Insight into the Wild Origin,Migration and Domestication History of the Fine Flavour Nacional Theobroma cacao L. Variety from Ecuador

Ecuador’s economic history has been closely linked to Theobroma cacao L cultivation, and specifically to the native fine flavour Nacional cocoa variety. The original Nacional cocoa trees are presently in danger of extinction due to foreign germplasm introductions. In a previous work, a few non-introgressed Nacional types were identified as potential founders of the modern Ecuadorian cocoa population, but so far their origin could not be formally identified. In order to determine the putative centre of origin of Nacional and trace its domestication history, we used 80 simple sequence repeat (SSR) markers to analyse the relationships between these potential Nacional founders and 169 wild and cultivated cocoa accessions from South and Central America. The highest genetic similarity was observed between the Nacional pool and some wild genotypes from the southern Amazonian region of Ecuador, sampled along the Yacuambi, Nangaritza and Zamora rivers in Zamora Chinchipe province. This result was confirmed by a parentage analysis. Based on our results and on data about pre-Columbian civilization and Spanish colonization history of Ecuador, we determined, for the first time, the possible centre of origin and migration events of the Nacional variety from the Amazonian area until its arrival in the coastal provinces. As large unexplored forest areas still exist in the southern part of the Ecuadorian Amazonian region, our findings could provide clues as to where precious new genetic resources could be collected, and subsequently used to improve the flavour and disease resistance of modern Ecuadorian cocoa varieties.     

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 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.     

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     

Development and characterization of novel tetra-, tri- and di-nucleotide microsatellite markers in cacao (<Emphasis Type="Italic">Theobroma cacao</Emphasis> L.)

The cacao plant, Theobroma cacao L., produces white seeds (beans) that form the major ingredient of processed chocolate. A great deal of research effort has been expended to the development of new genetically modified cacao plants with improved productivity and resistance and beans of good industrial quality. The availability of suitable genetic markers is an important aspect of the efficient selection and breeding of this perennial species. We describe the development of 123 microsatellite loci of cacao. An optimized protocol was used to construct and screen a microsatellite-enriched genomic library from which we isolated 64 di-nucleotide, 45 tri-nucleotide and 14 tetra-nucleotide microsatellite loci. The primers were tested on samples from five different T. cacao accessions, one accession from T. grandiflorum and one accession from Herranea sp. Among the 123 loci, 54 were polymorphic, 61 were monomorphic and eight did not present an amplification product. These new markers will be useful in future studies by increasing the accuracy of genotypic assessments in diverse cocoa tree populations as well as in other species of the Theobroma genus.     

Genetic diversity and structure of managed and semi-natural populations of cocoa (Theobroma cacao) in the Huallaga and Ucayali Valleys of Peru

BACKGROUND AND AIMS: Cocoa (Theobroma cacao) is indigenous to the Amazon region of South America, and it is well known that the Peruvian Amazon harbours a large number of diverse cocoa populations. A small fraction of the diversity has been collected and maintained as an ex-situ germplasm repository in Peru. However, incorrect labelling of accessions and lack of information on genetic diversity have hindered efficient conservation and use of this germplasm. This study targeted assessment of genetic diversity and population structure in a managed and a semi-natural population. METHODS: Using a capillary electrophoresis genotyping system, 105 cocoa accessions collected from the Huallaga and Ucayali valleys of Peru were fingerprinted. Based on 15 loci SSR profiles, genetic identity was examined for each accession and duplicates identified, population structure assessed and genetic diversity analysed in these two populations. KEY RESULTS: Ten synonymous mislabelled groups were identified among the 105 accessions. The germplasm group in the Huallaga valley was clearly separated from the group in Ucayali valley by the Bayesian assignment test. The Huallaga group has lower genetic diversity, both in terms of allelic richness and of gene diversity, than the Ucayali group. Analysis of molecular variance suggested genetic substructure in the Ucayali group. Significant spatial correlation between genetic distance and geographical distances was detected in the Ucayali group by Mantel tests. CONCLUSIONS: These results substantiate the hypothesis that the Peruvian Amazon hosts a high level of cocoa genetic diversity, and the diversity has a spatial structure. The introduction of exotic seed populations into the Peruvian Amazon is changing the cocoa germplasm spectrum in this region. The spatial structure of cocoa diversity recorded here highlights the need for additional collecting and conservation measures for natural and semi-natural cocoa populations.     

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.     

