Functional Genomics of Cacao

Functional Genomics of Cacao

FABIENNE MICHELI,*,{,1 MARK GUILTINAN,{ KARINA PERES GRAMACHO,} MIKE J. WILKINSON,¶ ANTONIO VARGAS DE OLIVEIRA FIGUEIRA,k ´ LIO CE´ZAR DE MATTOS CASCARDO,{ JU SIELA MAXIMOVA{ AND CLAIRE LANAUD*

*Cirad, UMR DAP, Avenue Agropolis TA96/03, Montpellier cedex 5, France { UESC, DCB, Laboratrio de Genoˆmica e Expressa˜o Geˆnica, Rodovia Ilhe´us-Itabuna, Ilhe´us-BA, Brazil { The Department of Horticulture, The Pennsylvania State University, University Park, Pennsylvania, USA } CEPLAC/CEPEC, Cacao Research Center, Molecular Plant Pathology Laboratory, Itabuna-BA, Brazil ¶ Institute of Biological, Environmental & Rural Sciences, Aberystwyth University Penglais, Aberystwyth, Wales, United Kingdom k Centro de Energia Nuclear na Agricultura, Universidade de Sa˜o Paulo, Avenida Centena´rio, Piracicaba-SP, Brazil

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Molecular Resources for Genomics Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. EST Resources ............................................................... B. Genome Sequencing Projects .............................................. C. BAC Library Resources ....................................................

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Corresponding author: Email: [email protected]

Advances in Botanical Research, Vol. 55 Copyright 2010, Elsevier Ltd. All rights reserved.

0065-2296/10 $35.00 DOI: 10.1016/S0065-2296(10)55003-1

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III. Genomics of Cacao Under Natural Conditions, Submitted to Mechanical Wounding or Challenged with Elicitors. . . . . . . . . . . . . . . . . . . . . . IV. Omics of the Cacao–M. perniciosa Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . A. Genomics of M. perniciosa ................................................. B. Genomics of the Cacao–M. perniciosa Interaction ..................... C. Proteomics of Cacao and M. perniciosa.................................. V. Genomics of the Cacao–Phytophthora Interaction . . . . . . . . . . . . . . . . . . . . . . . A. Phytophtora NEP1 Orthologues and Their Effects on Cacao ........ B. Genes Differentially Expressed During the Cacao–Phytophthora Interaction .................................................................... VI. Cacao Gene Expression Under Other Biotic and Abiotic Conditions. . . . A. Genomics of Cacao–Endophytic Interaction............................ B. Endophytic Colonisation of Cacao and Drought ...................... C. Regulation of PA Biosynthesis in Cacao ................................ VII. Gene Expression Related to Cacao Quality Flavour . . . . . . . . . . . . . . . . . . . . . VIII. In Vitro Culture and Genetic Transformation, and Their Applications in Functional Genomics Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Genetic Transformation of Cacao ........................................ B. RNA Interference in Cacao................................................ C. Model Plant Systems for Acceleration of Cacao Functional Genomics Studies............................................................ D. Characterisation of Leafy Cotyledon1-Like During Embryogenesis in Cacao.......................................................................... IX. Epigenetics and Regulation of the Cacao Genome . . . . . . . . . . . . . . . . . . . . . . . X. Molecular Genetic Studies of Important Cacao Traits as Support for Genomic Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Plant Genetic Resources.................................................... B. Molecular Markers .......................................................... C. Genetic Maps for QTLs and Association Studies ...................... XI. Bioinformatic Resources for Cacao Functional Genomics . . . . . . . . . . . . . . . XII. Cacao Genetics Research Community . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XIII. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgement. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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ABSTRACT Cacao (Theobroma cacao L.) is one of the main tropical crops worldwide. It is cultivated primarily to provide cacao liquor, butter, and powder for the chocolate industry, essentially due to its flavour properties. Unfortunately, destructive and newly encountered diseases have frequently been the major factors that limit cacao production in nearly all producing countries. The primary challenge is to develop improved cacao tree cultivars with durable and sustainable resistance to these diseases that at the same time have high bean quality for chocolate production. To achieve this goal, the use of functional genomics can be a key step to speed the development of such cultivars. During the last 10 years various functional genomics and some proteomic projects have been initiated, including expressed sequence tag and BAC libraries construction, cacao genome sequencing, expression studies of cacao tissues challenged with the main pathogens (Moniliophthora perniciosa, Phytophthora spp.) or subjected to other stress conditions (e.g. drought), and expression studies related to cacao quality flavour. Various others tools such as cacao in vitro culture, plant

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transformation or bioinformatics were developed to attend the necessity of cacao studies. Functional genomics research associated with genetics (molecular markers, maps and QTLs) will help to select new cacao varieties with agronomic characteristics demanded by farmers.

I. INTRODUCTION Theobroma cacao L. (cacao), a member of the Malvaceae sensu lato (Alverson et al., 1999), occurs naturally in Neotropical lowland rainforests as a small under-storey tree. The genus Theobroma contains 22 species classified into 6 sections, most native to the upper Amazon region in South America (Cuatrecasas, 1964), whereas only T. cacao and T. grandiflorum (cupuassu) are explored commercially on a large scale. T. cacao is preferentially outcrossing, diploid (2n ¼ 2x ¼ 20) and has a genome which was first estimated at 0.43 pg or 0.415  109 bp (Figueira et al., 1992) and at 0.40 pg or 0.388  109 bp (Lanaud et al., 1992). More recent evaluations inside T. cacao sp. indicated a range of genome size variations from 411 to 494 Mbp (Lanaud et al., in preparation). From the Amazon, cacao was initially introduced by ancient people into Meso-America (Motamayor et al., 2002), while after the Spanish Conquest, the species was spread to the Caribbean, West Africa and Southeast Asia. The traditional classification of cacao assumes three major groups with distinct historical, commercial and morphological features: the Forastero or the Amazon group, the ancient cultivar Criollo (‘native’), and the Trinitario (from Trinidad) group; presumably derived from crossings between the Forastero and Criollo types (Motamayor et al., 2002, 2003). Forastero genotypes are traditionally cultivated in Brazil and West African countries, and represent most of the commercial production of cacao. Criollos were originally cultivated in small areas of Central and northern South America (Wood and Lass, 1985). Cacao produces fruits (pods) along the trunk and branches (Fig. 1A) that contain an average of 20–40 seeds (also known as cocoa beans) embedded in sweet, mucilaginous pulp (Fig. 1H). Cacao beans are usually commercialised after a preliminary on-farm processing, which includes fermentation and drying (Figueira, 2008; Fig. 1I–K). Cacao butter and solids, including cacao powder and liquor, are the main products extracted from fermented and dried seeds, providing major raw materials for the chocolate, confectionary, cosmetic or pharmaceutical industries. Economically, cacao is considered as one of the main tropical crops worldwide, with a total bean production of 4,012,310 tonnes in 2007 (FAO, 2009). The fermentation step is necessary for full development of chocolate/cocoa flavour and aroma after roasting. The flavour precursors derive from enzymatic reactions

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Fig. 1. From the cacao tree to chocolate. (A) Cacao tree with pods. (B) Flower cushion. (C) Cacao flower. Real size: 1 cm. (D) Forastero pods. (E) Cherels. Real size: 5 cm. (F) and (G) Criollo pods. (H) Cacao beans and pulp inside of pod. (I) Fermentation boxes. (J) Cacao bean dryer (barcasse type). (K) Fermented and dried cacao beans. (L) Cacao bean conditioning in bags. (M) Chocolate powders. (N) Chocolate bite. Photographic credits: (A), (C) and (K): Didier Cle´ment#Cirad; (B) and (H): Claire Lanaud#Cirad; (D), (F), (G), (J), (L) and (N): chocolatitudes.com.; (E): Laurence Alemanno#Cirad; (I): Emile Cros#Cirad; (M): Barry Callebaut, (D) R. From Micheli (2009).

