Implication of the genetic structure of Theobroma cacao for breeding strategies

Diversity studies have been made by numerous authors using several kind of descriptors: morphological, enzymatic, molecular. A clear classification is difficult to obtain due to several reasons: - the genetic mixing happened during the three last centuries, - the samples studied are sometimes biased: eg. the Forastero were collected by Pound in Peru for resistance to witches broom, - the first Criollo diversity studies were first based on genotypes present in germplasm and corresponding to hybrids rather than pure types, - the results about the several populations are widespread in several different studies. However the main results indicate: -Forastero populations. An important diversity is observed between and within UpperAmazon Forastero populations with a continue variation. The highest diversity has been observed in Ecuadorian populations LCTEEN and lower diversity in some populations as NA, GU (French Guiana populations). However, the studied samples from Upper Amazon are biased and prevent to compare really the extent of diversity between populations. The Ecuadorian Allen collections have been made in a larger area, without criteria of selection, and on a larger number of trees than the Peruvian Pound samples. Very few samples from Colombia and Brazil have been analysed in these studies. - Criollo and Nacional varieties. "Ancestral" Criollo and Nacional have been identified to be -nearly unique homozygous genotypes varieties. Modem Criollo and Nacional are in fact hybrid types resulting from introgression of respectively Forastero (reduced number of genotypes) in the ancestral Criollo and of Trinitario in the ancestral Nacional genotype. - The specificity and differentiation of some populations / types as the French Guiana Forastero population, the ancestral Nacional and Criollo varieties could be explained by foundation effects or refuge areas. The consequences for breeding programmes is that a reduced number of Upper-Amazon Forastero have been used until now and mainly coming from Pound's collections. Trees from some populations have been never tested (French Guyana Forastero, Ecuadorian LCTEEN clones, Colombian EBC clones, etc.) and a limited. number of hybrid types have been tested. It will be useful to put in place prospective trials involving new clones and new hybrid types to exploit the diversity of natural populations of T. cacao not exploited until now. The narrow genetic base used in breeding programmes is favourable to exploit linkage desequilibriums in these populations combining information from diversity and from genome mapping studies. Genome analyses (QTL mapping) are generally made on specific progenies and the results concern the studied clones only. It is possible to analyse with markers what degrees of linkage between markers and traits of interest (linkage desequilibrium) could have been conserved during evolution and domestication processes. This linkage desequilibrium could allow to get a larger benefit of the information provided by markers to screen the genetic resources of some groups as WC, SCA, MO Forastero clones, Criollo, Trinitario, Nacional, when using markers closely linked to QTL identified in clones belonging to these groups. (Résumé d'auteur

Use of QTL detected for resistance to Phytophthora in Theobroma cacao L.

In the framework of a CAOBISCO sponsored project the genetic bases of resistance to several species of Phytophthora have been studied. Different significant QTL identified make it possible to accumulate various genes to improve varietal resistance. Markers closely linked to QTL could allow to control at early stages the presence of resistance alleles in progenies. Several applications or strategies using QTL analyses could be considered to improve the resistance level. - Creation of genotypes homozygous for resistance alleles. The most resistant clones, as SCA6 and SNK413, could have the resistance genes in a homozygous condition. The identification of a QTL means a heterozygous condition of the resistance gene identified in the parent studied. By selfing the genotypes and using Marker Assisted Selection (MAS) it is possible to produce and screen selfed progenies for plants that are homozygous for the resistance alleles. These clones will have a higher resistance level and a better combining ability to produce resistant hybrids. - Accumulation of various resistance genes and other genes of interest. Various resistance genes could be accumulated using MAS by crossing clones for which different QTL of resistance have been identified. It is also possible to use MAS to break linkage between favourable and unfavourable alleles located in the same chromosome region. - Early selection for resistance and other traits. Increased selection efficiency in pre-breeding, at the nursery stage, would be possible by applying MAS on a limited number of resistance QTL (the stronger QTL, QTL for resistance to several Phytophthora species...) and to have available more plants to apply selection for other traits of interest in the field. - Combined use of marker information and phenotypic selection to constitute a selection index. Information on a larger number of QTL could be combined with phenotypic selection related to resistance traits (e.g. intrinsic resistance revealed by leaf tests) or other traits of interest to constitute a selection index. - Application of MAS at other sites than those where QTL have been identified. It is possible to accumulate QTL identified at different sites for resistance to diseases not present in the country (examples: screening in Montpellier for resistance to P. palmivora, P. megakarya, P. capsici or possible selection of field resistance to P. megakarya on the basis of QTL identified in Cameroon). The first results obtained in the CAOBISCO project allow to put in place several experiments to test different MAS strategies (selfing clones to fix resistance genes in homozygous condition, accumulating various resistance genes). However the identification of QTL in important other resistant clones has to be continued to identify all major sources of resistance to Phytophthora in cocoa. (Texte intégral