Chloroplast microsatellite primers for cacao (Theobroma cacao) and other Malvaceae

? Premise of the study: Chloroplast microsatellites were developed in Theobroma cacao to examine the genetic diversity of cacao cultivars in Trinidad and Tobago. ? Methods and Results: Nine polymorphic microsatellites were designed from the chloroplast genomes of two T. cacao accessions. These microsatellites were tested in 95 hybrid accessions from Trinidad and Tobago. An average of 2.9 alleles per locus was found. ? Conclusions: These chloroplast microsatellites, particularly the highly polymorphic pentameric repeat, were useful in assessing genetic variation in T. cacao. In addition, these markers should also prove to be useful for population genetic studies in other species of Malvaceae.     

Neglected native or undesirable alien? Resolution of a conservation dilemma concerning the pool frog Rana lessonae

Introduced species often pose serious threats to biodiversity, but occasionally confusion arises as to whether a species really is introduced or is in fact an overlooked native. A recent UK conservation dilemma has centred on the status of the pool frog Rana lessonae. This species has been the subject of documented introductions from central and southern Europe since the early 1800s, the accepted position being that all UK R. lessonae populations are descended from these introductions. However, a closer examination of early UK literature sources, and recent discoveries of isolated, native R. lessonae populations in Sweden and Norway, led some herpetologists to question whether the species was in fact present as a native at some locations prior to the introductions. Research was initiated along four major lines of enquiry: genetic, bioacoustic, archaeozoological and archival. A high degree of convergence among the genetic and bioacoustic investigations demonstrated that the potentially native UK pool frogs were closely related to Scandinavian frogs, thus ruling out introductions from further south as a potential origin. Subfossil evidence of pool frogs was found from ca. 1000 years before present, demonstrating that the species occurred in the UK prior to known introductions. Archival sources produced no historical support for introductions from northern Europe. The postglacial history inferred for these northern populations is consistent with the known climatic and geographical conditions. Taken together, the evidence for the native status of the pool frog is compelling, and furthermore the UK population appears to be part of a distinct northern clade.     

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     

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.     

When invasion increases population genetic structure: a study with <Emphasis Type="Italic">Centaurea diffusa</Emphasis>

Biological invasions offer excellent systems to study the evolutionary processes involved in introductions of species to new ranges. Molecular markers can reveal invasion histories and the effects of introductions on amounts and structuring of genetic variation. We used five polymorphic microsatellite loci to elucidate genetic diversity and population structure between native range and introduced range populations of a prominent North American rangeland weed, Centaurea diffusa (Asteraceae). We found that the total number of alleles and the number of private alleles was slightly higher in the native Eurasian range, and that allelic richness did not differ between the ranges, indicating overall levels of diversity were similar in Eurasia and North America. It therefore seems unlikely that this invasion has been affected by genetic bottlenecks or founder effects. Indeed, results of assignment tests suggest that multiple introductions have contributed to North America’s C. diffusa invasion. Additionally, assignment tests show that both Eurasian and North American sites had a strong pattern of mixed genetic ancestry. This mixed assignment corresponded to a lack of geographic population structure among Eurasian samples. The lack of population structure in the native range conflicts with general expectations and findings to date for invasion genetics, and cautions that even species’ native ranges may show signs of recent ecological upheaval. Despite the mixed assignments, North American samples showed strong population structure, suggesting that the invasion has been characterized by long-range dispersal of genetically distinct propagules across the introduced range.     

Out of the Amazon: Theobroma cacao enters the genomic era

Theobroma cacao has long been a crop of near-mystical human importance among indigenous Mesoamerican cultures and its importance in modern culture is growing with new realizations of the potential health benefits of cocoa polyphenolic compounds. However, cultivated T. cacao is vulnerable to emerging disease pressures because it has a narrow genetic base, and systematic genetic improvement of the crop is imperative. A wide range of genomic tools and resources has been developed and are providing the basis for genome-based breeding and gene discovery.     

Fermentation of cacao (Theobroma cacao L.) seeds with a hybrid Kluyveromyces marxianus strain improved product quality attributes

Fermentation of Theobroma cacao (cacao) seeds is an absolute requirement for the full development of chocolate flavor precursors. An adequate aeration of the fermenting cacao seed mass is a fundamental prerequisite for a satisfactory fermentation. Here, we evaluated whether a controlled inoculation of cacao seed fermentation using a Kluyveromyces marxianus hybrid yeast strain, with an increased pectinolytic activity, would improve an earlier liquid drainage ('sweatings') from the fermentation mass, developing a superior final product quality. Inoculation with K. marxianus increased by one third the volume of drained liquid and affected the microorganism population structure during fermentation, which was detectable up to the end of the process. Introduction of the hybrid yeast affected the profile of total seed protein degradation evaluated by polyacrylamide gel electrophoresis, with improved seed protein degradation, and reduction of titrable acidity. Sensorial evaluation of the chocolate obtained from beans fermented with the K. marxianus inoculation was more accepted by analysts in comparison with the one from cocoa obtained through natural fermentation. The increase in mass aeration during the first 24 h seemed to be fundamental for the improvement of fermentation quality, demonstrating the potential application of this improved hybrid yeast strain with superior exogenous pectinolytic activity.     