involving hydrolysis of storage proteins (mainly vicilins), sugars, anthocyanins, purine alkaloids, and oxidation and condensation of polyphenols. The three main groups of cacao are distinguishable by the pod morphology (Fig. 1D–G). The Criollo group produces large pods with rough husk (Fig. 1F and G), containing white or violet cotyledons. Criollo is one of the two cacao varieties providing fine chocolate flavour highly sought for by chocolate

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manufacturers. Criollo is considered as a grand cru of chocolate such as in wine classification. The culture of Criollo represents an important economic niche for several countries like Venezuela, which has established the development of Criollo cultivation as a priority. However, Criollo is susceptible to many fungi diseases and insect attacks, and through time, its aromatic qualities have been diluted, because of various crossings with genotypes of various genetic origins, leading to hybrids with higher disease resistance (Motamayor et al., 2003). The current challenge is to cultivate high productive and resistant hybrid Criollo varieties, maintaining the original aromatic qualities. Forastero produces pods with highly variable shapes and number and size of seeds, which are generally violet (Rosa´rio et al., 1978; Fig. 1D). Within Forastero, considered as the group with the highest genetic diversity and better agronomic quality than Criollo, there is an Amelonado variety (oval pod with smooth bark; Fig. 1D) named cacao Comun da Bahia and cultivated in large areas in Brazil. Catongo is an albino mutant, from the Forastero group, with white staminodes and seeds that has originated from Bahia (Brazil) (Marita et al., 2001). Trinitario is a group with characteristics depending on the repartition and effects of alleles from the two founding groups (Forastero and Criollo). During the colonial period, most of the cacaos introduced in Africa and Asia were originated from Venezuela, Trinidad and Brazil (Wood, 1991), and corresponded, respectively, to Criollo, Trinitario and Amelonado cultivars. Destructive and newly encountered diseases have frequently been the major factors that limit cacao production in nearly all producing countries (Bowers et al., 2001). Besides fluctuations in production, important disease outbreaks have eliminated or strongly limited cacao cultivation in many tropical regions throughout the world. Cacao diseases represent an important factor in the economy of this crop, not only due to crop losses but also due to the high cost of control practices. Cacao trunk, branch, foliage, roots and pods may be affected by diseases. Cacao diseases are caused mainly by fungi (oomycetes such as Phytophthora spp., ascomycetes such as Ceratocystis cacaofunesta and basidiomycetes such as Moniliophthora roreri Cif. & Par and Moniliophthora perniciosa (Stahel) Aime & Phillips-Mora, among others); a single virus disease is known (cocoa swollen shoot virus, CSSV) with occurrence restricted to West Africa. Cacao is not affected by any serious bacterial disease. Cacao stems are also attacked by pests such as mirids (Sahlbergella singularis) or pod borer (Conopomorpha crammerella). On a global scale, pod diseases cause the greatest losses. Species of Moniliophthora and Phytophthora can reduce yields by up to 80–90% in some regions, resulting in abandonment of many production areas around the world.

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Phytophthora pod rot has been the primary fungal disease affecting global cacao production since the 1920s, causing pod losses in the order of 30% of the world production (Pereira et al., 1996). A high variability for resistance to Phytophthora pod rot has been described (Iwaro et al., 2006; Lawrence, 1978; Luz et al., 1996). Limited to South America and the Caribbean, witches’ broom disease, caused by M. perniciosa, is a severe constraint to cacao production and it has been responsible for the collapse of the cacao industry in Surinam (1900s), Trinidad, Ecuador (1920s), and more recently, in Brazil (1990s). Breeding for resistance started in the 1930s in Trinidad and Scavina 6 (SCA6), a Forastero from Upper Amazon (Peru) has been widely deployed in many programs as one major source of resistance. Genomic regions associated with M. perniciosa resistance (quantitative trait loci—QTLs) have also been identified (Brown et al., 2005; Faleiro et al., 2006; Queiroz et al., 2003). However, this resistance has been often overcome, posing a continuous demand for novel sources (Albuquerque et al., 2010). Frosty pod disease caused by M. roreri occurs in most producing countries in the Americas (Bolivia, Peru, Ecuador, Colombia, Venezuela, Panama, Costa Rica, Nicaragua and Mexico). Losses inflicted by frosty pod can be as high as 90% (Barros, 1977). Sources of resistance to frosty pod have been found (e.g. UF 273 cacao genotype) and QTLs for resistance have been identified (Brown et al., 2007), and it appears that the disease co-evolved with cacao in some regions of Colombia (Phillips-Mora et al., 2007). Fortunately, to date, the major devastating cacao pathogens have a restricted distribution. Phytophthora palmivora has a pantropical distribution, while P. megakarya, the most aggressive species, is confined to several countries of West Africa, and P. capsici occurs only in South and Central America and the Caribbean (Brasier and Griffin, 1979). There are two other Phytophthora species: P. heveae causing pod rot in Malaysia (Turner, 1968) and Mexico (LozanoTrevino and Romero-Cova, 1974) and P. megasperma Drechsler in Venezuela (Reyes et al., 1972). Another species, P. citrophthora, has been identified in Bahia, Brazil. P. citrophthora is more virulent on unwounded, detached pods than P. palmivora or P. capsici (Lawrence, 1978). The CSSV virus is confined to West Africa. M. roreri and M. perniciosa occur only in the Americas. Unfortunately, the various methods of disease control (chemical, biological, cultural) available to farmers are difficult to be applied for significantly reducing losses, either because of limited effectiveness or high cost. Thus, there is an urgent demand for the development of improved cultivars with durable and sustainable resistance to these diseases. Resistance is the method of choice as it is both economically and environmentally safe. The use of molecular biology can be a key step to speed up the development of resistant cultivars.

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The advent of genome sequencing has revolutionised the biology field, leading to a paradigm change in the way to conduct science. The advent of genome sequencing in the 1990s has been quickly followed by technological innovations, collectively known as Omics, which allow large-scale analysis of biomolecules present in the cell, including mRNA (transcriptomics), proteins (proteomics) and metabolites (metabolomics). Omics appeared as fundamental approaches in the post-genomic era to amplify the knowledge of biological processes at organism level by determining gene–protein– metabolite correlations. Genomics, defined as sequences and entire genome studies, is the most mature Omics. Transcriptomics research gives information about both presence and abundance of transcripts, highlighting the active genes of the cell. Macro- and micro-arrays (Hardiman, 2004) represent the approaches mostly used in a large variety of organisms. Even if these studies give crucial information related to cell expression stage, they do not evidence the various levels of post-transcriptional control (Mata et al., 2005). Proteomics research aims to identify and quantify the protein level in the cell based on two-dimensional (2D) electrophoresis and mass spectroscopy (Patterson and Aebersold, 2003). Described in this review are various cacao functional genomic and some proteomic initiatives that have emerged during last 10 years.

