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.     

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.     

Complex origin of Trinitario-type Theobroma cacao (Malvaceae) from Trinidad and Tobago revealed using plastid genomics

Trinidad and Tobago has a long history of producing high-quality cacao (Theobroma cacao L.). Cacao genotypes in Trinidad and Tobago are of a highly distinctive kind, the so-called “Trinitario” cultivar group, widely considered to be of elite quality. The origin of Trinitario cacao is unclear, although it is generally considered to be of hybrid origin. We used massive parallel sequencing to identify polymorphic plastidic single nucleotide polymorphisms (cpSNPs) and polymorphic plastidic simple sequence repeats (cpSSRs) in order to determine the origin of the Trinitario cultivar group by comparing patterns of polymorphism to a reference set of ten completely sequenced chloroplast genomes (nine T. cacao and one outgroup, T. grandiflorum (Willd. ex Spreng.) Schum). Only three cpSNP haplotypes were present in the Trinitario cultivars sampled, each highly distinctive and corresponding to reference genotypes for the Criollo (CRI), Upper Amazon Forastero (UAF) and Lower Amazon Forastero (LAF) varietal groups. These three cpSNP haplotypes likely represent the founding lineages of cacao to Trinidad and Tobago. The cpSSRs were more variable with eight haplotypes, but these clustered into three groups corresponding to the three cpSNP haplotypes. The most common haplotype found in farms of Trinidad and Tobago was LAF, followed by UAF and then CRI. We conclude that the Trinitario cultivar group is of complex hybrid origin and has derived from at least three original introduction events.     

Mapping quantitative trait loci for bean traits and ovule number in Theobroma cacao L.

Quantitative trait loci (QTL) mapping for bean traits and the number of ovules per ovary was carried out in cocoa (Theobroma cacao L.) using three test-cross progenies derived from crosses between a lower Amazon Forastero male parent (Catongo) and three female parents: one upper Amazon Forastero (IMC78) and two Trinitario (DR1 and S52). RFLP (restriction fragment length polymorphism), microsatellite, and AFLP (amplified fragment lengthpolymorphism) markers were used for mapping. Between one and six QTL for bean traits (length, weight, and shape index) and one and four QTL for the number of ovules per ovary were detected using composite interval mapping (CIM). Individual QTL explained between 5 and 24% of the phenotypic variation. QTL clusters were identified on several chromosomes, but particularly on chromosome 4. QTL related to bean traits were detected in the same region in both Trinitario parents and in a close region in the upper Amazon Forastero parent. In reference to a previous diversity study where alleles specific to Criollo and Forastero genotypes were identified, it was possible to speculate on the putative origin (Criollo or Forastero) of favorable QTL alleles segregating in both Trinitario studied.     

A new cacao linkage map based on codominant markers: development and integration of 201 new microsatellite markers

A linkage map of cacao based on codominant markers has been constructed by integrating 201 new simple sequence repeats (SSR) developed in this study with a number of isoenzymes, restriction fragment length polymorphisms (RFLP), microsatellite markers and resistance and defence gene analogs (Rgenes-RFLP) previously mapped in cacao. A genomic library enriched for (GA)_n and (CA)_n was constructed, and 201 new microsatellite loci were mapped on 135 individuals from the same mapping population used to establish the first reference maps. This progeny resulted from a cross between two heterozygous cacao clones: an Upper-Amazon Forastero (UPA 402) and a Trinitario (UF 676). The new map contains 465 markers (268 SSRs, 176 RFLPs, five isoenzymes and 16 Rgenes-RFLP) arranged in ten linkage groups corresponding to the haploid chromosome number of cacao. Its length is 782.8 cM, with an average interval distance between markers of 1.7 cM. The new microsatellite markers were distributed throughout all linkage groups of the map, but their distribution was not random. The length of the map established with only SSRs was 769.6 cM, representing 94.8% of the total map. The current level of genome coverage is approximately one microsatellite every 3 cM. This new reference map provides a set of useful markers that is transferable across different mapping populations and will allow the identification and comparison of the most important regions involved in the variation of the traits of interest and the development of marker-assisted selection strategies.Communicated by H. Nybom     

Dissecting Genetic Structure in Farmer Selections of Theobroma Cacao in the Peruvian Amazon: Implications for on Farm Conservation and Rehabilitation