Divers aspects de l'utilisation possible des cultures in vitro pour la multiplication végétative de Ananas comosus L. Merr, variété Cayenne Lisse

Deux types de multiplication végétative in vitro à partir de bourgeons axillaires de couronnes de Ananas comosus, variété "Cayenne Lisse". Il s'agit d'une part d'un bouturage simple afin d'obtenir le démarrage de bourgeons présents sur les boutures (fragments d'axes de jeunes plantes obtenues in vitro). D'autre part, on provoque une ramification intensive à partir d'un implant primaire, préalablement démarré in vitro et isolé sur un milieu de culture particulier. C'est cette dernière méthode qui est la plus efficace et doit pouvoir offrir des possibilités d'application. Franc

Rapport annuel d'activités du projet FIRC. Année 1 - 2001 : connaissance et maîtrise des composantes de la flaveur du cacao Forastero et Criollo

Indépendamment des mécanismes de maturation des cabosses qui peuvent dépendre des conditions culturales et de l'état sanitaire des arbres, l'objectif de ce projet est d'analyser 2 facteurs importants pouvant interagir sur la flaveur des fèves de cacao marchand: - l'influence des conditions de traitement post récolte sur un matériel génétique varié, il s'agira alors de définir sur différents types de cacao les conditions optima de traitement post-récolte qui garantissent une bonne qualité du cacao marchand, - et l'effet de la structure génétique des variétés sur l'expression de certains caractères de qualité. Cette étude devrait déboucher sur l'identification de marqueurs de sélection précoce permettant de sélectionner plus rapidement et plus efficacement des variétés productives et donnant un cacao de la qualité aromatique souhaitée. Ces marqueurs faciliteront aussi l'exploitation des ressources génétiques du groupe Criollo/Trinitario. En plus d'un financement Firc, ce projet bénéficie d'un financement du Ministère français des affaires étrangères dans le cadre d'un accord PCP (France-Venezuela) et d'un appui du Conicit. Cette première année du projet a été essentiellement consacrée à la préparation des échantillons de cacaos de type Criollo et Forastero et à la caractérisation moléculaire d'environ 300 arbres de type Criollo ou Trinitario. De plus, un important volet de formation de thésards vénézuéliens 1 aux techniques d'analyse biochimique et de marquage moléculaire ainsi que du transfert de ces techniques au Venezuela a été réalisé. (Résumé d'auteur

Analysis of QTL studies related to yield and vigour traits carried out with different cocoa genotypes

Theobroma cacao is mainly cultivated by small growers, especially in the main production area in West Africa. The sustainability of cocoa cultivation will be improved if farmers have access to new planting material with improved agronomic traits such as yield, vigour, pest, and disease resistance. Progress in breeding programmes to accumulate favourable alleles for these traits can be accelerated using molecular marker techniques which allow more direct access to the genome. The technology has developed rapidly and over the fast ten years studies have been carried out to map QTLs for agronomic traits in several plant species. In cocoa, a number of progenies have been mapped and several QTL related to resistance to Phytophthora spp. and to yield components have been detected. The comparison of the different linkage maps of cocoa is possible through specific markers (RFLP and microsatellites) mapped on to a reference map containing 473 markers. The purpose of this paper is to analyse available results on the detection of QTL for yield and vigour traits and the co-location of the QTL identified in different parental genotypes. Methodological approaches to the detection of QTL are also presented. Perspectives for further research on mapping of yield and vigour traits and the possible use of molecular markers in the selection for these traits in cocoa are discussed. (Résumé d'auteur

