A genetic linkage map of Theobroma cacao L.

A linkage map of the cocoa genome comprising 193 loci has been constructed. These loci consist of 5 isozymes, 101 cDNA/RFLPs, 4 loci from genes of known function, 55 genomic DNA/RFLPs and 28 RAPDs. A population of 100 individuals derived from a cross between two heterozygous genotypes was used. Segregation analyses were performed with the JoinMap program. Ten linkage groups, which putatively correspond to the ten gametic chromosomes of cocoa, were identified. The map covers a total length of 759 cM with a 3.9 cM average distance between 2 markers. A small fraction (9%) of the markers deviated significantly from the expected Mendelian ratios.     

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     

A new glycosylated dihydrophaseic acid from cacao germs (Theobroma cacao L.)

Cacao beans are composed of cacao nibs and germs. Although numerous chemical and physiological studies on cacao nib compounds have been reported, there is little information on cacao germ compounds. We therefore analyzed an extract from the cacao germ, and found two compounds that were specific to the germ. One of these two compounds was identified as the new glycosylated abscisic acid metabolite, dihydrophaseic acid-4'-O-6″-(β-ribofuranosyl)-β-glucopyranoside, and the other as the known compound, dihydrophaseic acid-4'-O-β-D-glucopyranoside.     

Microsatellite markers developed from Theobroma cacao L. expressed sequence tags

Theobroma cacao L. expressed sequence tags (ESTs) were converted into useful genetic markers for fingerprinting individuals and genetic linkage mapping. Primers were designed to microsatellite‐containing ESTs. Twenty‐two T. cacao accessions, parents of various mapping populations segregating for disease resistance and crop yield characteristics, were tested. Twenty‐seven informative loci were discovered with 26 primer pairs. The number of detected alleles ranged from two to 11 and averaged 4.4 per locus. All 27 markers could be mapped into at least one of the existing F₁ or F₂ populations segregating for agronomically important traits.     

Genetic Diversity and Natural Population Structure of Cacao (Theobroma cacao L.) from the Brazilian Amazon Evaluated by Microsatellite Markers

A sample of 94 accessions of Theobroma cacao L. (cacao), representing four populations from the Brazilian Amazon (Acre, Rondônia, lower Amazon and upper Amazon) were analyzed using microsatellite markers to assess the genetic diversity and the natural population structure. From the 19 microsatellite loci tested, 11 amplified scorable products, revealing a total of 49 alleles, including two monomorphic loci. The Brazilian upper Amazon population contained the largest genetic diversity, with the most polymorphic loci, the highest observed heterozygosity; and the majority of rare alleles, thereby this region might be considered part of the center of diversity of the species. The observed heterozygosity for all the Brazilian populations (H _o = 0.347) was comparable with values reported for other similar upper Amazon Forastero cacao populations, with the Acre and Rondônia displaying the lowest values. The lower Amazon population, traditionally defined as highly homozygous, presented an unexpectedly high observed heterozygosity (H _o = 0.372), disclosing rare and distinct alleles, with large identity with the upper Amazon population. It was hypothesized that part of the lower Amazon population might derive from successive natural or intentional introduction of planting material from other provenances, mainly upper Amazon. Most of the loci exhibited a lower observed heterozygosity than expected, suggesting that self-pollination might be more common than usually assumed in cacao, but excess of homozygotes might also derive from sub-grouping (Wahlund effect) or from sampling related individuals. Most of the gene diversity was found to occur within groups, with small differentiation between the four Brazilian Amazon populations, typical of species with high gene flow.     

Strcture of the Mature Embryo of Theobroma cacao L.

The mature embryo of Theobroma cacao L. consists of an embryonicaxis and two cotyledons. The cotyledonary tissue comprises thebulk of the embryo and contains mature and maturing vasculartissue surrounded by a bundle sheath. The cells of the groundtissue are filled with starch grains, anthocyanin pigments,or mucilage. The embryonic axis consists of epicotyl, hypocotyl,and root meristem. Maturation of the vascular tissue has begunin the procambium, where xylem maturation is ahead of phloemmaturation. Mucilage cells are found throughout the tissue of the axis.Most of the cells of the embryonic tissue contain starch grains.     

