacao, with the greatest amount of diversity found in cacao trees on the farms. Thus, a core collection to aid in prioritizing the plants to be sampled for Bahian cacao diversity is suggested. These results provide information that can be used to conserve Bahian cacao plants and applied in breeding programs to obtain more productive Bahian cacao with superior quality and tolerance to major diseases in tropical cacao plantations worldwide.     

OBSERVATIONS ON THE PATHOGENICITY OF CALONECTRIA RIGIDIUSCULA (BERK. & BR.) SACC. ON THEOBROMA CACAO L

Calonectria rigidiuscula has been found associated with an acute and a chronic form of dieback of cacao. It can also infect cankers caused primarily by Phytophthora palmivora , and the lesions following attack by the capsids Sahlbergella singidaris and Distantiella theobroma. The association with the capsid lesions is of great economic importance since it appears that capsids alone are capable of killing only green shoots, and that the severe damage caused to woody shoots follows C. rigidiuscula infection of the capsid lesions. Acute dieback occurs only in certain districts after exceptional drought and may be considered primarily the result of environmental conditions. The main importance of chronic dieback is that it prevents the recovery of cacao which has been seriously weakened from other causes. C. rigidiuscula infection retards the healing of P. palmivora cankers and also produces a lesion in the xylem tissue which may be extensive.
C. rigidiuscula has been established in wounded cacao stems of all ages without difficulty. The spread of the fungus is slow, but is more rapid in the xylem than in the cortical tissues. Spread is greatest in the unlignified tissues.     

Conservation Value of Cacao Agroforestry Systems for Terrestrial Herbaceous Species in Central Sulawesi,Indonesia

Tropical secondary forest and agroforestry systems have been identified as important refuges for the local species diversity of birds and other animal groups, but little is known about the importance of these systems for terrestrial herbs. In particular, few studies report how the conversion from tropical forest to technified cacao plantation affects the species richness and the community structure of herbs. We conducted surveys in 43 cacao plantations along the border of the Lore Lindu National Park in Central Sulawesi, ranging from agroforests to technified cacao, categorizing the plantations as rustic cacao, planted shade cacao, and technified cacao. We recorded 91 herb species. Of the 74 species determined to species level, 21 were also found in natural forests, while 53 were recorded only in agricultural habitats. Araceae was the most forest‐dependent plant family while Asteraceae included the highest number of nonforest species. Overall, the presence of forest species was confined to moderately intensively managed rustic and planted shaded plantations. Distance from the forest, which has been identified as a crucial parameter for the diversity and composition of other taxa in cacao agroforests, only played a minimal role for herbs. Our study suggests that native forest herbs maybe more vulnerable to forest conversion than animal groups. The intensification of cacao plantation management increases the presence of weedy species to the detriment of native forest species.     

Dispersal of a Human-Cultivated Crop by Wild Chimpanzees (<Emphasis Type="Italic">Pan troglodytes verus</Emphasis>) in a Forest–Farm Matrix

With the conversion of natural habitats to farmland, nonhuman primates (hereafter primates) are increasingly exposed to agricultural crops. Although frugivorous primates are important seed dispersers that sometimes feed on agricultural fruits, evidence for dispersal of crops by primates is lacking. Here, we examine flexible feeding on cacao (Theobroma cacao) fruit and seed dispersal patterns by chimpanzees (Pan troglodytes verus) at Bossou in Guinea, and consequent cacao germination and survival. From direct observations, we confirm that cacao fruit is not an important food to chimpanzees, representing 0.23 % of focal animal feeding time. Chimpanzees ingest cacao pulp and either spit out the large seeds intact from unripe cacao fruit or swallow the seeds from ripe cacao fruits, which are consequently deposited in feces. From ecological surveys we show that chimpanzees distributed cacao extensively throughout their home range, at a mean distance of 407 m?±?SE 0.6 (N?=?90 clusters, range: 4–1130 m) from cacao plantations. As distance from the cacao plantation increased, cacao plants were more likely to survive. Other factors, including number of cacao plants in a cluster, plant height, and openness of the understory did not predict short-term cacao survival. Cacao plants within the forest did not produce fruit. By contrast, when chimpanzees deposited seeds in a plantation, cacao plants produced fruits as a result of farmers’ maintenance of the area. Our local-scale findings emphasize the complex behavioral and ecological interconnections between coexisting humans and primates in agricultural landscapes and generate interesting questions regarding primate niche construction and crop “ownership” related to who “plants” the crop.     

Cacao domestication II: progenitor germplasm of the Trinitario cacao cultivar

Cacao (Theobroma cacao L.) has been cultivated in Central America since pre-Columbian times. The type of cacao cultivated in this region was called Criollo; cacao populations from the Amazon basin were called Forastero. The type of Forastero most commonly cultivated until 1950 was named Amelonado. Historical data show Trinitario cacao to have originated in Trinidad, resulting from natural hybridisation between Criollo and Amelonado Forastero. Doubts persist on the source of the Amelonado Forastero involved in the origin of Trinitario; the Amelonado parent may have come from the Lower Amazon, the Orinoco or the Guyanas. Most of the cacao cultivated worldwide until 1950 consisted of Criollo, Trinitario and Amelonado. From the early 1950s, Forastero material collected in the Upper Amazon region during the 1930s and 1940s began to be employed in breeding programmes. To gain a better understanding of the origin and the genetic basis of the cacao cultivars exploited before the utilisation of germplasm collected in the Upper Amazon, a study was carried out using restriction fragment length polymorphism and microsatellite markers. Trinitario samples from 17 countries were analysed. With molecular markers, it was possible to clearly identify three main genotypes (represented by clones SP1, MAT1-6 and SIAL70) implicated in the origin of most Trinitario clones.     

