David R. Butler

Towards the understanding of the cocoa transcriptome: Production and analysis of an exhaustive dataset of ESTs of Theobroma cacao L. generated from various tissues and under various conditions

BackgroundTheobroma cacao L., is a tree originated from the tropical rainforest of South America. It is one of the major cash crops for many tropical countries. T. cacao is mainly produced on smallholdings, providing resources for 14 million farmers. Disease resistance and T. cacao quality improvement are two important challenges for all actors of cocoa and chocolate production. T. cacao is seriously affected by pests and fungal diseases, responsible for more than 40% yield losses and quality improvement, nutritional and organoleptic, is also important for consumers. An international collaboration was formed to develop an EST genomic resource database for cacao.ResultsFifty-six cDNA libraries were constructed from different organs, different genotypes and different environmental conditions. A total of 149,650 valid EST sequences were generated corresponding to 48,594 unigenes, 12,692 contigs and 35,902 singletons. A total of 29,849 unigenes shared significant homology with public sequences from other species.Gene Ontology (GO) annotation was applied to distribute the ESTs among the main GO categories.A specific information system (ESTtik) was constructed to process, store and manage this EST collection allowing the user to query a database.To check the representativeness of our EST collection, we looked for the genes known to be involved in two different metabolic pathways extensively studied in other plant species and important for T. cacao qualities: the flavonoid and the terpene pathways. Most of the enzymes described in other crops for these two metabolic pathways were found in our EST collection.A large collection of new genetic markers was provided by this ESTs collection.ConclusionThis EST collection displays a good representation of the T. cacao transcriptome, suitable for analysis of biochemical pathways based on oligonucleotide microarrays derived from these ESTs. It will provide numerous genetic markers that will allow the construction of a high density gene map of T. cacao. This EST collection represents a unique and important molecular resource for T. cacao study and improvement, facilitating the discovery of candidate genes for important T. cacao trait variation.

Evaluation and utilisation of cacao (Theobroma cacao L.) germplasm at the International Cocoa Genebank, Trinidad

The aim of this study was to identify promising genotypes in the International Cocoa Gene bank, Trinidad (ICG,T) for use in cacao breeding. Subsets of the germ plasm collection were evaluated for bean number, bean weight, pod index (581 genotypes) and resistance to Phytophthora pod rot(500 genotypes). Among three groups of cacao assessed (For astero, Refractario and Trinitario), Trinitario had the highest percentage of genotypes (36.9%) with large bean weight (> 1.2 g), while For astero possessed the highest proportion of genotypes (22.6%) with a large number of beans (> 45). The ICS population had the highest percentage (44.1%) of genotypes with heavy beans, and IMC the highest percentage of genotypes with large bean number (68.6%). A low, but significant negative correlation (r = –0.19, p≤ 0.001) between bean number and bean weight suggests that an increase in bean number may repress an increase in bean weight and vice versa. However, inten genotypes good values of bean weight and number were combined demonstrating that selection for both large bean number and bean weight is possible. The highest percentage (28.1%) of genotypes with low pod index (< 20.1) was observed in the Trinitario group (mainly ICS). The Forastero group had the highest percentage of Phytophthora resistant accessions(18.0%). The PA population had the highest proportion of resistant (24.0%) and moderately resistant (38.0%)genotypes. Sixty-five and 99 genotypes were categorised with large bean weight and high bean number, respectively, and 60 genotypes were found with a low pod index. Seventy-eight genotypes were identified with resistance to Phytophthora podrot. A significant relationship was observed between resistance to Phytophthora pod rot (measured as the frequency of localised lesions) and bean number (r = –0.45, p ≤ 0.001)showing that the two traits may complement each other. The combination of low to intermediate pod index with moderate to high resistance to Phytophthora podrot was found in 87 genotypes, 12 of which were also reported to have resistance to witches’ broom disease. These genotypes are of high potential value in cacao breeding and their inclusion in working and corecollections would be justified.