Knowledge of genetic diversity in farmers?? selections is essential for planning on-farm conservation and rehabilitation. Using 15 microsatellite loci, we analyzed parentage and population structure in 220 farmer selections of cacao from the Huallaga valley in Peruvian Amazon. A high level of allele richness and heterozygosity were detected in these selections. Coordination analysis showed that these farmer selections are mainly comprised of hybrids derived from Trinitario and Upper Amazon Forastero germplasm. Bayesian clustering analysis assigned 54 selections as Trinitario and 166 as Upper Amazon Forastero hybrids. Parentage analysis identified 15 international clones as probable parents for 96 farmer selections, which corresponded to a fraction of the known hybrid families disseminated in this region in the late 1980s. Combined analysis of demographic and molecular data revealed a significant spatial autocorrelation (r?=?0.235; P?=?0.006) at short geographical distances (<5.0 km). This patch-like distribution of spatial heterogeneity suggests a significant ??neighborhood effects?? in seeds distribution or variety adoption, where closely related hybrid progenies were adopted in the neighboring farms or villages. The outcomes of this study indicate that in spite of the introgressions of exotic germplasm in the past, Upper Amazon Forastero is still the dominant component in the Huallaga valley of Peru. The high level of on-farm diversity can offer needed variability for participatory selection of superior clones in this low input, small-scale production system, where adaptability to specific environment is more preferred than in a high input, large scale production system.     

A genomewide admixture mapping study for yield factors and morphological traits in a cultivated cocoa (Theobroma cacao L.) population

The selection of productive varieties of modern Criollo cocoa, showing fine aromatic qualities in their beans, is of major interest for some producing countries, such as Venezuela. Cultivated populations of Modern Criollo or Trinitario varieties may be suitable for admixture mapping analysis, as large blocks of alleles derived from two identified divergent ancestors, recently admixed, are still preserved, after a few generations of recombination, similar to experimental mapping progenies. Two hundred and fifty-seven individuals from a cultivated population of Modern Criollo were selected and analysed with 92 microsatellite markers distributed along the genome. This population exhibited a wide range of variability for yield factors and morphological features. Population structure analysis identified two main subgroups corresponding to the admixture from the two ancestors Criollo and Forastero. Several significant associations between markers and phenotypic data (yield factors and morphological traits) were identified by a least squares general linear model (GLM) taking into account the population structure and the percentage of admixture of each individual. Results were compared with classical QTL analyses previously reported for other cacao populations. Most markers associated to quantitative traits were very close to QTLs detected formerly for the same traits. Associations were also identified between markers and several qualitative traits including the red pigmentation observed in different organs, mainly associated to common markers in linkage group 4.     

Mapping QTL for yield components, vigor, and resistance to Phytophthora palmivora in Theobroma cacao L.

Quantitative trait loci (QTL) mapping for agronomic traits was carried out in cocoa (Theobroma cacao L.). Regions of the genome involved in yield, vigor, and resistance to Phytophthora palmivora were identified. Three heterozygous clones, one upper Amazon Forastero (IMC78) and two Trinitario (DR1 and S52), were crossed with the same male parent, a lower Amazon Forastero (Catongo), known to be highly homozygous. Observations were made on progeny over nine consecutive years. One to three QTL related to yield were detected in each of the three populations, located on chromosomes 1, 2, 4, 5, 9, and 10. They explained between 8.1 and 19.3% of the phenotypic variation and showed various levels of repeatability. In IMC78, the QTL detected on chromosome 5 was the most repeatable over years. The QTL for the average individual pod weight on chromosome 4 was the most significant with an LOD of 17.3 and an R2 of 43.7. QTL related to these traits were identified in the same region of the genome in clones of different genetic groups. This suggests that molecular markers can be used to improve cocoa varieties.     

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.     