II. MOLECULAR RESOURCES FOR GENOMICS STUDIES A. EST RESOURCES

Genes potentially associated with resistance and/or defence response have been sought from expressed sequence tag (EST) collections, derived from unchallenged leaves and seeds (Jones et al., 2002; see Section III), from leaves treated with response elicitors such as ethylene, methyl jasmonate, and the fungal necrosis and ethylene inducing protein 1 (NEP1) (Verica et al., 2004; Bailey et al., 2005a,b; see Section III). Full-length cDNA and subtractive and suppressive hybridisation (SSH) libraries were also obtained from distinct cacao accessions infected by M. perniciosa. The studied accessions were TSH1188 (derived from SCA6) and CAB 214 as M. perniciosa as resistant, and Catongo and ICS 39 as susceptible (Gesteira et al., 2007; Leal et al., 2007). The work of Gesteira et al. (2007) and Leal et al. (2007) included the first efforts in sequencing of the expressed genome in response to M. perniciosa infection (see Section IV.B). More recently, an international

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collaboration was established to develop a large EST genomic resource from a wide range of cacao organs (flowers, leaves, roots, meristems, embryos) during normal plant development or submitted to biotic and abiotic stresses (Argout et al., 2008). For this study, 56 cDNA libraries (full-length and SSH) were constructed from two main genotypes representing mainly three contrasting genetic origins: ICS1 (a hybrid between Criollo and Forastero from Lower Amazon of Brazil) and SCA6 (a Forastero from Upper Amazon of Peru). A few other genotypes belonging to various genetic origins and characterised by specific resistance or quality traits were also included. Twenty five libraries corresponding to tissues subjected to different biotic stresses were obtained: pods inoculated with P. palmivora, P. megakarya, M. perniciosa and M. roreri; leaves inoculated with P. palmivora and P. megakarya; stems inoculated with M. perniciosa and C. cacaofunesta; and stems attacked by mirids. Two libraries corresponding to cacao tissues submitted to drought stress, and 11 corresponded to seed development and fermentation stages, were also obtained. The sequencing was conducted using Sanger methodology. A total of 149,650 valid ESTs were generated corresponding to 48,594 unigenes (12,692 contigs and 35,902 singletons). A total of 29,849 unigenes (61.4%) shared significant homology with public sequences from other species. Surprisingly, although the evolutionary distance between Vitis vinifera and cacao was higher than between Arabidopsis and cacao (Zhu et al., 2007), more similarities between the cacao sequences and V. vinifera ones were identified than between cacao and Arabidopsis. Indeed, among the 25,049 cacao sequences (56%) presenting at least one significant hit with an Arabidopsis or V. vinifera sequence, 18,643 cacao sequences showed similarity with V. vinifera, while only 6406 with Arabidopsis, in spite of fewer sequences from V. vinifera than from Arabidopsis present in the non-redundant database used for the analysis. One explanation suggested by the authors was the fact that V. vinifera and cacao are both ‘tree crops’. The large amount of hits (8605) found with Populus trichocarpa, another tree crop (despite the small number of nonredundant proteins available for this species) also supports this hypothesis. The Argout et al. (2008) initiative also provided an important resource to study plant–pathogen interactions by identifying 1001 sequences classified as ‘response to stress’ with AmiGO browser, or similar to known proteins involved in resistance or defence mechanisms such as Leucine Repeat Region-Nucleotide-Binding Site (LRR-NBS), chitinase, serine–threonine kinase or pathogenesis-related (PR) protein. Similarly, a large representation of genes potentially involved in the different metabolic pathways related to cacao qualities (flavonoids, terpenes, purines, fat and sugars) was observed in this collection. Because of theimportance of flavonoids and terpenes in cacao

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flavours, and the presence, in this cacao EST collection, of all the genes encoding the enzymes known to be involved in the flavonoids and terpenes pathways, these two metabolic pathways were more precisely analysed using micro-arrays (see Section VII). The different public EST sets contribute, in complementary ways (genotypes and methods used to obtain the libraries), to increase the genomic resources necessary to understand the cacao molecular biology, to develop genetic approaches and functional studies related to important agronomic or economic traits. Large-scale expression study using macro- and/or micro-arrays—generally associated to real-time reverse transcription polymerase chain reaction (RT-qPCR) validation—constitute the first step towards functional genomics. One of the approaches consists on developing thematic arrays containing selected genes related to a given biological process such as resistance/defence mechanisms (see Sections IV.B and V.B), gene regulation (see Section IV.B), or cacao flavour (see Section VII). These EST sets also constitute a valuable resource to provide genetic markers (simple sequence repeats (SSRs), single nucleotide polymorphisms (SNPs)) defined in genes (see Section X.B). B. GENOME SEQUENCING PROJECTS

Two genome sequencing projects are currently underway and will be the basis for new functional genomic studies. One of them, coordinated by the Centre de Cooperation Internationale en Recherche Agronomique pour le De´veloppement (Cirad, France) team, is focused on a Criollo variety (Lanaud, 2009; Lanaud et al., 2008). The Criollo variety has been chosen because of its aromatic qualities. The pure and homozygous Criollo material is rarely available. However, some clones were collected in Belize (Mooleedhar et al., 1995) and are available in the International Collection of Cacao Research Unit (CRU, University of the West Indies, St. Augustine, Trinidad and Tobago; see Section X.A). The sequencing strategy proposed by the Cirad consortium relies on a whole-genome shotgun approach of the ‘B97-61/B2’ Criollo genotype, combining Sanger BAC End Sequencing (BES) with 454 Roche and Solexa nuclear sequencing. A high-density genetic map will allow anchoring the scaffolds produced. The second genome sequencing project is coordinated by a MARS/United States Department of Agriculture (USDA) team and aims to sequence the genome of a Forastero cultivated genotype (‘Matina 1-6’), originated from Brazil (Motamayor et al., 2009). This genotype is the second ancestor of the Trinitario trees (hybrid between Criollo and Forastero) (Motamayor et al., 2002, 2003), widely cultivated worldwide. The chosen genotype is also nearly completely homozygous. The sequencing strategy relies also on a

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whole-genome shotgun approach, combining Sanger BES with 454 Roche and Solexa nuclear sequencing. A physical map based on three new BAC libraries (see Section II.C) and on a genetic map will allow to ordinate and orientate the scaffolds produced. The availability of the cacao genome sequence will accelerate the discovery of candidate genes underlying the QTL identified in previous genetic studies and in relation to functional genomics. The genome sequencing of both contrasting Criollo and Forastero genotypes, originated from distinct genetic groups, will allow to produce a wide SNP resource useful for all genetic and genomics studies. C. BAC LIBRARY RESOURCES

Initially, two cacao Bacterial Artificial Chromosome (BAC) libraries were constructed. A first library was obtained from SCA6 and contains approximately 11 genome equivalents, with an average insert size of 120 kb (Cle´ment et al., 2004). A second BAC library was created in the frame of collaboration between the USDA Subtropical Horticultural Research Station (Miami, Florida, USA) and the Clemson BAC Resource Center (University of Clemson, South Carolina, USA), which also distributes this resource (http://www. genome.clemson.edu). This library obtained from ‘LCT-EEN37’ genotype, collected in Ecuador, represents approximately 11 genome equivalents with an average insert size of 120 kb. Other BAC libraries were recently constructed to support the two genome sequencing projects under progress (see Section II.B). For this purpose, a Criollo genotype from Belize (‘B97-61’) and a Forastero (‘Matina 1-6’) from lower Amazon were used. The clones from the three BAC libraries constructed with the ‘Matina 1-6’ clone have been fingerprinted to establish a cacao physical map (Motamayor et al., 2009).

III. GENOMICS OF CACAO UNDER NATURAL CONDITIONS, SUBMITTED TO MECHANICAL WOUNDING OR CHALLENGED WITH ELICITORS Jones et al. (2002) undertook a cacao gene-discovery programme and demonstrated its use in gene-expression arrays. Sequencing and assembling bean and leaf cDNA library inserts produced a unigene set of 1380 members, whose 75% were annotated. This study was the first to identify the types of gene expressed in cacao seeds and leaves, and to analyse gene expression in these organs from five cacao varieties using micro-arrays.

