Deciphering the genome structure and paleohistory of Theobroma cacao

We sequenced and assembled the genome of Theobroma cacao, an economically important tropical fruit tree crop that is the source of chocolate. The assembly corresponds to 76% of the estimated genome size and contains almost all previously described genes, with 82% of them anchored on the 10 T. cacao chromosomes. Analysis of this sequence information highlighted specific expansion of some gene families during evolution, for example flavonoid-related genes. It also provides a major source of candidate genes for T. cacao improvement. Based on the inferred paleohistory of the T. cacao genome, we propose an evolutionary scenario whereby the ten T. cacao chromosomes were shaped from an ancestor through eleven chromosome fusions. The T. cacao genome can be considered as the simplest living relic of higher plant evolution identified to date. INTRODUCTION Theobroma cacao L., is a diploid tree fruit species (2n = 2x = 20; Davie, 1933) that is endemic to the South American rainforests. Its seeds are used in a wide range of products, the most popular being chocolate. There is also an increasing appreciation of its value for environmental preservation because the cocoa tree can be cultivated under forest shade, allowing for land rehabilitation and enrichment of biodiversity, while providing income for many subsistence farmers1 . T. cacao was first domesticated by Mesoamerican natives approximately 3000 years ago2. Criollo, the first domesticated variety, provides white beans highly appreciated for making fineflavored aromatic chocolate (Supplementary Data 1 online). Relics of the ancestral Criollo, first cultivated by Olmec or Mayan people, can still be encountered in old Mesoamerican plantations or in forests where Mayan people lived3. Our genome sequence is derived from a Belizean Criollo plant collected in the Mayan mountains4 (Supplementary Data 1). Cocoa culture has now expanded to all humid tropical countries, providing 3.7 million of tons of cocoa annually (http://www.icco.org/economics/market.aspx) and income for millions of small-scale farmers. However these farmers face increasing threats from fungal diseases and insect pests, which are globally responsible for 30% of harvest losses (http://www.dropdata.org/cocoa/cocoa_prob.htm). Like many other tropical crops, knowledge of T. cacao genetics and genomics is limited. Therefore, to accelerate progress in cocoa breeding and understanding of its biochemistry, we sequenced and analyzed the genome of a Belizean Criollo genotype (B97-61/B2), which has large white beans suitable for producing a high quality and fine-flavored chocolate. This genotype is suitable for high quality genome sequence assembly because it is highly homozygous as a result of the many generations of self-fertilization that occurred naturally during the domestication process. CONCLUSION Theobroma cacao is the first long-generation-time, tropical tree fruit crop that has been sequenced. We were able to assemble 76% of its genome and identify 28,798 N at ur e P re ce di ng s : d oi :1 0. 10 38 /n pr e. 20 10 .4 90 8. 1 : P os te d 15 S ep 2 01 0 protein-coding genes among which 23,529 (82%) could be anchored in the ten cocoa chromosomes. We found that 682 gene families are specific to T. cacao. Only 20% of the genome consisted of transposable elements, a significantly lower percentage than in other genomes of similar size. The analysis of specific gene families that are potentially linked to cocoa qualities and disease resistance, two important traits for cocoa consumption and cultivation, revealed that particular expansion or reduction of some gene families had occurred during evolution. The mapping of these gene families along the cocoa chromosomes and comparison with the genome regions involved in trait variation (QTLs) constitutes an invaluable source of candidate genes for further functional studies that aim to discover the specific genes directly involved in trait variation. This genome sequence will facilitate a better understanding of trait elaboration and will accelerate T. cacao selection through efficient marker-assisted selection and exploitation of genetic resources. This study has highlighted the close evolutionary distance of the T. cacao genome from the eudicot putative ancestor, showing a limited number of recombinations between ancestral chromosomes, as was also observed in grape7. T. cacao, which has only ten pairs of chromosomes, is easily propagated by both sexual and vegetative methods, and can be transformed; it represents a new and perhaps the simplest model to study evolutionary processes, gene functions and tree fruit crop genetics and biochemistry. The large amount of information generated by this project dramatically changes the status of this tropical plant and its potential interest for the scientific community. We hope this situation will encourage greater investment in research with Theobroma cacao, the "food of the Gods" whose magic flavor has spread worldwide since the time of the Maya and Aztec civilizations, and whose continued study will benefit developing countries for which cocoa is of high economic importance. Acknowledgements We would like to thank CIRAD, the Agropolis foundation, the Region Languedoc Roussillon, Agence Nationale de la Recherche (ANR), Valrhona and the Venezuelan Ministry of Science, Technology and Industry for their financial contribution to this project. We thank the Toulouse Midi-Pyrenees bioinformatic platform for providing us with computational resources. Activities at The Pennsylvania State University were supported by a gift from the Hershey Corp., by funds from the American Cocoa Research Institute Endowment, and through support from the Schatz Center for Tree Molecular Genetics in The School of Forest Resources. Acquisition of an Illumina sequencer by the CSHL was supported by National Science Foundation grant DBI0923128 to WRM. We would like to thank F. C. Baurens, Y. Jiao, O. Garsmeur and C. dePamphilis for helpful advice and assistance with bioinformatics. N at ur e P re ce di ng s : d oi :1 0. 10 38 /n pr e. 20 10 .4 90 8. 1 : P os te d 15 S ep 2 01 0