Confocal observations of late-acting self-incompatibility in Theobroma cacao L.

Cocoa (Theobroma cacao) has an idiosyncratic form of late-acting self-incompatibility that operates through the non-fusion of incompatible gametes. Here, we used high-resolution confocal microscopy to define fine level changes to the embryo sac of the strongly self-incompatible cocoa genotype SCA 24 in the absence of pollination, and following compatible and incompatible pollination. All sperm nuclei had fused with the female nuclei by 48?h following compatible pollinations. However, following incompatible pollinations, we observed divergence in the behaviour of sperm nuclei following release into the embryo sac. Incomplete sperm nucleus migration occurred in approximately half of the embryo sacs, where the sperm nuclei had so far failed to reach the female gamete nuclei. Sperm nuclei reached but did not fuse with the female gamete nuclei in the residual cases. We argue that the cellular mechanisms governing sperm nucleus migration to the egg nucleus and those controlling subsequent nuclear fusion are likely to differ and should be considered independently. Accordingly, we recommend that future efforts to characterise the genetic basis of LSI in cocoa should take care to differentiate between these two events, both of which contribute to failed karyogamy. Implications of these results for continuing efforts to gain better understanding of the genetic control of LSI in cocoa are discussed.     

A comparison between Theobroma cacao L. zygotic embryogenesis and somatic embryogenesis from floral explants

Summary In order to improve the late phases of Theobroma cacao L. embryogenesis from tissues of maternal origin, zygotic embryogenesis and somatic embryogenesis were compared, with respect to morphological, histological, and physiological parameters. Zygotic embryogenesis could be divided into three steps: (a) embryogenesis sensu stricto, (b) a growth period in which cotyledonary embryos reached their final dimensions, and (c) a maturation period in which embryos accumulated protein and starch reserves, dehydrated to a water content equal to 30%, and underwent a modification in soluble sugar composition. Monosaccharides and sucrose contents decreased to the benefit of the oligosaccharides raffinose and stachyose. The formation of somatic embryos by use of basic protocols was studied to define the limiting factors that could lie behind their poor development. Morphological abnormalities of somatic embryos, which represented 80% of the total population, were described. A histological study showed that somatic embryos lacked starch and protein reserves; moreover, their water content was much higher than that of their zygotic counterparts. Introducing a growth period into the culture protocol made for better embryo development. Adding sucrose and abscisic acid to the maturation medium was effective in increasing reserve synthesis and resulted in higher germination, conversion, and acclimatization rates.     

Numerous Clones Resistant to Phytophthora palmivora in the "Guiana" Genetic Group of Theobroma cacao L

Cocoa black pod rot, a disease caused by Stramenopiles of the genus Phytophthora, and particularly by the pan-tropical species P. palmivora, causes serious production losses worldwide. In order to reduce the impact of these pests and diseases, preference is given to genetic control using resistant varieties and, to that end, breeders seek sources of resistance in wild cocoa trees. For instance, surveys of spontaneous cocoa trees in French Guiana between 1985 and 1995 led to the collection of abundant plant material forming a particular genetic group (the "Guiana" group). Following numerous one-off studies demonstrating the merits of this group as a source of resistance to Phytophthora, this article presents the results of a comprehensive study assessing the resistance of 186 "Guiana" clones in relation to the Guianan strain (GY 27) of P. palmivora. This study, undertaken in French Guiana, using an efficient methodology (ten series of tests and a statistical test adapted to the ordinal nature of the data) confirmed that the "Guiana" genetic group does indeed constitute an important source of resistance to P. palmivora, though with some variations depending on the demes of origin. Numerous clones (59) proved to be as resistant as the SCAVINA 6 resistance control, whilst nine were statistically more resistant. The "Resistant" and "Moderately Resistant" Guianan clones totalled 108 (58% of the total tested). Some of the clones more resistant than SCAVINA 6 could be incorporated into numerous cocoa breeding programmes, particularly those that also display other notable qualities. The same applies for numerous other clones equivalent to SCAVINA 6, especially the "elite"' clones GU 134-B, GU 139-A and GU 285-A.     