Use of microsatellites for identification and genome analysis of cocoa genotypes

The developement and application of molecular genetic markers provide the opportunity to established and evaluate measure of quality for genetic ressources collections. In particular DNA-based polymorphisms are a powerful] tool in the assesment of the genetic caracterisation. Among the various molecular markers, RFLPs were the first to be used for plant genome studies, on mapping and diversity analysis. However RFLPs are labour intensive and time consuming, and require a large quantity of DNA and for cocoa a purification :by ultracentrifugation. PCR based techniques can be used to detect polymorphism, these methods do not require a so large quantity of DNA that RFLPs, and are convenient for genetic analysis on plant at early stage. Between PCR -techniques, all advantage of the microsatellites, is the codominant mode of inheritance permitting easy transfer of markers between genetic maps of different crosses in contrast to the dominant PCR markers type based on arbitrary primer. Compared to the RFLPs, microsatellites detected more alleles and a higher level of polymorphism within cultivar variation, they are a powerfull tool for estimation of heterozygosity. Some results obtained on cocoa clones included in the CFC/ICC0/IPGRI project on Cocoa Germplasm Utilization and Conservation will be discussed. In a future, there is a possibility to construct a database for all microsatellite alleles on cocoa clones. (Résumé d'auteur

Implications of new insight into the genetic structure of Theobroma cacao L. for breeding strategies

The genetic diversity of cocoa has been studied using morphological, enzymatic and molecular descriptors. It has often proved difficult to obtain a clear classification due to factors including the significant genetic mixing that has occurred over the past three centuries, bias in the samples analysed (e.g. the limited number of Forastero samples collected in Peru for Witches' broom resistance), the uncertain origin of some accessions (e.g. some early studies were based on material believed to be Criollo, but which was in fact of hybrid origin), different populations have been used in different studies. Breeding has been hampered by a lack of knowledge of the genetic diversity and level of heterozygosity of the accessions. The main results of the diversity studies are: - In Forastero populations there is significant diversity between and within populations, with continuous variation between them. The greatest diversity was observed among Ecuadorian LCTEEN populations, and the least among a few populations such as Peruvian NA or GU from French Guiana. However, the Ecuadorian populations studied by Allen were collected from a larger number of trees and from a wider area than those collected by Pound, and very few Colombian and Brazilian samples were used in these studies. - Almost completely homozygous "ancestral" Criollo and Nacional genotypes that were probably at the origin of "modern" Criollo and Nacional varieties were identified. Modern Criollo and Nacional varieties are hybrid types resulting from introgression of a few Lower Amazon Forastero genotypes into ancestral Criollo, and of Trinitario into ancestral Nacional varieties, respectively. - The specificity of some populations or varieties has been recognised, for example wild French Guiana, ancient Criollo and Nacional varieties. The founder effect or refuge areas may be responsible for the differences between these populations. - The narrow genetic base of cocoa genotypes used in breeding programs is well known. - The level of heterozygosity of several hundreds of clones has recently been established (data presented here) and this new information may be very useful to breeders. Many breeding programmes have only used a limited number of Upper Amazon Forastero types collected by Pound. Genotypes from other populations have been used very little or not at all (e.g. wild French Guiana, LCT EEN, Colombian EBC types, etc). It would be particularly interesting to set up prospective trials of crosses between genotypes from these different populations. This would exploit the diversity of natural T. cacao populations that have not previously been used and may result in new heterotic combinations, Secondly, the genetic diversity studies have given useful information for population breeding approaches, such as reciprocal recurrent selection. Thirdly, the narrow genetic basis used in many cocoa breeding programmes to date is favourable for the exploitation of the expected linkage disequilibria within such populations. QTL mapping is generally done on a few specific progenies, and the results only relate to the clones involved. It is possible to enlarge such studies to analyse the degree to which genetic linkage between markers and traits of interest has been maintained during the evolution and domestication processes in genetic groups such as IMC, SCA and MO, Forastero, Criollo, Trinitario or Nacional. (Résumé d'auteur

