Deciphering the genome structure and paleohistory of _Theobroma cacao_

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Abstract

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_ disease resistance and quality 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 a simple living relic of higher plant evolution.

Deciphering the genome structure and paleohistory of Theobroma cacao Xavier Argout*1, Jerome Salse*2, Jean Marc Aury*3, Gaetan Droc1, Jerome Gouzy4, Mathilde Allegre1, Cristian Chaparro5, Thierry Legavre1, Mark Guiltinan6, Siela Maximova6, Michael Abrouk2, Florent Murat2, Olivier Fouet1, Julie Poulain3, Manuel Ruiz1, Yolande Roguet1, Maguy Rodier-Goud1, Jose Fernandes Barbosa-Neto5, Francois Sabot5, Dave Kudrna7, Jetty Ammiraju7, Stephan Schuster8, John Carlson9, Erika Sallet4, Thomas Schiex10, Anne Dievart1, Melissa Kramer11, Laura Gelley11, Zi Sh12, Aurélie Bérard13, Christopher Viot1, Michel Boccara1, Ange Marie Risterucci1, Valentin Guignon1, Xavier Sabau1, Michael Axtell14, Zhaorong Ma14, Yufan Zhang12, Spencer Brown15, Mickael Bourge15, Wolfgang Golser7, Xiang Song7, Didier Clement1, Ronan Rivalan1, Mathias Tahi16, Joseph Moroh Akaza16, Bertrand Pitollat1, Karina Gramacho17, Angélique D’Hont1, Dominique Brunel13, Diogenes Infante18, Ismael Kebe16, Pierre Costet19, Rod Wing7, William McCombie11, Emmanuel Guiderdoni1, Francis Quetier20, Olivier Panaud5, Patrick Wincker3, Stephanie Sidibe-Bocs1, Claire Lanaud1. *These authors contributed equally to this work1 CIRAD - Biological Systems Department – UMR DAP TA A 96/03- 34398, Montpellier, cedex 5- France 2 Institut National de la Recherché Agronomique UMR 1095, 63100 Clermont-Ferrand, France 3 Genoscope (CEA) and UMR 8030 CNRS-Genoscope-Université d'Evry, 2 rue Gaston Crémieux, BP5706, 91057 Evry, France 4 INRA-CNRS LIPM Laboratoire des Interactions Plantes Micro-organismes, BP 52627, 31326 Castanet Tolosan Cedex, France 5 UMR 5096 CNRS-IRD-UPVD, Laboratoire Génome et Développement des Plantes, Université de Perpignan, 52 Avenue Paul Alduy, 66860 Perpignan Cedex, France 6 Penn State University, Department of Horticulture and the Huck Institutes of the Life Sciences, University Park, PA 16802, USA 7 Arizona Genomics Institute and School of Plant Sciences, University of Arizona, Tucson AZ 85721, USA 8 Penn State University, Department of Biochemistry and Molecular Biology, University Park, PA 16802, USA 9 Penn State University, The School of Forest Resources and the Huck Institutes of the Life Sciences, University Park, PA 16802, USA and The Department of Bioenergy Science and Technology (WCU), Chonnam National University, 333 Yongbongro, Buk-Gu, Gwangju, 500-757, Korea 10 Unité de Biométrie et d’Intelligence Artificielle (UBIA), UR875 INRA, F-31320 Castanet Tolosan France 11 Cold Spring Harbor Laboratory, NY 11723, USA 12 Penn State University, Plant Biology Graduate Program and the Huck Institutes of the Life Sciences, University Park, PA 16802, USA 13 INRA, UR 1279 Etude du Polymorphisme des Génomes Végétaux, CEA Institut de Génomique, Centre National de Génotypage, 2, rue Gaston Crémieux, CP5724, 91057 Evry, France 14 Penn State University, Bioinformatics and Genomics Ph.D. Program &Department of Biology, University Park, PA 16802, USA 15 Institut des Sciences du Végétal, UPR 2355, CNRS, 91198 Gif-Sur-Ivette, France 16 Centre national de la recherche agronomique (CNRA), B.P. 808, Divo, Côte d’Ivoire 17 CEPLAC, Km 22 Rod. Ilheus Itabuna, Cx. postal 07, Itabuna 45600-00, Bahia, Brazil 18 Centro Nacional de Biotecnología Agrícola, Instituto de Estudios Avanzados, Caracas 1015-A, Venezuela 19 Chocolaterie VALRHONA, 8, quai du général de Gaulle, 26600 Tain l'Hermitage, France 20 Département de Biologie, Université d'Evry Val d'Essonne, 25 boulevard François Mitterrand, 91025 Evry, France*ABSTRACT 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 disease resistance and quality 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 a simple living relic of higher plant evolution.INTRODUCTION Theobroma cacao L., is a diploid tree fruit species (2n = 2x = 20) 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 farmers . T. cacao was first domesticated by Mesoamerican natives approximately 3000 years ago. Criollo, the first domesticated variety, provides white beans highly appreciated for making fine flavored aromatic chocolate. 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 lived. Our genome sequence is derived from a Belizean Criollo plant collected in the Mayan mountains. Cocoa culture has now expanded to all humid tropical countries, providing 3.7 million of tons of cocoa annually and income for millions of smallscale farmers. However these farmers face increasing threats from fungal diseases and insect pests, which are globally responsible for 30% of harvest losses. 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 We sequenced Theobroma cacao, a long-generation-time tropical tree fruit crop. We were able to assemble 76% of its genome and identify 28,798 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 markerassisted 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 grape. 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 simple 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. DATA ACCESS Data will be released to public access on the date of publication. Sequence reads are hosted at: http://cocoagendb.cirad.fr/gbrowse Additional Project Data: http://cocoagendb.cirad.fr The Theobroma cacao whole genome sequences are deposited in the EMBL/Genbank/DDBJ databases under accession numbers CACC01000001-CACC01025912. Any use of this data should be acknowledged by citation of the published manuscript and through use of the EMBL/Genbank/DDBJ databases accession numbers. ACKNOWLEDGEMENTS We would like to thank CIRAD, the Agropolis foundation, the Région 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-Pyrénées 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 advices and assistance with bioinformatics.*

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Correspondence to Cristian Chaparro or Florent Murat or John Carlson or Valentin Guignon or Mathias Tahi or Emmanuel Guiderdoni or Thierry Legavre or Olivier Fouet or Erika Sallet or Xavier Sabau or Joseph Moroh Akaza or Francis Quetier or Mark Guiltinan or Julie Poulain or Schiex T. or Michael Axtell or Bertrand Pitollat or Siela Maximova or Manuel Ruiz or Anne Dievart or Zhaorong Ma or Karina Gramacho or Patrick Wincker or Yolande Roguet or Melissa Kramer or Yufan Zhang or Angélique D’Hont or Stephanie Sidibe-Bocs or Claire Lanaud or Maguy Rodier-Goud or Laura Gelley or Spencer Brown or Dominique Brunel or Diogenes Infante or Jerome Salse or Jose Fernandes Barbosa-Neto or Zi Sh or Mickael Bourge or Wolfgang Golser or Ismael Kebe or Jean Marc Aury or Francois Sabot or Aurélie Bérard or Christopher Viot or Xiang Song or Pierre Costet or Gaetan Droc or Dave Kudrna or Jetty Siva Ammiraju or Michel Boccara or Didier Clement or Rod Wing or Stephan Schuster or Ange Marie Risterucci or Ronan Rivalan or W. Richard McCombie or Mathilde Allegre.

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Keywords

  • cocoa
  • cacao
  • genome
  • sequence
  • Theobroma cacao
  • Evolution