Somatic embryogenesis and plant regeneration from floral explants of cacao (Theobroma cacao L.) using thidiazuron

Summary A procedure for the regeneration of cacao (Theobroma cacao) plants from staminode explants via somatic embryogenesis was developed. Rapidly growing calli were induced by culturing staminode explants on a DKW salts-based primary callus growth (PCG) medium supplemented with 20 g glucose per L, 9 μM 2,4-D, and thidiazuron (TDZ) at various concentrations. Calli were subcultured onto a WPM salts-based secondary callus growth medium supplemented with 20 g glucose per L, 9 μM 2,4-D, and 1.4 nM kinetin. Somatic embryos were formed from embryogenic calli following transfer to a hormone-free DKW salts-based embryo development medium containing sucrose. The concentration of TDZ used in PCG medium significantly affected the rate of callus growth, the frequency of embryogenesis, and the number of somatic embryos produced from each responsive explant. A TDZ concentration of 22.7 nM was found to be the optimal concentration for effective induction of somatic embryos from various cacao genotypes. Using this procedure, we recovered somatic embryos from all 19 tested cacao genotypes, representing three major genetic group types. However, among these genotypes, a wide range of variation was observed in both the frequency of embryogenesis, which ranged from 1 to 100%, and the average number of somatic embryos produced from each responsive explant, which ranged from 2 to 46. Two types of somatic embryos were identified on the basis of their visual appearance and growth behavior. A large number of cacao plants have been regenerated from somatic embryos and established in soil in a greenhouse. Plants showed morphological and growth characteristics similar to those of seed-derived plants. The described procedure may allow for the practical use of somatic embryogenesis for clonal propagation of elite cacao clones and other applications that require the production of a large number of plants from limited source materials.     

Physiological and biochemical responses of Theobroma cacao L. genotypes to flooding

Flooding is common in lowlands and areas with high rainfall or excessive irrigation. A major effect of flooding is the deprivation of O₂ in the root zone, which affects several biochemical and morphophysiological plant processes. The objective of this study was to elucidate biochemical and physiological characteristics associated with tolerance to O₂ deficiency in two clonal cacao genotypes. The experiment was conducted in a greenhouse with two contrasting clones differing in flood tolerance: TSA-792 (tolerant) and TSH-774 (susceptible). Leaf gas exchange, chlorophyll (Chl) fluorescence, chemical composition and oxidative stress were assessed during 40 d for control and flooded plants. Flooding induced a decrease in net photosynthesis, stomatal conductance and transpiration of both genotypes. In flood conditions, the flood-susceptible clone showed changes in chlorophyll fluorescence, reductions in chlorophyll content and increased activity of peroxidase and polyphenol oxidase. Flooding also caused changes in macro- and micronutrients, total soluble sugars and starch concentrations in different plant organs of both genotypes. Response curves for the relationship between photosynthetically active radiation (PAR) and net photosynthetic rate (P _N) for flooded plants were similar for both genotypes. In flood conditions, the flood-susceptible clone exhibited (1) nonstomatal limitations to photosynthesis since decreased in maximum potential quantum yield of PSII (F_v/F_m) values indicated possible damage to the PSII light-harvesting complex; (2) oxidative stress; (3) increased leaf chlorosis; and (4) a reduction in root carbohydrate levels. These stresses resulted in death of several plants after 30 d of flooding.     

Effect of Fruiting Traits on the Field Resistance of Cocoa (Theobroma cacao L.) Clones to Phytophthora megakarya

The effects of some traits of field resistance (precocity and duration of the fruiting cycle, age of diseased fruit and vertical pod distribution on the tree) to Phytophthora megakarya of four known cocoa clones were studied in an on‐station clonal plot planted in 1982 in the south‐west of Cameroon. Weekly observations of fruit set and development, black pod and rainfall were carried out during three growing seasons (1999, 2000 and 2001). The study confirmed the previous field and laboratory assessments of resistance of these clones based on the mean percentages of rotten pods obtained annually. The present study has permitted the identification of fruit aged 2–3 months as the highly susceptible stage of development in the most susceptible clone. In addition, precocity and pod cycle duration varied significantly among the clones. The earlier the pod cycle began, the more susceptible was the clone: the most resistant clone started flowering 1 month after the most susceptible clone and therefore escaped the peak of disease severity. Rainfall intensity greatly modified the incidence of the disease in 2001, with high yield losses occurring in all four clones (70–93%), but their ranking remained stable over the 3 years. The spatial distribution of pods on the trees showed that pods on the trunk were more likely to become diseased than those on the branches, but its effect as a clone resistance component is variable among the four clones; the resistant clone producing more pods on the trunk and the susceptible clone more in the canopy.     

