PRESENTATION SUMMARIES PLANT SCIENCE: ENSURING THE SURVIVAL OF A FRAGILE CROP Integrating genomics into an applied cacao breeding program. R. J. Schnell1, J. C. Motamayor2, J. S. Brown1, D. N. Kuhn3, C. Cervantes-Martinez1, C. T. Olano1, R. C. Ploetz4, and E. Rosenquist5 1USDA, ARS, SHRS, Miami FL, USA 2Mars Inc., Miami FL, USA 3Florida International University, Miami FL, USA 4University of Florida, TREC, Homestead FL, USA 5USDA-ARS, National Program Staff, Beltsville MD, USA Abstract Theobroma cacao L. is an under story tree from the Amazon basin that can be cultivated in a sustainable agro-forestry system, providing income to small farmers while maintaining biodiversity. During the seventeenth and eighteenth centuries, plants derived from a small number of parents were distributed to many tropical regions of the world, resulting in commercial plantings with a relatively narrow genetic base. Production of cacao in tropical America has been severely affected by three fungal pathogens causing diseases known as witches(cid:146) broom (WB), frosty pod (FP) and black pod (BP). Currently, WB and FP are confined to Central and South America; however, commercial populations in West Africa and South Asia are highly susceptible to both diseases. Traditional cacao breeding programs have only been marginally successful in producing resistant material with suitable commercial characteristics. In 1999, the USDA-ARS in collaboration with Mars Inc. initiated a project to apply modern genomic techniques to cacao breeding. The objectives were to develop an international Marker Assisted Selection (MAS) breeding program focusing on disease resistance. Over 400 micro satellite and 50 candidate gene markers have been used to map families segregating for resistance to WB, FP, and BP diseases. Quantitative Trait Loci (QTL) has been identified for resistance to WB, FP BP and productivity traits. These are being employed in the newly developed accelerated MAS cacao breeding program using a two stage selection technique that greatly improves genetic gain in selection cycles. The application of new genomic technologies along with the accelerated MAS will result in significant cost savings and enhanced efficiency in the development of new cultivars. Large evaluation trials are located in Costa Rica, Ecuador, and Papua New Guinea with additional QTL evaluation studies in Ghana, Nigeria, Costa Rica, and Ecuador. All these projects are collaborations with national agricultural institutes in the respective countries. The accelerated MAS project is expected to produce new disease resistant cultivars by 2010. The utility of Association Mapping for productivity traits has been demonstrated to provide an alternative method to traditional mapping and allow the mining of current germplasm collections for novel alleles. Genetic stocks developed in this program will be distributed to areas currently free of WB and FP in anticipation of the arrival of these diseases. International collaboration and sharing of genetic resources will ensure that crop losses due to these pathogens are manageable and will contribute to stability in the supply of cocoa beans. Reconstructing the natural history of Theobroma cacao L. geographic origins and domestication through molecular genetics and its consequences on modern breeding Juan C. Motamayor1, Jonathan K. Pritchard2 , Nannette Langevin3, Cuahtemoc Cervantes2, Jay Wallace, Freddy Amores and Raymond Schnell3 1MARS Inc., 800 High Street, NJ 07840, USA 2Department of Human Genetics, University of Chicago, 920 East 58th Street, Chicago, IL 60637, USA 3National Germplasm Repository, US Department of Agriculture, Agricultural Research Service, Subtropical Horticulture Research Station, Miami, FL 33129, USA. 4CEPLAC/SUPOR (AMAZONIA), Rod. Augusto Montenegro Km 07, Belem-Pa, Brasil 5INIAP, Estacion Experimental Pichilingue, Km 5, Quevedo, Los Rios, Ecuador. Traditionally, two main genetic groups, also called morphogeographic groups, have been defined within Theobroma cacao L. according to morphological traits and geographic origins. As a result of this division, some authors recognized two subspecies: cacao and sphaeorocarpum, corresponding to the morphogeographic or genetic groups Criollo and Forastero respectively. It was proposed that subspecies cacao evolved in Central America and subspecies sphaeorocarpum in South America. Cacao breeding schemes were developed to maximize genetic gain through hybridization between these two groups. A number of germplasm collecting expeditions have been