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

Molecular markers for cacao traits

Seventy-one microsatellites and 82 SNP markers were examined for their association with flush colour, shell content, seed butterfat content, fruit butterfat content, dry mass of cotyledons in a seed and dry mass of cotyledons from a fruit in 145 cacao accessions drawn from Refractario and Forastero groups. The more reliable markers were obtained when a vector matrix of multidimensional similarities rather than a matrix of population composition was utilised as a system screen. Fifteen markers (five SNPs and 10 SSRs) were found over six linkage groups (chromosomes 1, 2, 4, 5, 7 and 9). Each marker accounted for 10 - 43% of the phenotypic variation. Three markers (mTcCIR250, mTcCIR251 and SNP836) appeared fairly robust. The microsatellite mTcCIR250, present on chromosome nine, accounted for a substantial 34.1% of variation in the cotyledonary mass of a fruit. The microsatellite mTcCIR251, located on the same chromosome, tagged both the cotyledonary mass and butterfat content of a fruit explaining 43.4% and 41.7% of the variation respectively. The marker SNP836, present on chromosome 2, tagged both fruit butterfat content and cotyledonary dry mass in a seed explaining 12% and 11% of the total variation respectively. (Texte intégral

Identification of Theobroma cacao genes differentially expressed during Phytophthora infection

Pod rot, caused by different species belonging to the genus Phytophthora, is the main cause of harvest losses worldwide for cocoa production. Between 15% to 80% of losses could be observed according to the Phytophthora species, P. megakarya being the most aggressive. Varieties¿ improvement with a sustainable resistance has been identified as a priority of research programs of producer countries with about 14 millions of workers getting their income from the cocoa cultivation. Cocoa resistance to Phytophthora is quantitative and polygenic. The objectives of this project is to improve our knowledge of molecular mechanisms involved in the partial cocoa resistance in order to develop efficient tools of breeding to increase the resistance level of cocoa trees. This work aims to develop functional genomic approaches to identify candidate genes involved in this partial resistance. Suppression subtractive hybridisation (SSH) was used to generate cDNA libraries representing genes differentially expressed in response to cocoa/Phytophthora interactions. More than 15,000 ESTs were sequenced (in the frame of a GENOSCOPE project) and used for these studies. ESTs were analyzed by Blastn and/or Blastx search against NCBI data base. A little part of the clones had no homology with sequences and/or function already describe. The other part had significant matches to known genes. Among them, sequence homologies were found with pathogenesis related function knowledge, as PR protein (PR-1, glucanase, chitinase...), kinases, receptors (LRR), and transduction factors. Gene expression was conducted on leaf tissues of a progeny created in Papoua Nouvelle Guinea derived from a cross implied forastero and haut amazonien. Two resistant individuals with the better allele combination and two susceptible individuals with the less allele combination were kept and used for this work. Nylon cDNA macro arrays were used to assess the differentially expressed genes of the resistant and the susceptible cocoa clones infected by Phytophthora megakarya.. Libraries were screened with the inoculated-subtracted probes and non-inoculated reverse-subtracted probes, to reduce the candidate clones. We developed a novel set of macro arrays and obtained expression profiles during the several steps of phytophthora infection kinetic. Several genes differentially regulated between resistant/susceptible individuals revealed in this study are already known as integrated in signal transduction or in plant defence responses of other species. (Texte intégral