Biodiversity in tropical agroforests and the ecological role of ants and ant diversity in predatory function

Abstract.  1. Intensive agricultural practices drive biodiversity loss with potentially drastic consequences for ecosystem services. To advance conservation and production goals, agricultural practices should be compatible with biodiversity. Traditional or less intensive systems (i.e. with fewer agrochemicals, less mechanisation, more crop species) such as shaded coffee and cacao agroforests are highlighted for their ability to provide a refuge for biodiversity and may also enhance certain ecosystem functions (i.e. predation).
2. Ants are an important predator group in tropical agroforestry systems. Generally, ant biodiversity declines with coffee and cacao intensification yet the literature lacks a summary of the known mechanisms for ant declines and how this diversity loss may affect the role of ants as predators.
3. Here, how shaded coffee and cacao agroforestry systems protect biodiversity and may preserve related ecosystem functions is discussed in the context of ants as predators. Specifically, the relationships between biodiversity and predation, links between agriculture and conservation, patterns and mechanisms for ant diversity loss with agricultural intensification, importance of ants as control agents of pests and fungal diseases, and whether ant diversity may influence the functional role of ants as predators are addressed. Furthermore, because of the importance of homopteran-tending by ants in the ecological and agricultural literature, as well as to the success of ants as predators, the costs and benefits of promoting ants in agroforests are discussed.
4. Especially where the diversity of ants and other predators is high, as in traditional agroforestry systems, both agroecosystem function and conservation goals will be advanced by biodiversity protection.     

Microsatellite-aided detection of genetic redundancy improves management of the International Cocoa Genebank, Trinidad

Cacao (Theobroma cacao L.), the tree from which cocoa butter and chocolate is derived, is conserved in field genebanks. The largest of these ex situ collections in the public domain is the International Cocoa Genebank, Trinidad (ICG,T). Reduction of genetic redundancy is essential to improve the accuracy and efficiency of genebank management. This study examined the pedigree and genetic diversity in a subset of 387 accessions in this collection. Sibship reconstruction of this subset revealed 56 full-sib families nested within 189 half-sib families. Sixteen centers of interconnectivity were identified, which suggested a high level of genetic redundancy in the collection. Generally, consistent phylogenetic trees were obtained using different genetic distance measures. However, a principal coordinate analysis of the D _est differentiation measure elicited the best representation of accession group clustering, and we recommend this approach when probing fine-scale genetic differentiation among cacao accessions. The composite genetic diversity of 414 cacao accessions was contained in a core set of 59 unique accessions. These results have significant implications in the conservation of genetic resources of the ICG,T and other cacao genebanks. The approach developed in this study is recommended as a strategy to curators in guiding conservation management practices of cacao and other similar ex situ genebanks.     

Gene discovery and microarray analysis of cacao (Theobroma cacao L.) varieties

The cacao bean harvest from the relatively under developed tropical tree cacao (Theobroma cacao L.) is subject to high losses in potential production due to pests and diseases. To discover and understand the stability of putative natural resistance mechanisms in this commodity crop, essential for chocolate production, we undertook a gene-discovery program and demonstrated its use in gene-expression arrays. Sequencing and assembling bean and leaf cDNA library inserts produced a unique contig set of 1,380 members. High-quality annotation of this gene set using Blast and MetaFam produced annotation for 75% of the contigs and allowed us to identify the types of gene expressed in cacao beans and leaves. Microarrays were constructed using amplified inserts of the uni-gene set and challenged with bean and leaf RNA from five cacao varieties. The microarray performed well across the five randomly chosen cacao genotypes and did not show a bias towards either leaf or bean tissues. This demonstrates that the gene sequences are useful for microarray analysis across cacao genotypes and tissue types. The array results, when compared with real-time PCR results for selected genes, showed a correlation with differential gene-expression patterns.We intend that the resultant DNA sequences and molecular microarray platform will help the cacao community to understand the basis, likely stability and pathotype resistance range of candidate cacao plants.     

Incorporating livelihoods in biodiversity conservation: a case study of cacao agroforestry systems in Talamanca,Costa Rica

Over the past two decades, various organizations have promoted cacao agroforestry systems as a tool for biodiversity conservation in the Bribri-Cabécar indigenous territories of Talamanca, Costa Rica. Despite these efforts, cacao production is declining and is being replaced by less diverse systems that have lower biodiversity value. Understanding the factors that influence household land use is essential in order to promote cacao agroforestry systems as a viable livelihood strategy. We incorporate elements of livelihoods analyses and socioeconomic data to examine cacao agroforestry systems as a livelihood strategy compared with other crops in Talamanca. Several factors help to explain the abandonment of cacao agroforestry systems and their conversion to other land uses. These factors include shocks and trends beyond the control of households such as crop disease and population growth and concentration, as well as structures and processes such as the shift from a subsistence to a cash-based economy, relative prices of cacao and other cash crops, and the availability of market and government support for agriculture. We argue that a livelihoods approach provides a useful framework to examine the decline of cacao agroforestry systems and generates insights on how to stem the rate of their conversion to less diverse land uses.