The regulation of triacylglycerol biosynthesis in cocoa (Theobroma cacao) L.

Developing cocoa cotyledons accumulate initially an unsaturated oil which is particularly rich in oleate and linoleate. However, as maturation proceeds, the characteristic high stearate levels appear in the storage triacylglycerols. In the early stages of maturation, tissue slices of developing cotyledons (105 days post anthesis, dpa) readily accumulate radioactivity from [¹⁴C]acetate into the diacylglycerols and label predominantly palmitate and oleate. In older tissues (130 dpa), by contrast, the triacylglycerols are extensively labelled and, at the same time, there is an increase in the percentage labelling of stearate. Thus, the synthesis of triacylglycerol and the production of stearate are co-ordinated during development. The relative labelling of the phospholipids (particularly phosphatidylcholine) was rather low at both stages of development which contrasts with oil seeds that accumulate a polyunsaturated oil (e.g. safflower). Microsomal membrane preparations from the developing cotyledons readily utilised an equimolar [¹⁴C]acyl-CoA substrate (consisting of palmitate, stearate and oleate) and glycerol 3-phosphate to form phosphatidate, diacylglycerol and triacylglycerol. Analysis of the [¹⁴C]acyl constituents at the sn-1 and sn-2 positions of phosphatidate and diacylglycerol revealed that the first acylase enzyme (glycerol 3-phosphate acyltransferase) selectively utilised palmitate over stearate and excluded oleate, whereas the second acylase (lysophosphatidate acyltransferase) was highly selective for the unsaturated acyl-CoA. On the other hand, the third acylase (diacylglycerol acyltransferase) exhibited an almost equal selectivity for palmitate and stearate. Thus, stearate is preferentially enriched at position sn-3 of triacylglycerol at 120–130 dpa because of the relatively higher selectivity of the diacylglycerol acyltransferase for this fatty acid compared with those of the other two acylation enzymes.Abbreviation dpa days post anthesis We are grateful to Drs. G. Pettipher (Cadbury-Schweppes, Reading, UK), M. End and P. Hadley (Department of Horticulture, University of Reading) for the supply of cocoa pods and to the Agricultural and Food Research Council for financial support. We also wish to thank Dr. S. Stymne (Swedish University of Agricultural Sciences, Uppsala, Sweden) for a generous gift of acyl-CoA substrates.     

Localization and identification of phenolic compounds in Theobroma cacao L. somatic embryogenesis

Cocoa breeders and growers continue to face the problem of high heterogeneity between individuals derived from one progeny. Vegetative propagation by somatic embryogenesis could be a way to increase genetic gains in the field. Somatic embryogenesis in cocoa is difficult and this species is considered as recalcitrant. This study was conducted to investigate the phenolic composition of cocoa flowers (the explants used to achieve somatic embryogenesis) and how it changes during the process, by means of histochemistry and conventional chemical techniques. In flowers, all parts contained polyphenolics but their locations were specific to the organ considered. After placing floral explants in vitro, the polyphenolic content was qualitatively modified and maintained in the calli throughout the culture process. Among the new polyphenolics, the three most abundant were isolated and characterized by 1H- and 13C-NMR. They were hydroxycinnamic acid amides: N-trans-caffeoyl-l-DOPA or clovamide, N-trans-p-coumaroyl-l-tyrosine or deoxiclovamide, and N-trans-caffeoyl-l-tyrosine. The same compounds were found also in fresh, unfermented cocoa beans. The synthesis kinetics for these compounds in calli, under different somatic embryogenesis conditions, revealed a higher concentration under non-embryogenic conditions. Given the antioxidant nature of these compounds, they could reflect the stress status of the tissues.     

Phenol content, acidic peroxidase and IAA-oxidase during somatic embryogenesis in Theobroma cacao L.