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Verica et al. (2004) employed SSH libraries, macroarray hybridisation analysis, high-throughput DNA sequencing and bioinformatics to identify cacao genes induced by signalling molecules such as salicylic acid, jasmonic acid and ethylene, known to induce resistance to disease in other plant species. A unigene set of 1256 members, including 330 members representing genes induced during the defence response, was identified. In a subsequent work, Bailey et al. (2005b) characterised the responses of cacao to wounding and to a treatment by ethylene and methyl jasmonate. The authors reviewed the effects of jasmonate and ethylene, which are able to induce signal transduction pathways leading to resistance to plant–pathogens and insects. On the other hand, wounding itself can induce the formation of jasmonate and ethylene. The gene expression was analysed by Northern blot in response to the three treatments (wounding, jasmonate and ethylene) applied at different stages of leaf development of cacao seedlings. Differential expression was observed for putative genes encoding a DNA-binding protein (TcWRKY-1), a protein regulating cell division (TcORFX-1), a type III peroxidase (TcPer-1), a endo-1,4-beta-glucanase (TcGlu-1), a class VII chitinase (TcChiB), a caffeine synthase (TcCaf-1) and a light harvesting complex protein (TcLhca-1). The induction of gene expression varies according to the genes considered and according to the delay after wounding or ethylene/ methyl jasmonate treatment. TcWRKY-1 and TcORFX-1 are rapidly induced, and expressed 15 min after the beginning of the wounding treatment. There was evidence of genetic crosstalk between the actions of ethylene and methyl jasmonate on gene expression in cacao leaves, with both synergistic and antagonistic interactions.

IV. OMICS OF THE CACAO–M. PERNICIOSA INTERACTIONS A. GENOMICS OF M. PERNICIOSA

Due to the impact of the introduction of the witches’ broom disease in southern Bahia, Brazil and the limited biological knowledge of the pathogen, a full genome sequencing project of M. perniciosa, involving several Brazilian institutions (www.lge.ibi.unicamp.br/vassoura) was initiated in 2000 (Mondego et al., 2008). The pathogen contains a 28-Mbp genome organised in 8 chromosomes, with around 8000 genes predicted. The analysis allowed a general overview of the M. perniciosa genome, and highlighted a number of important genes involved in stress adaptation and plant necrosis induction (two of the steps necessary for the life cycle of a hemibiotrophic fungus), and

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genes associated with traits that may play a role in pathogenesis mechanisms (Mondego et al., 2008). Other complementary M. perniciosa genomic information previously available included: (i) analysis of the M. perniciosa karyotype (Rincones et al., 2003); (ii) analysis of genetic and chromosomal variation among 38 isolates of biotype-C, -S and -L, sampled from various regions of Brazil and Ecuador (Rincones et al., 2006); (iii) molecular epidemiological studies revealing that the fungus may be adapted to and overcome SCA6 resistance (Gramacho et al, 2008; Pires, 2003) and (iv) sequence of the mitochondria M. perniciosa genome (Formighieri et al., 2008). In parallel, a comparative transcription analysis between biotrophic and saprophytic M. perniciosa phases was conducted (Rincones et al., 2008). Genes specific to each life-stage were identified (e.g. oxaloacetate acetylhydrolase in biotrophic phase), as well as putative virulence genes (e.g. glucuronyl hydrolase; putative chitinase) and transposons (induced in the biotrophic phase), which suggested that the activation of the different types of transposable elements may be regulated through the fungal life cycle (Rincones et al., 2008). A non-normalised cDNA library from various fruiting stages of M. perniciosa was constructed and analysed to elucidate gene function and regulation associated with basidiocarp formation and development (Pires et al., 2009). A macroarray prepared with 192 selected clones from this library was hybridised with two RNA pools from mycelium at distinct basidiocarp formation phases. It was observed that genes coding for hydrophobin, glucose transporter, Rho-GEF, Rheb, extensin precursor and cytochrome p450 monooxygenase were up-regulated in primordial phases of development, while others, such as calmodulin, lanosterol 14 alpha demethylase and PIM1 were down-regulated. The macroarray data were validated by RT-qPCR. Glucose transporter gene expression increased in mycelium after water stress, coinciding with a decrease of adenylate cyclase gene transcription, suggesting that nutrient uptake may be an important signal to trigger fruiting in this fungus (Pires et al., 2009). Based on these genomic data, some genes were chosen for further functional analyses, including two genes encoding NEP (MpNEP1 and MpNEP2), identified in the genome, as well as in the cacao–M. perniciosa interaction library in the case of MpNEP2 (Gesteira et al., 2007). MpNEP1 and MpNEP2 proteins, expressed in the bacterial system, purified and infiltrated in tobacco leaves or in cacao meristems, produced a localised necrosis and induced ethylene production (Garcia et al., 2007). Ethylene production has been associated with hypertrophy (Orchard et al., 1994), chlorophyll degradation, and petiole, leaf and stem epinasty (Woodrow et al., 1989); all symptoms observed during the infection of cacao by M. perniciosa (Scarpari et al., 2005; Silva et al., 2002). MpNEP1 and MpNEP2 transcription analysis

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indicated that MpNEP2 was mainly expressed in biotrophic mycelium, whereas MpNEP1 was expressed in biotrophic as well as saprotrophic mycelium (Garcia et al., 2007). These gene products, in particular from MpNEP2, may act as elicitor of necrosis observed in cacao during infection (Ceita et al., 2007). Another family of possible elicitors expressed by M. perniciosa was described by Zaparoli et al. (2009): at least five sequences encoding putative proteins similar to cerato-platanin (CP)-like proteins were identified in the fungal genome. The MpCP1 gene was expressed in vitro and the corresponding protein showed ability in inducing necrosis in tobacco and cacao leaves. Transcription analysis ex planta showed that MpCP1 was more expressed in monokaryotic mycelium than in dikaryotic one and that the provoked necrosis was different from the one caused by MpNEPs. Moreover, a mixture of MpCP1 with MpNEP2 led to a synergistic necrosis effect, highly similar to the one occurring in naturally infected plants (Zaparoli et al., 2009). Functional analysis of potentially pathogenesis-associated genes may be facilitated by gene silencing using dsRNA. Transfection of an in vitro synthesised gfpdsRNA successfully silenced a reporter gfp gene, stably introduced into the M. perniciosa genome (Caribe´ dos Santos et al., 2009). Similarly, the endogenous genes coding for hydrophobins and a peroxiredoxin were also silenced by transfection with specific dsRNA, indicating that this method can be used to assess gene function in this pathogen (Caribe´ dos Santos et al., 2009). Genomic studies and gene identification from M. perniciosa may be also used for a further understanding of fungal life cycle and/or for biotechnological applications. Souza et al. (2009) identified and characterised, from basidiocarp and second mycelium stages of M. perniciosa, the first class III chitin synthase, which may play an important role in basidioma formation. DNA and RNA polymerases of M. perniciosa mitochondrial plasmid were completely sequenced and their structural models were carried out by comparative homology approach (Andrade et al., 2009). Because plasmid insertions into host mitochondrial genomes are probably associated with modifications in host generation time, which can be involved in fungal aging, such polymerases can be used as new targets for drugs against mitochondrial activity of fungi (Andrade et al., 2009). In the same way, biochemical analysis, modelling and crystal structure of M. perniciosa acyl-coA binding protein (ACBP) may help to understand M. perniciosa cell organisation (Monzani et al., 2010). In other organisms, such as Saccharomyces cerevisiae, ACBP is a critical protein involved in growth and changes in vacuoles and plasma membrane, vesicular trafficking, organelle biogenesis and membrane assembly, among other.