accomplished over the past 50 years in several countries of South and Central America. The germplasm collected has had little impact on cacao breeding because its relationship with the exploited germplasm was not understood. Molecular genetic analysis has had a major influence on our understanding of the origin and domestication of many important crops. In cacao, a clonally propagated species, these types of studies have been problematic due to the low reliability of field maps and tagging systems used in germplasm collections. Clones collected decades ago and introduced after a long quarantine into different locations were often misidentified in the process. This problem of mislabeling has limited molecular studies in cacao as the geographic origin of the plants analyzed is not known or is wrong. With the goal of resolving the origin, classification and process of domestication of Theobroma cacao L., molecular analyses were performed on 1241 accessions covering a very large geographic sampling area from South and Central America. These included individuals from the most important collection trips. Using Bayesian statistics we identified and eliminated mislabeled samples and optimally performed subsequent analyses. Instead of two major genetic groups, at least 13 major ones were found by fingerprinting with 97 micro satellite markers. We propose a new classification of the germplasm that will enhance management, conservation and breeding. Our data clearly demonstrate that the germplasm present in Central America was introduced by humans, giving rise to a single cultivar, which corresponds to what is called Criollo. The origin of two other traditionally exploited cultivars, Nacional and Amelonado, is also discussed. The analysis revealed gaps in the current germplasm diversity and led to a new collecting expedition in the Ucayali river valley in Peru that yielded new, unique genetic resources. Important information for breeding was obtained, more specifically for the production of homogeneous hybrid seeds and for research on genetic gain through heterosis. Implications of Different Mechanisms by which endophytic fungi protect Theobroma cacao from pathogens Edward Allen Herre Smithsonian Tropical Research Institute Abstract: Cultivation of Theobroma cacao constitutes one of the most ecologically beneficial agricultural uses of tropical lands. However, losses due to pathogens undermine the economic incentives to maintain cacao where it is currently cultivated. Recent laboratory, greenhouse, and field studies indicate that naturally occurring endophytic fungi isolated from healthy T. cacao leaf tissues can be used to protect the host from pathogen damage. Further, we have evidence suggesting that different endophytic fungi protect the host via different mechanisms. I will discuss the very different ecological and applied implications of the different mechanisms that potentially underlie host protection. Thoughts on Crinipellis: Defeating the Disease that Destroyed Brazil’s Cocoa Crop Jœlio C M Cascardo1, Abelmon S Gesteira1, Fabienne Micheli1,2, Nicolas Carels1; Gon(cid:231)alo G A Pereira3. 1UESC-DCB, IlhØus, BA-Brazil; 2CIRAD-CP, Montpellier, France; 3UNICAMP-IB, Campinas, SP-Brazil. Crinipellis perniciosa (Stahel) Singer is a fungus basidiomycete that causes witches(cid:146) broom disease in members of the plant families Sterculiaceae, Solanaceae, and Bixaceae. The economic impact of the pathogen is mainly on Theobroma cacao L. Basidiospores infect meristematic tissues (shoots, flower cushions, single flowers, and developing fruits), inducing a range of symptoms depending on the organ infected and stage of development. Hypertrophy growth of infected buds ((cid:145)brooms(cid:146)) is the most dramatic symptom. Flower cushion infection usually leads to the production of vegetative shoots and abnormal flower development. Pod infections can directly result in seed losses reaching up to 90% of potential annual production in some areas and seasons. Pod set is further reduced indirectly by the infection of flower cushions and the general debilitation of the tree. The life cycle of the pathogen is completed by the production of basidiocarps on necrotic brooms and dried pods releasing basidiospores, the unique source of inoculum. Since 1989, witches(cid:146) broom disease, caused by Crinipellis, has spread throughout Brazil, destroying cacao tree plantations, and leading to important economical and social changes in specific areas such as the State of Bahia. In order to recover cacao