Patterns of Morphological Variation in a Sample of Cacao (Theobroma Cacao L.) Germplasm from the International Cocoa Genebank, Trinidad

The International Cocoa Genebank, Trinidad (ICG,T) contains 2300 accessions representing a broad range of diversity and geographic origins. This study was undertaken to examine the phenotypic relationships in a diverse sample of 600 cacao accessions from the Genebank. The major objectives were to test the applicability of the accepted nomenclature, viz., the definition of classes, as well as to elucidate the relationships between wild and cultivated germplasm and among accession groups. This will facilitate efficient utilisation, management and improvement of cacao genetic resources through the identification of potentially heterotic groups. In addition, information on morphological variation may be considered along with allelic richness or gene frequencies in selecting core collections, and when formulating strategies for future collections in the wild. Considerable phenotypic variation (high coefficients of variation and Shannon Weaver Diversity Index values) in the germplasm studied was found, and the main groups of cacao were clearly separated. Wild cacao was differentiated from cultivated types by all of the quantitative descriptors except pod length. Recognised genetic groups (Trinitario and Forastero) were discriminated by several quantitative traits including sepal length, cotyledon weight, length and width, and pod index. Principal Component Analysis differentiated among 14 accession groups according to geographic origin and genetic grouping. Cotyledon weight, length and number, pedicel column colour, mature pod ridge colour, sepal length, pod basal constriction and surface texture, and ovule number accounted for most of the variation recorded. The observed improvement in the cultivated germplasm, in terms of agronomic traits such as seed weight and pod index, attests to the success of past selection activities. This apparent improvement (particularly among Refractario germplasm) appears to be accompanied by increased style length, a finding that warrants further study. The reduced diversity in sepal length, ligue width, ovule number, and cotyledon length in the cultivated compared to the wild germplasm is consistent with the narrowing of the genetic base in the former. These results have significance for cacao breeding programmes, conservation and future collecting strategies.

Molecular characterization of an earliest cacao (Theobroma cacao L.) collection from Upper Amazon using microsatellite DNA markers

Cacao (Theobroma cacao L.) is indigenous to the Amazon region of South America. The river basins in the Upper Amazon harbor a large number of diverse cacao populations. Since the 1930s, several numbers of populations have been collected from the present-day Peruvian Amazon and maintained as ex situ germplasm repositories in various countries, with the largest held in the International Cacao Genebank in Trinidad. The lack of information on population structure and pedigree relationship and the incorrect labeling of accessions are major concerns for efficient conservation and use of cacao germplasm. In the present study, we assessed the individual identity, sibship, and population structure in cacao populations collected from the present-day Loreto Region, Peru in the 1930–1940s. Using a capillary electrophoresis genotyping system, we analyzed the simple sequence repeat variation of 612 cacao accessions collected from the Marañon, Nanay, and Ucayali river systems. A total of 180 cases of mislabeling were identified using a Bayesian clustering method for admixture detection. Using maximum likelihood-based methods, we reconstructed 78 full-sib families nested in 48 half-sib families, indicating that the pods collected in the 1930s were from 48 mother trees, maximum. Likelihood simulation also identified eight probable parents that are responsible for 117 pairs of mother–offspring relationships in this collection. Principal coordinate analysis (PCoA) and the Bayesian clustering method cohesively demonstrated a pronounced structure of genetic diversity, stratified by the river systems of the Peruvian Amazon. Our results also show that, in spite of the high level of allelic diversity in this collection, it was composed of a large number of related family members collected from a relatively small area, including a couple of sites in the Ucayali and Nanay rivers, as well as the lower Marañon river near Iquitos. The vast majority of the Peruvian Amazon, especially the upper Marañon River and its tributaries, have not been sampled by collecting expeditions. The improved understanding of the individual identities, genealogical relationships, and geographical origin of cacao germplasm in this collection will contribute to more efficient conservation and utilization of these germplasm. Additionally, this study also provides more baseline information to help guide future collecting expeditions in the Peruvian Amazon.

High throughput, high resolution selection of polymorphic microsatellite loci for multiplex analysis

BackgroundLarge-scale genetic profiling, mapping and genetic association studies require access to a series of well-characterised and polymorphic microsatellite markers with distinct and broad allele ranges. Selection of complementary microsatellite markers with non-overlapping allele ranges has historically proved to be a bottleneck in the development of multiplex microsatellite assays. The characterisation process for each microsatellite locus can be laborious and costly given the need for numerous, locus-specific fluorescent primers.ResultsHere, we describe a simple and inexpensive approach to select useful microsatellite markers. The system is based on the pooling of multiple unlabelled PCR amplicons and their subsequent ligation into a standard cloning vector. A second round of amplification utilising generic labelled primers targeting the vector and unlabelled locus-specific primers targeting the microsatellite flanking region yield allelic profiles that are representative of all individuals contained within the pool. Suitability of various DNA pool sizes was then tested for this purpose. DNA template pools containing between 8 and 96 individuals were assessed for the determination of allele ranges of individual microsatellite markers across a broad population. This helped resolve the balance between using pools that are large enough to allow the detection of many alleles against the risk of including too many individuals in a pool such that rare alleles are over-diluted and so do not appear in the pooled microsatellite profile. Pools of DNA from 12 individuals allowed the reliable detection of all alleles present in the pool.ConclusionThe use of generic vector-specific fluorescent primers and unlabelled locus-specific primers provides a high resolution, rapid and inexpensive approach for the selection of highly polymorphic microsatellite loci that possess non-overlapping allele ranges for use in large-scale multiplex assays.