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The genomics of the cacao–M. perniciosa interaction has been investigated using two types of libraries; full-length cDNA (Gesteira et al., 2007) and SSH (Leal Jr et al., 2007). Gesteira et al. (2007) developed two meristems cDNA libraries, both inoculated with M. perniciosa, one from resistant cacao genotype TSH1188 (RT library), another from the susceptible genotype Catongo (SP library). A total of 6884 ESTs were obtained, corresponding to 2926 nonredundant sequences (2585 singletons plus 341 contigs). The redundancy of the RT and SP libraries was low, while their specificity was high when compared with other cacao cDNA libraries previously published (Jones et al., 2002; Verica et al., 2004; see Section III). Sequence analysis allowed the assignment of a putative functional category for 54% of sequences. Despite the overall similar distribution of the sequences in functional categories between the two libraries, qualitative differences were observed. The TSH1188 unigene presented a high amount of potential resistance genes (e.g. PR proteins), while the Catongo unigene presented numerous genes related to programmed cell death (PCD). Leal et al. (2007) constructed two SSH libraries from inoculated meristems collected from the resistant CAB 214 accession and the susceptible ICS39, subtracting common transcripts in both directions: 104 and 187 unique sequences were obtained, respectively, from each of these two libraries. Twenty three genes related to resistance or defence mechanisms were analysed for subsequent validation by RT-qPCR. From the 23 transcripts, 21 were induced in the resistant genotype CAB. From these, 14 were present at both early accumulation stage (48–72 h after inoculation—hai) and late accumulation stage (120–240 hai). Seven transcripts (short vegetative phase, two peroxidases, caffeine synthase, anthocyanin reductase, leucoanthocyanidin dioxygenase, cytochrome oxidase P450 and EIG7) appeared only at early accumulation stage. More generally, most of the 23 genes seemed to be up-regulated by pathogen inoculation in the evaluated genotypes, but differed for induction kinetics. The resistant CAB displayed a stronger induction at 48 and 72 hai for some of the genes evaluated, while in the susceptible ICS 39, a peak of transcript accumulation occurred only later (at 120 and/or 240 hai). Qualitative differences for specific transcripts between two resistant genotypes tested (CAB 214 and CAB 208) were also observed. From the 23 genes evaluated, only 16 were induced in the susceptible genotype, while 21 were induced in the resistant one (Leal et al., 2007). The results obtained with cacao meristems infected by M. perniciosa were compared with those obtained with healthy plants (leaves and beans; Jones et al., 2002) or plants treated with plant defence inducers (Verica et al., 2004).

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The full-length cDNA libraries, as well as the SSH, showed specific sequences related to M. perniciosa infection, which did not match to sequences from the libraries published by Verica et al. (2004) and Jones et al. (2002). The RT library showed 34.2% and 38.6% of sequences different from plants treated with defence inducers and healthy leaves and beans, respectively. According to Leal et al. (2007), from the 127 sequences compared with the cacao genes available in databank, 51 presented positive matches, and from these 36 were different from the library of induced defence genes (Verica et al., 2004); 25 were different from the leaf and seed EST library (Jones et al., 2002), whereas 41 were different from both libraries. In cacao–M. perniciosa interaction studies, special emphasis was given to cDNA sequences related to resistance and to necrosis, and death of infected tissues as probable components of the defence and susceptibility reactions occurring in cacao after infection by M. perniciosa. The search for resistance gene candidates identified sequences implicated in pathogen detection (e.g. Cf9 protein, receptor kinase, RGC2), signal transduction (e.g. MAP kinase, calmodulin-binding protein), regulation events (e.g. transcription factors— TFs), and defence (e.g. peroxidase). In particular, Lopes et al. (2010) focussed on cacao TFs by developing a macroarray with 88 TF cDNA from interaction libraries (Gesteira et al., 2007). Seventy-two TFs were found differentially expressed between the susceptible (Catongo) and resistant (TSH1188) genotypes and/or during the disease time course—from 24 hai to 30 days after infection (dai). Most of the TFs differentially expressed belonged to bZIP, MYB and WRKY families, and presented opposite expression patterns in susceptible and resistant cacao–M. perniciosa interactions. The results of the macroarray were confirmed by RT-qPCR for bZIP and WRKY TFs (Lopes et al., 2010). On the other hand, SVP (short vegetative phase), which shared similarity with a Populus tomentosa MAD-Box transcription factor, was up-regulated in resistant CAB plants (Leal et al., 2007). The presence of apoptosis and oxidative burst-related genes (apoptosis inhibitor, senescence-associated protein, genes related to oxidative burst) in the SP inoculated library (Gesteira et al., 2007) strengthens the hypothesis that the susceptible cacao–M. perniciosa interaction involves in PCD, initially occurring in the plant as a defence mechanism, which then is diverted by the fungus for its own profit, allowing its sporulation and further propagation (Ceita et al., 2007). It has been shown that oxalate oxidase and ascorbate peroxidase genes, which were present in the SP library, participated in the compatible interaction (Ceita et al., 2007; Gesteira et al., 2007). But other plant genes are under investigation, which may contribute to understand the mechanisms of fungus transition from the biotrophic to the necrotrophic

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phase, and to relate them with biochemical changes occurring in the green broom (Scarpari et al., 2005), or to in vitro observations (Meinhardt et al., 2006). Other genes related to PCD were also found in cacao–M. perniciosa libraries, such as those encoding a protease, in particular plant vacuolar protease, metallothionein and ankyrin-repeat protein (Gesteira et al., 2007). TcPR10 gene was identified in the SP library and shown to be differentially expressed at 60 dai in the susceptible interaction by in silico analysis (Gesteira et al., 2007). The TcPR10 protein was over-expressed in heterologous system and its activity tested under various conditions: it has an in vitro and in vivo ribonucleasic activity against fungal and plant RNA, as well as an antifungal activity against M. perniciosa (Pungartnik et al., 2009). Because TcPR10 was highly expressed in susceptible plants at 60 dai, the corresponding protein may be involved in acting directly on the pathogen, which might have become intracellular as an ultimate attempt to impede fungal development. On the other hand, because TcPR10 was demonstrated to have a RNase activity against plant RNA, it may directly participate in PCD, degrading plant RNA, thus allowing necrosis and death and thereby supporting fungal phase transition and basidiocarp production (Pungartnik et al., 2009). Genes involved in biosynthesis pathways of molecules, such as purine alkaloids and tannins, were also found among unigenes from interaction libraries, such as caffeine synthase, caffeic acid 3-O-methyltransferase, chalcone syntase, flavonol synthase and flavanone-3-hydroxylase. The presence of these transcripts might be associated with changes in the content of caffeine/theobromine and tannins, being higher in infected plant than in non-infected, as observed by Scarpari et al. (2005). A good candidate gene could be caffeine synthase that was induced early in the resistant CAB 214 genotype, compared to later up-regulation in the susceptible genotype. Caffeine was accumulated during the cacao–M. perniciosa interaction (Aneja and Gianfagna, 2001; Scarpari et al., 2005) and was demonstrated to have in vitro activity against the pathogen (Aneja and Gianfagna, 2001). A negative relationship between caffeine content and resistance against diseases has been described in Coffea arabica (Guerreiro Filho and Mazzafera, 2000, 2003), but this association could not be demonstrated in cacao, because only plants with symptoms were analysed (Aneja and Gianfagna, 2001; Scarpari et al., 2005). The increase in caffeine might have resulted in response to biotic or abiotic stresses (Guerreiro Filho and Mazzafera, 2000). Induction of caffeine synthetase by elicitors, such as benzothiadiazole or methyl jasmonate, suggested that different forms of this enzyme might occur in cacao (Aneja and Gianfagna, 2001; Bailey et al., 2005b). An alternative function of this putative caffeine synthetase could include methyl transferase