Use of microsatellite markers of germplasm identity analysis in cocoa

Molecular genetic markers provide, among other applications, the opportunity to verify identity in germplasm collections. DNA-based polymorphisms are a powerful tool in genetic characterisation. RFLP markers were the first to be used for plant genome studies, for mapping and for diversity analyses. However, RFLPs are labour intensive, time consuming, require a large quantity of DNA and purification by ultra centrifugation. The PCR based techniques, including microsatellite analysis, require less DNA than RFLP markers and are therefore convenient for genetic analysis on young plants. An additional advantage of the use of microsatellites is the codominant mode of inheritance, which in contrast to the dominant PCR markers based on arbitary primers, allows easy transfer of markers between genetic maps of different crosses. Compared to RFLPs, microsatellites detect more alleles and a higher level of polymorphism and are equally powerful tools for estimation of heterozygosity. Results obtained on cocoa accessions included in the CFC/ICCO/IPGRI project on "Cocoa Germplasm Utilization and Conservation, a Global Approach" are presented here, including about 150 comparisons of DNA samples from nine collections concerning 28 different accessions. It is concluded that identification problems occur frequently, on average in about 30% of the samples examined in our study. These identification problems occur both in comparisons between accessions from different collections and between trees within accessions from the same collection. Such identification problems constitute a serious problem for comparative analysis of clones obtained from different sites or even from different trees within the same accession. Convenient loci number for identification analyses using microsatellites, sample collection methodology as well as conditions required for comparisons between different microsatellite analyses or between laboratories are briefly discussed. (Résumé d'auteur

Identification de gènes de Theobroma cacao différemment exprimés pendant une infection par Phytophthora megakarya

La pourriture des cabosses, provoquée par différentes espèces appartenant au genre Phytophthora, est la principale cause de pertes de récolte dans la production cacaoyère au niveau mondial. On a pu observer entre 15 et 80 % de pertes selon les espèces de Phytophthora, P. megakarya étant la plus agressive. L'amélioration des variétés présentant une résistance durable a été identifiée comme une priorité pour les programmes de recherche des pays producteurs, dans lesquels environ 14 millions de travailleurs tirent leurs revenus de la culture du cacao. La résistance du cacaoyer à Phytophthora est quantitative et polygénique. L'objectif de ce projet est de faire progresser notre connaissance des mécanismes moléculaires intervenant dans la résistance partielle du cacaoyer, de façon à développer des outils efficaces de sélection pour augmenter le degré de résistance des cacaoyers. Ces travaux visent à développer des approches génomiques fonctionnelles pour identifier les gènes candidats impliqués dans cette résistance partielle. L'hybridation soustractive suppressive (SSH) a été utilisée pour constituer les bibliothèques d'ADNc représentant des gènes différemment exprimés en réponse aux interactions cacaoyer/Phytophthora. Les EST ont été séquencées et analysées par recherche Blastn et/ou Blastx par comparaison à la base de données NCB!. Des homologies de séquence ont été découvertes avec les gènes liés à la pathogénie avec une fonction connue et identifiée à partir de la plante, comme les protéines PR (PR-l, glucanase, chitinase...), les kinases, les récepteurs (LRR), les facteurs de transduction et aussi à partir du pathogène, comme la protéine inhibitrice du glucanase. Nous avons mis au point une puce cADN nylon avec ces séquences EST candidates pour évaluer les gènes qui s'expriment différemment entre les clones de cacaoyers résistants et sensibles infectés par Phytophthora megakarya. L'expression génique a été menée sur des tissus foliaires d'un descendant créé en Papouasie Nouvelle Guinée issu d'un croisement faisant intervenir des génotypes Trinitario et Upper Arnazon Forastero du Pérou. Deux individus résistants avec la meilleure combinaison d'allèles et deux individus sensibles avec la moins bonne combinaison d'allèles ont été conservés et utilisés pour ce travail. Plusieurs gènes régulés différemment entre les individus résistants/sensibles ont été révélés par cette étude. Les résultats ont été confirmés par des expériences de RT-PCR quantitative et ont révélé l'induction précoce d'un gène de kinase pendant les premières heures de l'infection et une expression ultérieure d'une protéine PRo L'expression des différentes protéines inhibitrices de glucanase, exprimée par l'agent pathogène, dans les clones de cacaoyer sensibles a également été discutée. (Texte intégral