Sorghum head-bugs and grain molds in West and Central Africa: II. Relationships between weather, head-bug and mold damage on sorghum grains

Abstract A regional Sorghum Head-Bug and Grain Mold Trial was conducted in 1996 and 1997 by WCASRN in, respectively, 15 and 13 research stations in ten west and central African countries. Empirical relationships between weather factors and head-bug damage on the one hand, and between weather factors and grain mold damage on the other hand, were examined using the “Window” computer program. No significant correlation was found between head-bug damage and those weather factors examined. In the case of grain mold, high relative humidity (RH) during early plant growth (5–40 days after sowing, DAS) on the one hand, and between end of flowering and harvest (65–125 DAS) on the other hand, were the most strongly correlated with mold incidence. The relationships between maximum RH and grain mold scores in the scatter diagrams were clearly non-linear, showing a marked increase in grain mold scores when the RH exceeded a threshold of about 95%. These results are discussed and future research directions are proposed.

Sources of Resistance to Phytophthora Pod Rot at the International CocoaGenebank, Trinidad

The International Cocoa Genebank, Trinidad (ICG,T) contains about 2000 cacao (Theobroma cacao L.) accessions. The purpose of the collection is to provide a source of genes for the genetic improvement of cacao through breeding. In fulfilling this objective, it is imperative that this collection be evaluated to identify major sources of genes for the genetic improvement of important agronomic–economic traits. In this study, 816 cacao accessions were evaluated for resistance to Phytophthora pod rot (black pod disease), which causes enormous loss of yield in cocoa production throughout the world.The study identifies promising resistant genotypes that could be exploited in cacao breeding programmes. It further compares the levels of resistance to Phytophthora pod rot among two cacao types (wild and cultivated types), three major groups (Forastero, Trinitario and Refractario) and 11 accession groups (B, AM, CL, ICS, IMC, JA, LP, MOQ, NA, PA and TRD) . The distribution of scores for the 816 accessions showed skewness towards the susceptible end of the disease rating scale, indicating that a large proportion (68.9%) of the sample was susceptible (disease rating 6–8) to Phytophthora pod rot. However, 12.9% of the sample population was found to be resistant (disease rating 1–3) and 18.2% moderately resistant (disease rating 4–5). Significant differences were observed between the wild and cultivated accessions and among the Forastero, Refractario and Trinitario groups. Marked differences were also observed among 11 accession groups, each of which was represented by at least 20 genotypes. Higher proportions of resistant (17.7%) and moderately resistant (22.6%) genotypes were observed in the wild accessions than in the cultivated varieties (9.4% resistant and 14.4% moderately resistant). The Forastero group, consisting of many wild accessions, was found to contain more resistant (18.0%) and moderately resistant (23.1%) genotypes than either the Trinitario (4.8% resistant and 13.6% moderately resistant) or Refractario (11.3% resistant and 15.4% moderately resistant). Among 11 accession groups, the largest percentage of resistant (24.2%) and moderately resistant (28.8%) genotypes were found in the PA (Forastero). Different accession groups had varying proportions of resistant and moderately resistant genotypes. This is not unexpected in an out-breeding crop such as cacao with a high level of heterogeneity. This finding reinforces the idea of a pre-breeding programme (germplasm enhancement) to accumulate resistance genes over several populations as a strategy for improving the genetic base of resistance in national cacao breeding programmes. One hundred and five promising resistant genotypes were identified among the 816 accessions evaluated. These accessions are potential sources of resistance genes for the development of high-yielding resistant varieties in cacao.