plantations, numerous efforts have been made such as the development of new cacao varieties, use of cloned resistant plant material or biological control of the disease. Although these technical procedures have been efficient, they do not represent an adequate method of control. The variability of the fungus, associated with several possible genetic recombinations, in a short period of time, could break the cacao resistance, as observed for some cacao tree hybrids which contain the SCA-6 resistant parent. For many pathogenic systems in plants, study of gene interaction is a suitable approach to discover plant defense mechanisms. The identification of differential expressed genes between cacao trees susceptible and resistant to witches(cid:146) broom disease is essential to understand biological events of the cacao x Crinipellis interactions. For that purpose we generated two cDNA libraries corresponding to resistant (TSH1188) and susceptible (Catongo) cacao trees inoculated by a pool of Crinipellis perniciosa spores. The sequencing of 10,000 ESTs is completed. Fungus and cacao sequences could be discriminated according to their codon usage using a new bioinformatics tool developed in the LABBI (http://labbi.uesc.br). This algorithm is able to discriminate cacao from Crinipellis sequences with 95% confidentiality. The libraries presented two distinct gene expression patterns, with the susceptible genotype undergoing a programmed cell death triggered by the pathogen and the resistant genotype expressing several defense related genes. We also found, in the susceptible library) key genes expressed by the fungus that are clearly related to the infection success and disease progression. The collections of ESTs obtained are providing new data about the physiology of the interaction between Theobroma cacao and Crinipellis perniciosa. We are currently performing functional analyzes of some candidate genes. Validated genes could also be used for mapping, aiming to facilitate introgression strategies and to create new varieties resistant to Crinipellis perniciosa. These data may also be related to research on gene expression of cacao challenged with other pathogens, such as Phytophthora spp and Monillia. Acknowledgment: Work supported by CNPq, CAPES, FINEP, FAPESB and BNB. BIOMEDICAL SCIENCE: FLAVANOLA AND CARDIOVASCULAR HEALTH COCOA FLAVANOLS, NITRIC OXIDE AND ENDOTHELIAL FUNCTION Norman Hollenberg, M.D., Ph.D., Professor of Medicine, Brigham and Women’s Hospital, Howard Medical School Diet patterns are widely recognized as contributors to hypertension. Widely studied potential contributors include intake of sodium, potassium, magnesium, calcium, soluble fiber, Omega- 3 fatty acids, alcohol, protein, and calories. We add to that list the effect of dietary flavanols present in certain cocoas, which have sufficient activity on vascular nitric oxide to influence blood pressure control. Kuna Indians who live on islands near Panama have little age-related rise in blood pressure or hypertension. On migration to Panama City, blood pressure raises with age and the frequency of essential hypertension matches urban levels elsewhere. We have identified a specific food that probably makes an important contribution to cardiovascular status. Island-dwelling Kuna drink more than five cups of flavanol-rich cocoa per day and incorporate that cocoa into many recipes. Mainland Kuna ingest little cocoa and what they do take is commercially available and flavanol-poor. The flavanol-rich cocoa activates nitric oxide synthase in vitro and in intact humans in the doses that the Kuna employ. Island-dwelling Kuna have a 3-fold larger urinary nitrate: nitrite than do Mainland dwellers. As endothelial dysfunction is central to current thinking on cardiovascular pathophysiology, a food that enhances endothelial function could have broad implications. The list of candidate conditions that might be influenced is impressive, ranging from atherosclerosis and diabetes mellitus to hypertension and pre-eclampsia, to vascular dementias and end-stage renal disease. All share as a common feature endothelial dysfunction with loss of either production or action of nitric oxide. If restoration of nitric oxide production can be achieved by ingestion of flavonoid-rich cocoa, as commonly it can, does this improve natural history? We reduced blood pressure with medications not to make the numbers nicer but because doing so reduces morbidity and mortality. The same logic applies to treatment of high blood cholesterol or a high blood sugar. Does