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activity resulting in synthesis of methyl salicylate or methyl jasmonate, which could participate in plant defence responses. Genomic studies have identified several candidate genes for cacao resistance to M. perniciosa. Functional studies of these genes and in planta analysis become a natural sequence for understanding the complex mechanisms leading to plant resistance. Cacao transformation process and in planta analysis are discussed in Section VIII. ESTs are also a good source of polymorphic markers (such as SSRs and SNPs) for genetic studies. Several SSR and SNP markers were identified from cacao–M. perniciosa ESTs (Gesteira et al., 2007; Karina P. Gramacho, unpublished results). These results are presented in Section X.B. C. PROTEOMICS OF CACAO AND M. PERNICIOSA

In parallel to genomic studies, some proteomic analyses have been conducted to investigate the cacao–M. perniciosa interaction. As observed during nucleic acids isolation (Gesteira et al., 2003), cacao tissues contain very high amount of polyphenols and polysaccharides (Figueira et al., 1994) that are not easily removed by conventional extraction procedures. For these reasons, it was first necessary to establish reliable methods for cacao protein extraction and purification from tissues. Similar problems have been faced for quality analysis of cacao seeds (A. Possignolo and collaborators, unpublished results). Three protocols were developed: one for apoplastic washing fluid extraction and two for protein extraction (under denaturing and non denaturing conditions) (Pirovani et al., 2008). On the other hand, the secretome of M. perniciosa cultivated on different mediums was analysed (Alvim et al., 2009), evidencing quantitative and qualitative relationships between secreted proteins and their activity, and the hyphal morphology of M. perniciosa. It appeared that the carbon source-dependent energy metabolism of M. perniciosa results in physiological alterations in protein expression and secretion, which may affect not only M. perniciosa growth, but also its ability to express pathogenicity proteins (Alvim et al., 2009).

V. GENOMICS OF THE CACAO–PHYTOPHTHORA INTERACTION Black pod, caused by several species of Phytophthora, is one of the most important diseases affecting cacao and is responsible for important yield losses. Between 15% and 80% of losses could be observed depending on the Phytophthora species, with P. megakarya, being the most aggressive.

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The search for a sustainable Phytophthora resistance is one of the major goals of most breeding programmes around the world. Until now, cacao resistance to Phytophthora spp. has been described as a quantitative resistance and ‘race nonspecific’-like type (Cilas and Despreaux, 2004). A recent meta-QTL analysis has identified a large number of genomic regions involved in Phytophthora resistance (Lanaud et al., 2009). This situation offers the possibility to improve the cacao resistance durability by a possible accumulation of many different resistance genes located in different chromosome regions using marker assisted selection (MAS). However, the molecular basis of this resistance is poorly understood and functional genomics studies are being carried out to improve our knowledge of the molecular processes involved in the cacao–Phytophthora interaction. A. PHYTOPHTORA NEP1 ORTHOLOGUES AND THEIR EFFECTS ON CACAO

Phytophthora species produce a protein that has a similar sequence to the NEP1 of Fusarium oxysporium. Multiple copies of NEP1 orthologues have been identified in a P. megakarya strain and in four other Phytophthora species (P. citrophthora, P. capsici, P. palmivora and P. sojae) (Bae et al., 2005). From the nine different NEP1 orthologues from P. megakarya identified by Bae et al. (2005), six were expressed in mycelium and in zoosporeinfected cacao leaf tissue. Sequence analysis revealed that six NEP1 orthologues were organised in two clusters of three orthologues each in the P. megakarya genome. The existence of a NEP1 multigene family in Phytophthora species suggests that this gene could have an important function in microbial biology. Bailey et al. (2005a) compared the effect of the NEP1 protein and cacao infection by P. megakarya on the expression of 10 cacao genes involved in defence, gene regulation, cell wall development or energy production. Seven of the 10 genes studied were responsive to the infection of cacao leaf disc by P. megakarya; the main expression variation occurring between 24 and 48 hai, period which corresponded to the beginning of tissue necrosis. Five of the six genes that were responsive to NEP1 were also responsive to infection by P. megakarya: TcWRKY-1 involved in gene regulation; TcPer-1 and TcGlu1,3 involved in defence mechanisms, and TcLhca-1 and TcrbcS involved in energy production. Unfortunately, these genes are not directly involved in plant defence, but are related to cellular effects, such as membrane damage, altered chloroplast functions and reduced energy and carbohydrate production. The data indicate that the constitutive defence mechanisms used by cacao leaves differ according to the developmental stage. The proteins produced from NEP1 and its orthologues in Phytophthora spp. have similar activities in a broad range of dicot species (Qutob et al.,

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2002), and the response of cacao to NEP1 mimics in some ways the response of cacao to infection by P. megakarya in a susceptible interaction. B. GENES DIFFERENTIALLY EXPRESSED DURING THE CACAO– PHYTOPHTHORA INTERACTION

A set of genes known to play a crucial role in plant–pathogen resistance and defence mechanisms (De Young and Innes, 2006; Mishra et al., 2006; Walters et al., 2007; Wrbel-Kwiatkowska et al., 2004) was selected from Argout et al. (2008) libraries (development libraries or libraries produced from cacao tissues infected by pathogens; see Section II.A), and used for developing macro- and micro-arrays. Genes similar to LRR-NBS (8 contigs and 32 singletons), chitinase (19 contigs and 37 singletons), 1-3 beta-glucanase (5 contigs and 7 singletons) and PR protein (24 contigs and 24 singletons) were identified. Other sequences involved in regulation of pathogen-induced genes like TFs (6 contigs and 7 singletons), in signal transduction like MAPkinase (5 contigs and 3 singletons), and in PCD (13 contigs and 20 singletons) were also identified. This tool is presently being used to study cacao–Phytophthora spp. interactions (Argout et al., 2008; Legavre et al., 2006). For macroarray analysis, 90 genes were selected and amplified using specific primers (Legavre et al., 2006). Leaves were collected on resistant and susceptible cacao genotypes from a progeny segregating for Phytophthora resistance and inoculated by P. megakarya strains. Samples were collected from 1 hai to 7 dai for probe preparation and macroarray hybridisations. Confirmation of genes differentially expressed between susceptible and resistant cacao clones was conducted by RT-qPCR. Twelve genes appeared differentially expressed between resistant and susceptible plants. The genes up-regulated in resistant clones were similar to those involved in classical model of signal transduction, including: (i) gene defence response like protein kinase (early expressed, i.e. 2 hai) and (ii) transcriptional regulator, PR proteins (PR-1, PR-5), chitinase and protease inhibitors (expressed 24 hai) (Legavre et al., 2009). In susceptible cacao clones, Legavre et al. (2006) observed an over-expression of a glucanase inhibitor protein (GIP), known to be secreted by Phytophthora (Rose et al., 2002), and to specifically inhibit the endoglucanase activity of the plant host. GIP activity may represent a defence mechanism used by the pathogen to suppress a plant defence response, and it could be also involved in the cacao–Phythophthora interaction. Several genes differentially expressed in this study are in common with those identified by Verica et al. (2004) on leaves treated with artificial inducers, such as protein kinase and DNAK-type molecular chaperone precursor.