Abundance of sorghum panicle-feeding bugs (Hemiptera : Miridae) in Mali and empirical relationships with weather

In West and Central Africa, panicle-feeding mirid bugs, particularly Eurystylus oldi Poppius (Hemiptera: Miridae), are major pests of improved sorghum cultivars. Population dynamics of panicle-feeding bugs were studied on the sorghum cultivar ICSV 197, sown on eight dates each year from 1992 to 1994 at Samanko, Mali. Relationships between weather factors and abundance of panicle-feeding bugs were examined for the three most commonly found mirids, E. oldi, Campylomma spp. and Creontiades pallidus Rambur (representing on average respectively 58, 39 and 2% of all mirid species). Although only one generation of E. oldi could develop between flowering and grain maturity on a particular panicle, there were three successive generations each year during the extended grain-maturing period in the experiment with eight sowing dates, with a major peak in abundance in early October, followed by a gradual decrease until late November. Abundance of Cr. pallidus peaked in late October, while that of Campylomma spp. showed no relationship with date of sowing.An increase in abundance of E. oldi nymphs (y1) was favoured by high precipitation, reduced maximum temperature and high minimum relative humidity during and 2–3 weeks before the grain-filling period [a strong negative relationship was found with the number consecutive days without precipitation from 78–88 days after sowing (das) (x1): y1 = -11.7x1 + 149.3 (r2 = 0.67)]. The abundance of Cr. pallidus (y2) was correlated with wide temperature ranges and low precipitation during the same periods (notably precipitation frequency from 75–91 das: x2): y2 = -2.28x2 + 24.6 (r2 = 0.65). The abundance of Campylomma spp. (y3) was negatively correlated with amount of rainfall between 19–32 das (x3): y3 = -3.82x3 + 705.9 (r2 = 0.49). Reasons for these trends are suggested and their possible application for management of panicle-feeding bugs discussed.RésuméEn Afrique Centrale et de l’Ouest, les punaises mirides des panicules, particulièrement Eurystylus oldi Poppius, sont des ravageurs-clés des variétés améliorées de sorgho. Les populations de punaises ont été suivies sur la variété de sorgho ICSV 197, semée à huit dates différentes chaque année de 1992 à 1994 à Samanko au Mali. Les relations entre facteurs climatiques et populations ont été examinées pour les trois principaux mirides, £. oldi, Campylomma spp. et Creontiades pallidus Rambur (représentant en moyenne respectivement 58, 39 et 2% des espèces de mirides). Bien qu’une seule génération d’E. oldi puisse se développer entre la floraison et la maturité du grain sur une panicule, trois générations se sont succédées chaque année pendant la longue période de maturation du grain sur l’essai avec huit dates de semis, avec un pic principal de pullulation début octobre, suivi d’une baisse régulière jusqu’à fin novembre. Les populations de Cr. pallidus ont culminé fin octobre; celles de Campylomma spp. n’ont présenté aucune relation avec la date de semis. Une faible température maximum, une forte humidité relative minimum et de fortes précipitations pendant les 2–3 semaines précédents le remplissage du grain ont favorisé l’augmentation des populations larvaires d’E. oldi (yl)[forte corrélation négative notamment avec le nombre de jours sans pluies de 78 à 88 jours après semis (JAS) (x1): y1= 11.7x1+ 149.3 (r2 = 0.67)]. De fortes populations de Cr. pallidus (y2) sont associées à de grandes amplitudes thermiques et à de faibles précipitations simultanées (notamment la fréquence des précipitations de 75 à 91 JAS: x2): y2 = 2.28x2 + 24.6 (r2 = 0.65). Les nombres de Campylomma spp. (y3) sont corrélés négativement avec la pluviométrie 19–32 JAS (x3): y3 = -3.82x3 + 705.9 (r2 = 0.49). Des explications permettant d’interpréter ces tendances sont avancées, et on discute de leur application possible pour la lutte contre les punaises.

The Impact of Pollen Donor on Flavor in Cocoa

Past studies have shown evidence of pollen parent effects on yield, bean size and pod characteristics but its effect on flavor attributes is not clearly understood. An incomplete diallel mating design involving five cacao cultivars (West African Amelonado (WAA), Imperial College Selection (ICS) 1, Iquitos Mixed Calabacillo (IMC) 67 and two Trinidad Selected Hybrids (TSH) coded as CCL 200 and CCL 201) with widely differing flavor attributes were used to investigate the magnitude of female and male parent effects on key intrinsic flavor attributes. The seeds derived from pods arising from these pollinations were fermented, dried and made into cocoa liquor according to standardized methods. Flavor evaluations were carried out by a trained sensory panel for nine flavor attributes with five repetitions and hidden flavor reference controls. The study was conducted over two cocoa crop years. The results failed to detect dominant xenia effects for important ancillary flavor attributes i.e., cocoa flavor, acidity, fruitiness and floral flavors but showed significant female parent effects for cocoa and floral flavors. Small but inconsistent male parent effects were seen for astringency. Lack of xenia effect for the major flavor attributes implies that the flavor quality of cocoa beans is determined principally by the genotype of the female parent.