Calli were induced in cacao cotyledon explants on a half-strength Murashige and Skoog medium containing 6 × 10-2 g m-3 saccharose and various combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) with kinetin (kin), benzylaminopurine (BAP) or 2-isopentenylphosphate (2-iP). Experiments were carried out on two clones of cacao differing in their susceptibility to black pod disease. The highest percentage of explants forming callus and the most rapid callus development were obtained with 10-6 g m-3 2,4-D and 0.5× 10-6 g m-3 kin. Somatic embryogenesis and rhizogenesis were induced by transferring 3-week-old callus in a half strength Murashige and Skoog medium containing 3 × 10-2 g m-3 saccharose and NAA or IBA in the 0 to 5 × 10-6 g m-3 concentration range. No differentiation could be observed when the medium was supplemented with kin or BAP. The conversion of callus into somatic embryos and roots was accompanied by a drop in phenol content and an increase in peroxidase and IAA-oxidase activities. Moreover, cell differentiation was characterized by the persistence in the callus of one acidic soluble isoperoxidase which was not detected in nondifferentiating callus. Although some differences were noticed between the clones, alterations responsible for cell differentiation were the same in both genotypes. This revised version was published online in July 2006 with corrections to the Cover Date.     

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

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

Genetic diversity assessment of sub-samples of cacao, Theobroma cacao L. collections in West Africa using simple sequence repeats marker

Knowledge of genebank and on-farm genetic diversity, particularly in an introduced crop species, is crucial to the management and utilization of the genetic resources available. Microsatellite markers were used to determine genetic diversity in 574 accessions of cacao, Theobroma cacao L., representing eight groups covering parental populations in West Africa, genebank, and farmers’ populations in Nigeria. From the 12 microsatellite markers used, a total of 144 alleles were detected with a mean allelic richness of 4.39 alleles/locus. The largest genetic diversity was found in the Upper Amazon parent population (H _nb  = 0.730), followed by the 1944 Posnette’s Introduction (H _nb  = 0.704), and was lowest in the Local parent population (H _nb  = 0.471). Gene diversity was appreciably high in the farmers’ populations (H _nb  = 0.563–0.624); however, the effective number of alleles was lower than that found in the genebank’s Posnette’s population. Fixation index estimates indicated deficiency of heterozygotes in the Upper Amazon and the Local parent populations (F _is  = 0.209 and 0.160, respectively), and excess of heterozygotes in the Trinitario parent population (F _is  = −0.341). The presence of inbreeding in the Local parent populations and substructure (Wahlund effect) in the Upper Amazon were suggested for the deficiency of heterozygotes observed. Non-significant genetic differentiation observed between the genebank’s and farmers’ populations indicated significant impact of national breeding programs on varieties grown in farmers’ plantations. From this study, we showed that appreciable genetic diversity was present in on-farm and field genebank collections of cacao that can be exploited for crop improvement in West Africa. Suggestions for future conservation of on-farm genetic diversity and local landraces are further discussed.     

Characterization of the exopeptidase activity existing in Theobroma cacao L. during germination

The present work reports exopeptidase activity existing in cacao (Theobroma cacao L.) during germination. Aminopeptidase (APE), carboxypeptidase (CP) and Xaa-Prolyl dipeptidyl aminopeptidase (Xaa-Pro-DAP) membrane-bound enzymes have been identified. The Xaa-Pro-DAP enzyme (E.C. 3.4.14.5) had not been previously detected in germinating cacao seeds. Xaa-Pro-DAP was partially purified and characterized, and the highest activity was found after 10 days of germination. Xaa-Pro-DAP was isolated by precipitation with 40% ammonium sulfate and partially purified with two chromatographic steps. The enzyme had a relative molecular weight of 80 kDa as determined by Native-PAGE and was, able to use Ala-Pro-4μβNA as substrate. In the presence of SDS, this enzyme did not show activity because it must be in a trimer to be functional. Its activity was inhibited 44% by the chelating agent EDTA and 48% by the serine peptidase inhibitor PMSF at 0.1 mM, indicating that the partially purified Xaa-Pro-DAP is a serine metallopeptidase. The cations Cu²⁺ and Cd²⁺ caused 44% and 67% inhibition, respectively, while the other divalent cations tested had no significant effect on the activity of the partially purified enzyme. The enzyme showed a high specificity for Ala-Pro-pNA as a proteolytic substrate.