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VI. CACAO GENE EXPRESSION UNDER OTHER BIOTIC AND ABIOTIC CONDITIONS A. GENOMICS OF CACAO–ENDOPHYTIC INTERACTION

In their native areas, many endophytic fungi are associated with cacao and among them many species of Trichoderma (Holmes et al., 2004; Samuels et al., 2000). These Trichoderma species inhabit different plant tissues, including roots, trunks, stems, leaves and pods. Trichoderma species have the potential to control plant or specifically cacao diseases (Bastos, 1996; Harman et al., 2004; Samuels et al., 2000). Bailey et al. (2006) analysed fungal and plant gene expression during the colonisation of cacao seedlings by isolates of four Trichoderma species. Differential display analysis was carried out using total RNA from cacao seedlings individually colonised by Trichoderma isolates. One hundred and sixty-four EST clones were selected, sequenced and analysed for their putative function. A putative function or conserved domain was identified for 59 ESTs, including 39 more probable plant genes and 16 more probable fungal genes. These genes plus additional ESTs identified from previous studies of cacao stress responses (Bailey et al., 2005a,b) were included in a macroarray and used to analyse gene expression in cacao tissues colonised by Trichoderma strains. The differential expression of some genes was then validated by RT-qPCR. Several cacao ESTs induced during colonisation by Trichoderma isolate shared homology with genes known to respond to abiotic and biotic stresses. In particular plant diseases: (i) ornithine decarboxylase, involved in polyamine (PA) biosynthesis (see Section VI.C) (Walters, 2000; Yoo et al., 2004); (ii) zinc finger proteins (Kim et al., 2004) and (iii) glutathione-S-transferase (GST)-like proteins known to play a broad role in protecting cells from oxidative injury by detoxifying compounds that would otherwise damage plant cells (Dixon et al., 2002). Trichoderma stromaticum is a mycoparasite of the cacao witches’ broom pathogen, M. perniciosa. This beneficial fungus is being used in Bahia, Brazil, to control the witches’ broom disease under field conditions. De Souza et al. (2008) studied the effect of an endophytic colonisation of cacao plants by T. stromaticum on genes involved in defence and plant growth. The data indicated that the activity of T. stromaticum in the biocontrol of M. perniciosa did not promote plant growth, nor induced resistance against M. perniciosa on seedlings that had been treated 30 days prior to inoculation with the pathogen. These results were confirmed by Northern blot, where the fungus was unable to alter the expression of selected genes involved in: (i) plant defence, such as ChiB, encoding a putative class VII chitinase, Glu-1, encoding a putative endo-1,4-beta-glucanase, and Caf-1, encoding a putative

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caffeine synthase; (ii) genes involved in growth regulation, such as Per-1 encoding an apoplastic quiacol peroxidase and (iii) genes involved in energy production, such as TcORFX-1 (fw2.2-like) and TcLhca-1 (photosystem I 24 kDa protein). B. ENDOPHYTIC COLONISATION OF CACAO AND DROUGHT

Bailey et al. (2006) also identified another class of protein, potentially involved in drought stress, repressed during the cacao endophyte colonisation. This class of protein is closely related to the major intrinsic protein (MIP) superfamily, also called aquaporins, which work as membrane channels that selectively transport water, small neutral molecules and ions out of and between cells. The repression of MIP gene expression in drought stress may reduce membrane water permeability and stimulate water conservation during periods of drought (Smart et al., 2001). Enhanced drought tolerance is commonly associated with endophyte-colonised grasses (Schardl et al., 2004), and it could help cacao cultivation during drought seasons. Bae et al. (2009) characterised the effect of endophytic colonisation by T. hamatum (isolate DIS 219b) on the responses of cacao to drought. Changes of transcript levels were monitored during drought treatment in leaves and roots of cacao seedlings. The ESTs analysed were chosen based on their relatedness to orthologues in other plants with characterised involvement in various biological processes, including drought. The altered expression of 19 ESTs (7 in leaves and 17 in roots, with some overlap) by drought was detected using RT-qPCR. Roots tended to respond earlier to drought than leaves, with the drought-induced changes in expression of 7 ESTs. The majority of the drought-responsive ESTs (16 of 19) were identified in previous studies, characterising the responses of cacao to other biotic and abiotic stresses (Bailey et al., 2006; Verica et al., 2004). Several enzymes involved in signal transduction, transcription and post-transcriptional regulation or in the production of osmoprotectants and/or regulatory metabolites were identified: (i) trehalose-6-phosphatase, which confers drought tolerance to microorganisms and various higher plants (Garg et al., 2002); (ii) sorbitol transporter, important in stress tolerance; (iii) osmotin-like protein, commonly associated with tolerance to osmotic stress and in plant defence (D’Angeli and Altamura, 2007) and (iv) alkaline/neutral invertases, participating in the hydrolysis of sucrose and providing a source of carbon for the biosynthesis of other osmoprotective substances. Colonisation of cacao seedlings with T. hamatum delayed the drought-altered expression of all 7 ESTs responsive to drought in leaves by 3 days, but had less influence on the expression pattern of the drought-responsive ESTs in roots. T. hamatum

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colonisation had minimal direct influence on the expression of droughtresponsive ESTs in 32-day-old seedlings. By contrast, the colonisation of 9-day-old seedlings altered expression of drought-responsive ESTs, sometimes in patterns opposite of that observed in response to drought. The transcript level of TcRbcS (putative Rubisco small subunit) declined from 74% at 10 days post-watering in non-colonised seedlings, compared with a decline of only 39% in colonised seedlings, suggesting drought-induced changes in net photosynthesis and stomatal conductance were delayed in T. hamatum-colonised seedlings. Colonisation of cacao seedlings resulted in a delay in many aspects of the drought response. It was proposed that this effect is mediated through enhanced root growth, resulting in an improved water status, allowing cacao seedlings to tolerate drought stress (Bae et al., 2009). C. REGULATION OF PA BIOSYNTHESIS IN CACAO

PAs have been associated with response to drought and many other biotic and abiotic stresses in plants, in addition to their roles in physiological and developmental processes. The most common PAs in higher plants are putrescine (Put), spermidine (Spd) and spermine (Spm). In higher plants, there are two pathways for PA biosynthesis (Illingworth et al., 2003): (i) Put synthesis from ornithine by ornithine decarboxylase (ODC) and (ii) Put synthesis from arginine by arginine decarboxylase (ADC). Spd and Spm are synthesised by either spermidine synthase (SPDS) or spermine synthase (SPMS) from Put and decarboxylated S-adenosylmethionine, which donates the aminopropyl groups. A full-length ODC (TcODC) was cloned from cacao and its expression together with four other ESTs associated with PA biosynthesis, ADC (TcADC), SAMDC (TcSAMDC), SPDS (TcSPDS) and SPMS (TcSPMS) were studied by Bae et al. (2008) after drought treatment and other stresses made on cacao leaves, including wounding, Phytophthora inoculations and F. oxysporum NEP1 treatment. Expression analysis using RT-qPCR results showed that the PA biosynthesis genes were expressed in all plant tissues examined. Expression of TcODC, TcADC and TcSAMDC was induced with the onset of drought. Elevated levels of Put, Spd and Spm were detected in cacao leaves 13 days after the onset of drought. Genes encoding putative ODC, ADC and SAMDC were also responsive to mechanical wounding, infection by P. megakarya and NEP1 treatment. TcODC was induced approximately 100-fold by NEP1 and P. megakarya, and it was constitutively expressed at much lower levels than TcADC, TcSAMDC, TcSPDS and TcSPMS. In comparison, genes encoding SPDS and SPMS were induced 3.5-fold after 24 h treatment with P. megakarya. The results indicated that TcODC, TcADC and TcSAMDC are co-regulated by both abiotic and biotic

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stresses. The stresses responsible for the induction of TcODC, TcADC and TcSAMDC may share signal transduction pathways, and/or the stress induced signal induction pathways may converge at these three genes, leading to their coordinated induction. According to Bae et al. (2008), it is possible that alteration of PA levels in cacao will result in enhancing the tolerance of cacao to multiple stresses, including drought and disease, therefore, it may be possible to improve tolerance of cacao to stresses by modifying PA production in cacao through plant breeding or genetic engineering.