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.     

Establishment of a Core Collection of Traditional Cuban <Emphasis Type="Italic">Theobroma cacao</Emphasis> Plants for Conservation and Utilization Purposes

Presently, Theobroma cacao L. (cacao) in Cuba is mainly cultivated in the eastern region where plantations comprise a mixture of clonal varieties, hybrids, progeny of Trinidad Selected Hybrids, and traditional—also known as ancient—cacao. The ancient genetic resources, probably the plants most closely related to the original introductions, are endangered by their progressive replacement by modern clones. To promote the conservation and utilization of these genetic resources, a representative sample of 537 traditional Cuban cacao plants was used to develop a core collection. Core collections based on 15 simple sequence repeat (SSR) markers were generated using five different sampling algorithms: random sampling, simulated annealing, stepwise clustering with random sampling, the M strategy, and maximum genetic diversity. The five core collections were designed to capture 95 % of the SSR alleles in the complete collection. The genetic, morphological, and geographical diversity of each core collection was compared with that of the entire collection. The entire collection contained 139 alleles, including 104 rare ones, with the 95 % allelic coverage threshold achieved with 133 alleles. The core collection generated by the maximum genetic diversity algorithm had the lowest number of accessions (185), the highest mean genetic distance (0.486), the lowest morphological character redundancy, and the highest diversity as assessed by the mean Shannon-Weaver diversity index (0.757). This core collection can thus serve as the basis of future improvement programs based on local genetic resources.     

Patterns of introduction and adaptation during the invasion of Aegilops triuncialis (Poaceae) into Californian serpentine soils

Multiple introductions can play a prominent role in explaining the success of biological invasions. One often cited mechanism is that multiple introductions of invasive species prevent genetic bottlenecks by parallel introductions of several distinct genotypes that, in turn, provide heritable variation necessary for local adaptation. Here, we show that the invasion of Aegilops triuncialis into California, USA, involved multiple introductions that may have facilitated invasion into serpentine habitats. Using microsatellite markers, we compared the polymorphism and genetic structure of populations of Ae. triuncialis invading serpentine soils in California to that of accessions from its native range. In a glasshouse study, we also compared phenotypic variation in phenological and fitness traits between invasive and native populations grown on loam soil and under serpentine edaphic conditions. Molecular analysis of invasive populations revealed that Californian populations cluster into three independent introductions (i.e. invasive lineages). Our glasshouse common garden experiment found that all Californian populations exhibited higher fitness under serpentine conditions. However, the three invasive lineages appear to represent independent pathways of adaptation to serpentine soil. Our results suggest that the rapid invasion of serpentine habitats in California may have been facilitated by the existence of colonizing Eurasian genotypes pre‐adapted to serpentine soils.     

Microsatellite analysis reveals a progressive widening of the genetic basis in the elite durum wheat germplasm

It has been argued that the level of genetic diversity in the modern durum wheat (Triticum turgidum L. var. durum) elite germplasm may have declined due to the high selection pressure applied in breeding programs. In this study, 58 accessions covering a wide spectrum of genetic diversity of the cultivated durum wheat gene pool were characterized with 70 microsatellite loci (or simple sequence repeats, SSRs). On average, SSRs detected 5.6 different allelic variants per locus, with a mean diversity index (DI) equal to 0.56, thus revealing a diversity content comparable to those previously observed with SSRs in other small-grain cereal gene pools. The mean genetic similarity value was equal to 0.44. A highly diagnostic SSR set has been identified. A high variation in allele size was detected among SSR loci, suggesting a different suitability of these loci for estimating genetic diversity. The B genome was characterized by an overall polymorphism significantly higher than that of the A genome. Genetic diversity is organised in well-distinct sub-groups identified by the corresponding foundation-genotypes. A large portion (92.7%) of the molecular variation detected within the group of 45 modern cvs was accounted for by SSR alleles tracing back to ten foundation-genotypes; among those, the most recent CIMMYT-derived founders were genetically distant from the old Mediterranean ones. On the other hand, rare alleles were abundant, suggesting that a large number of genetic introgressions contributed to the foundation of the well-diversified germplasm herein considered. The profiles of recently released varieties indicate that the level of genetic diversity present in the modern durum wheat germplasm has actually increased over time.Communicated by F. Salamini