Molecular characterization of an international cacao collection using microsatellite markers

Plant germplasm collections invariably contain varying levels of genetic redundancy, which hinders the efficient conservation and utilization of plant germplasm. Reduction of genetic redundancies is an essential step to improve the accuracy and efficiency of genebank management. The present study targeted the assessment of genetic redundancy and genetic structure in an international cacao (Theobroma cacao L.) collection maintained in Costa Rica. A total of 688 cacao accessions maintained in this collection were genotyped with 15 simple sequence repeat (SSR) loci, using a capillary electrophoresis genotyping system. The SSR markers provided a high resolution among the accessions. Thirty-six synonymously labeled sets, involving 135 accessions were identified based on the matching of multilocus SSR profiles. After the elimination of synonymous sets, the level of redundancy caused by closely related accessions in the collection was assessed using a simulated sampling scheme that compared allelic diversity in different sample sizes. The result of the simulation suggested that a random sample of 113 accessions could capture 90% of the total allelic diversity in this collection. Principal Coordinate Analysis revealed that the Trinitario hybrids from Costa Rica shared a high similarity among groups as well as among individual accessions. The analysis of the genetic structure illustrated that the within-country/within-region difference accounted for 84.6% of the total molecular variation whereas the among-country/among-region difference accounted for 15.4%. The Brazilian germplasm contributed most to this collection in terms of total alleles and private alleles. The intercountry/interregion relationship by cluster analysis largely agreed with the geographical origin of each germplasm group and supported the hypothesis that the Upper Amazon region is the center of diversity for cacao. The results of the present study indicated that the CATIE International Cacao Collection contains a high level of genetic redundancy. It should be possible to rationalize this collection by reducing redundancy and ensuring optimal representation of the genetic diversity from distinct germplasm groups. The results also demonstrated that SSR markers, together with the statistical tools for individual identification and redundancy assessment, are technically practical and sufficiently informative to assist the management of a tropical plant germplasm collection.     

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.     

A high-density linkage map of Theobroma cacao L.

The first linkage map established by Lanaud et al. (1995) was used as a starting point to produce a high-density molecular linkage map. A mapping population of 181 progenies resulting from a cross between two heterozygous genotypes, a Forastero and a Trinitario (hybrid between Forastero and Criollo), was used for the linkage analysis. A new DNA isolation protocol was established, which allows enough good quality DNA to construct a genetic map with PCR-based markers. The map comprises 424 markers with an average spacing between markers of 2.1 cM. The marker types used were five isozymes, six loci from known function genes, 65 genomic RFLPs, 104 cDNA RFLPs, three telomeric probes, 30 RAPDs, 191 AFLPs and 20 microsatellites. The use of new marker types, AFLP and microsatellites, did not disturb the original order of the RFLP loci used on the previous map. The genetic markers were distributed over ten linkage groups and cover 885.4 cM. The maximum distance observed between adjacent markers was 16.2 cM, and 9.4% of all loci showed skewed segregation. Received: 2 January 2000 / Accepted: 12 February 2000     

Adding value to cocoa (Theobroma cacao L.) germplasm information with domestication history and admixture mapping

A sound understanding of crop history can provide the basis for deriving novel genetic information through admixture mapping. We confirmed this, by using characterization data from an international collection of cocoa, collected 25 years ago, and from a contemporary plantation. We focus on the trees derived from three centuries of admixture between Meso-American Criollo and South American Forastero genomes. In both cacao sets of individuals, linkage disequilibrium extended over long genetic distances along chromosome regions, as expected in populations derived from recent admixture. Based on loose genome scans, genomic regions involved in useful traits were identified. Fifteen genomic regions involved in seed and fruit weight variation were highlighted. They correspond to ten previously identified QTLs and five novel ones. Admixture mapping can help to add value to genetic resources and thus, help to encourage investment in their conservation. Maria Marcano and Tatiana Pugh contributed equally to this work.     

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

Quantitative data are provided for the occurrence of Calonectria rigidiuscula and other fungi in occluded Mirid lesions and other sites in shoots of cacao plants. Mirid lesions appear to be particularly favourable for the development of C. rigidiuscula. The fungus also occurs as a saprophyte on cacao pods, and as a wound parasite in woody plants other than cacao; it was successfully introduced into plants of varied affinities by inoculation. C. rigidiuscula spreads from inoculations in cacao stems much more rapidly than other fungi. The results confirm that it is the most important fungus infecting Mirid lesions and causing dieback of cacao in West Africa.
Inoculation tests with a range of Amelonado and Trinitario clones suggested that the clones vary in their susceptibility to C. rigidiuscula , but the plant-to-plant variation was too great to conclude that any one is highly resistant. Various types of introduced cacao were also tested; preliminary experiments indicate that certain types of Upper Amazon cacao may be resistant, but they need further investigation.     