restoration of nitric oxide production improve natural history in these conditions? Only a partial answer is available, but it is very promising. We are at the stage where epidemiology usually plays a crucial role. In the case of flavonoids, efforts to use epidemiology have been limited by the variability in the content of flavonoids in foods, largely representing processing or handling. Grapes grown in the same vineyard have a very different flavonoid content in different years, and so does the red wine produced from these grapes. When an individual eats applesauce or apple pie, he or she rarely knows whether the skin of the apple was included. Essentially all of the flavonoids in apples come from the skin. Flavonoid content in chocolate and cocoa is equally or even more variable. In the island-dwelling Kuna, conversely, cocoa intake is very high, and the cocoa is always rich in flavonoids. Thus, the indigenous Kuna provide an opportunity to address this issue. In data obtained from death certificates, mainland Panama residents who died had disease of the circulation as the most common cause and cancer as a close third. The frequency of these two problems was much lower in the San Blas where the indigenous Kuna live. Indeed, the relative risk of death from heart disease on the Panama mainland was 1,280% of that in the island, and a cancer death was 630%. Thus, the preliminary data available indicate that flavonoid-rich foods may provide an extraordinary benefit in the management of the two most common causes of death in today(cid:146)s world. Cocoa Flavanols: Bioactive Nutrients Beyond Antioxidants? Hagen Schroeter*, Christian Heiss§, Carl L. Keen*, Malte Kelm(cid:135) * Department of Nutrition, University of California Davis, One Shields Avenue, 3150E Meyer Hall, Davis, CA 95616, USA; § Division of Cardiology, University of California San Francisco, 513 Parnassus Ave Room S-1136, San Francisco, CA 94143-0124; (cid:135) Medical Clinic I, Division of Cardiology, Pulmonology, Vascular Medicine, University Hospital RWTH Aachen, Germany Previous studies, as well as epidemiological and medical anthropological investigations suggest that the consumption of flavanol-rich cocoa is associated with cardiovascular health benefits. In order to further investigate the biomedical properties of cocoa we aimed at identifying specific, bioactive cocoa constituents, and at elucidating potential mechanisms that underlie the effects on the cardiovascular system that are associated with certain cocoas. We show that the ingestion of flavanol-rich cocoa leads to enhanced vascular function as demonstrated by an increased flow-mediated dilation (FMD) response of conduit arteries, and an augmented microcirculation. Applying previously established causality criteria we demonstrate that: 1) certain cocoa flavanols are absorbed following ingestion, and the kinetics of their appearance in plasma temporally parallels the vascular effects observed. 2) The circulating concentrations of specific flavanols in plasma are sufficient to mediate ex vivo vasodilation in isolated aortic rings, mimicking a physiological function of the endogenous mediator acetylcholine. 3) The ingestion of pure (-)epicatechin, a flavanol abundant in certain cocoas, by humans closely and quantitatively mimics the vascular effects of flavanol-rich cocoa consumption. 4) The increase in plasma flavanol concentration after consumption of flavanol-rich cocoa is paralleled by increases in plasma levels of the vasodilator nitric oxide (NO). Furthermore, the direct inhibition of the NO-producing enzyme, nitric oxide synthase (NOS), in humans, and in isolated aortic rings, abolishes the vascular effects related to the consumption of flavanol-rich cocoa. 5) The concept that a chronic intake of high-flavanol diets is associated with prolonged, augmented NO synthesis and cardiovascular health is supported by data from the Kuna Indians that indicate a correlation between the chronic consumption of a cocoa flavanol-rich diet and the augmented urinary excretion of NO metabolites. 6) Data obtained in endothelial cell culture models in vitro demonstrate that (-) epicatechin, and (-)epicatechin-related cocoa flavanol metabolites increase cellular NO production by modulating the activity of NOS. In addition, data from in vitro studies, and results obtained following the ingestion of flavanol- rich cocoa by human subjects indicate that the efficacy of cocoa flavanols in the context of cardiovascular function is not solely dependent on antioxidant effects.