Isolation of ESTs from cacao (<Emphasis Type="Italic">Theobroma cacao</Emphasis> L.) leaves treated with inducers of the defense response

Pathogenic diseases represent a major constraint to the growth and yield of cacao (Theobroma cacao L.). Ongoing research on model plant systems has revealed that defense responses are activated via signaling pathways mediated by endogenous signaling molecules such as salicylic acid, jasmonic acid and ethylene. Activation of plant defenses is associated with changes in the expression of large numbers of genes. To gain a better understanding of defense responses in cacao, we have employed suppressive subtractive hybridization (SSH) cDNA libraries, macroarray hybridization analysis, high throughput DNA sequencing and bioinformatics to identify cacao genes induced by these signaling molecules. Additionally, we investigated gene activation by a phytotoxic elicitor-like protein, Nep1. We have identified a unigene set of 1,256 members, including 330 members representing genes induced during the defense response.Electronic Supplementary Material Electronic supplementary material is available in the online version of this article at Sequences presented here are deposited with GenBank under accession numbers CF972636–CF974749     

Selection of international molecular standards for DNA fingerprinting of <Emphasis Type="Italic">Theobroma cacao</Emphasis>

A collaborative international program was initiated to identify and describe the genetic diversity of living germplasm collections of Theobroma cacao genotypes that are maintained in several international collections scattered throughout tropical cacao growing countries of the world. Simple sequence repeat (SSR) DNA analysis was identified as the most appropriate molecular tool for DNA fingerprinting these collections during an international forum representing academic, government and industry scientists in the cacao community. Twenty-five SSR primers, which had been previously described, were evaluated as potential candidates to define an efficient, standardized, molecular fingerprinting protocol for T. cacao accessions. These primers have been evaluated for reliability, widespread distribution across the cacao genome, number of alleles produced by the SSR primers in cacao and their ability to discriminate between cacao accessions. Approximately 690 cacao accessions were used to evaluate the utility of these SSR primers as international molecular standards, and a small number of test samples of T. cacao were sent to two other independent laboratories for verification. DNA fragments were selectively amplified by PCR, using the SSR primers labeled with fluorescent dyes, and separated by capillary electrophoresis. Based on this study, the 15 SSR primers that had the highest reproducibility and consistency within a common genotype, while allowing the differentiation of separate divergent genotypes, were selected as international molecular standards for DNA fingerprinting of T. cacao.     

Evaluation of the extent of genetic variability among Theobroma cacao accessions using RAPD and RFLP markers

 Random amplified polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP) markers were used to evaluate genetic relationships within the Theobroma cacao species and to assess the organization of its genetic diversity. Genetic variability was estimated with 18 primers and 43 RFLP probes on 155 cocoa trees belonging to different morphological groups and coming from various geographic origins. The majority of the RFLP probes issued from low-copy DNA sequences. On the basis of on the genetic distance matrices, the two molecular methods gave related estimates of the genetic relationship between genotypes. Although an influence of cocoa morphological groups and geographical origins of trees was observed, a lack of gene differentiation characterized the T. cacao accessions studied. The continuous RFLP variability observed within the species may reflect the hybridization and introgressions between trees of different origins. Nevertheless, the Nacional type was detected to be genetically specific and different from well-known types such as Forastero, Criollo and Trinitario. Some of those genotypes were characterized by a low heterozygosity rate and may constitute the original Nacional pool. These results also provide information for the constitution of a cocoa tree core collection. Received: 10 June 1996/Accepted: 11 October 1996     

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.     

Histology of somatic embryogenesis from floral tissues cocoa

Somatic embryogenesis fromTheobroma cacao L. flower buds, as previously reported on five Forastero hybrid genotypes, was tested on several other genotypes, belonging to the three cocoa-tree groups: Forastero, Trinitario and Criollo. The results gave evidence of genotypic efficiencies. Explants were cultivated under two successive conditions: callogenesis and expression media. The morphological and histological responses were different for embryogenic or non-embryogenic genotypes. For embryogenic genotypes, only staminodes and stamen filaments were able to produce somatic embryos: after a few days on the expression medium, groups of callus cells went through the meristematic and then embryonic stages, and finally formed somatic embryos. Many of them showed abnormalities. Simultaneously, some embryogenic cells were visible. These started to divide to form pro-embryos which however were unable to evolve into proper somatic embryos.