VII. GENE EXPRESSION RELATED TO CACAO QUALITY FLAVOUR Flavour is among the main criterion of quality for chocolate manufacturers. Flavour components not only depend strongly on conditions of post-harvest processing (environmental effects, storage, fermentation, drying, roasting) (Chanliau and Cros, 1996) but also depend strongly on the genetic origin, regardless of post-harvest processing conditions (Clapperton et al., 1994). Only a small number of studies are related to gene expression regarding biochemical compounds involved in cacao quality; they are related to gene expression of linalool synthase and other genes involved in terpen synthesis during seed development and fermentation (Sabau et al., 2006a,b). Linalool is known as a major constituent of floral scent of many species. It has been detected in larger amounts among ‘Nacional’ varieties rather than in other cacao varieties and therefore, it may be involved in the floral notes that characterise chocolate made from the Nacional cultivars (Chanliau, 1998). Linalool is an acyclic monoterpene alcohol and linalool synthase catalyses the formation of S-linalool from the monoterpene precursor geranyl diphosphate. A cacao cDNA homologous to Arabidopsis linalool synthase (TcLIS) was identified in a cDNA library constructed from the testa of fermented seeds from the ‘ICS1’ and ‘Nacional’ genotypes (Argout et al., 2008; Sabau et al., 2006a,b). Sabau et al. (2006a,b) analysed by RT-qPCR the expression of linalool synthase in the testa and cotyledons of ‘ICS1’ and ‘Nacional’ seeds collected at various stages of development (18–27 weeks), and during the first 4 days of fermentation. For both cacao genotypes, the relative expression of TcLIS increased in the cotyledons during the fermentation steps. An increase of TcLIS expression was observed until the 47 h of fermentation, at a temperature of 44 8C in ‘ICS1’. This expression decreased afterwards during fermentation, probably due to RNA degradation under elevated temperature. During the first 48 h of fermentation, higher expression of TcLIS was also observed: (i) in the testa of fermented seeds compared to unfermented

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ones and (ii) in the testa of fermented seeds compared the cotyledons. In the following 48 h, the TcLIS expression increased in the cotyledons, but decreased in the testa. In seeds from ‘Nacional’, the TcLIS activity was constant in both cotyledons and testa during the first 96 h of fermentation, and the TcLIS expression was always higher in the testa than in the cotyledons. According to Sabau et al. (2006a,b), the increase in the linalool synthase expression observed during the fermentation steps could be a response of cacao seeds to stresses induced by the fermentation, involving yeasts and bacteria. Indeed, terpenes are recognised as plant defence responses towards aggressions (Schnee et al., 2002). Preliminary analyses were also carried out with a micro-array to study the expression of other genes involved in the terpene and polyphenol pathways during seed development and fermentation (Sabau et al., 2009). This microarray was constructed from Argout et al. (2008) libraries and contains a set of genes potentially related to cacao quality. Eight hundred and sixty-five oligonucleotides were designed from genes encoding enzymes from 10 metabolic pathways potentially involved in cacao quality, and related to several biochemical compounds synthesis (polyphenol, fat, sugar, pectin, terpenes and purines). This set was completed with oligonucleotides corresponding to 715 unique sequences of TFs and about 450 P450 cytochrome homologues.

VIII. IN VITRO CULTURE AND GENETIC TRANSFORMATION, AND THEIR APPLICATIONS IN FUNCTIONAL GENOMICS STUDIES A. GENETIC TRANSFORMATION OF CACAO

One of the major tools available for functional analysis of genes and proteins is genetic transformation. The ability to isolate, modify and reintroduce genes of interest to plants allows the systematic dissection of gene and protein structure/ functional relationships and provides a means to assess the activity of a gene product within a living cell. A genetic transformation system for cacao has been developed in the Guiltinan lab (Antunez de Mayolo et al., 2003; Maximova et al., 2003, 2006, 2008b). Transformation of cacao, regeneration of plantlets and their subsequent analysis require about 6 months from construct to small plantlet. Although the frequencies are low compared to other species, which increases the time and cost to produce sufficient numbers of independent transformation events, the transformation system is reproducible and has resulted in the accumulation of multiple transgenic lines carrying reporter genes and genes potentially important in disease resistance.

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The system is based on somatic embryogenesis as an in vitro plant regeneration system. A pipeline of primary somatic embryo cultures is developed by initiating new cultures every 2 weeks with immature floral explants of the SCA6 variety by published methods (Li et al., 1998; Maximova et al., 2002, 2005, 2008a; Traore and Guiltinan, 2006; Traore et al., 2003). Good results were also obtained using petal explants from other cacao genotypes such as TSH565 and TSH1188 (Silva et al., 2008). Cotyledons excised from primary somatic embryos are used as explants for co-cultivation with Agrobacterium harbouring binary plasmids containing a green fluorescence protein (GFP) gene, a kanamycin resistance gene and the gene under investigation. After several days of co-cultivation of Agrobacterium and primary somatic embryo cotyledons, the explants are washed free of Agrobacterium then plated on selection medium with antibiotics to eliminate growth of the bacteria and select for transgenic cells. Explants are cultured on somatic embryo induction media for several days then transferred to embryo development media containing geneticin as a positive selection for transgenic plant cells as described by Maximova et al. (2003). Secondary embryogenesis is induced and embryos begin to develop over the next few months of culture in the dark. While geneticin is used as a lethal selection against non-transformed cells, it is thought to also inhibit regeneration so antibiotic concentration is kept at a minimal level. Therefore, GFP is used as a visible selection marker, by screening cultures using a fluorescence stereo microscope. Transgenic embryos are identified by visual screening using a stereo fluorescence microscope, removed to selection free media and cultured to the plantlet stage. Several months later, plantlets will be acclimated then grown to maturity. Using this method, between 1 and 10 transgenic plants are routinely obtained for each transformation attempt. Using fluorescence imaging techniques, images of transgenic cacao plant tissues were obtained, demonstrating the stable expression of the GFP transgene in plants grown in a greenhouse for many years, and the sexual transmission of the GFP gene to seeds developing from the plants (Fig. 2). Silva et al. (2009) evaluated the effect of PAs and
-lactam antibiotics on somatic embryogenesis, hygromycin as selective agent, and different factors affecting uidA gene transfer of genotype TSH 565. It was demonstrated that the PAs Put, Spd and Spm significantly improved secondary somatic embryogenesis in cacao without Agrobacterium infection. Although no transgenic plants were produced as a result of this study, the results indicated that the
-lactam antibiotics timentin and meropenem, used for Agrobacterium tumefaciens counter-selection, had a non-detrimental effect on secondary somatic embryogenesis, whereas the commonly used
-lactam cefotaxime inhibited it. Hygromycin showed a strong inhibitory effect on secondary somatic embryogenesis of cacao (Silva et al., 2009).

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Fig. 2. Transgenic cacao flower, pod and seeds visualised by fluorescence imaging. Left; transgenic flower (centre) from a mature GFP expressing cacao plant. Nontransgenic flowers (outer circle) do not fluoresce but are slightly illuminated with light from the transgenic flower. Centre: transgenic cacao fruit expressing GFP; right: seeds from transgenic pod segregating for GFP and anthocyanins. Left petri dish contains transgenic seeds, upper group of seeds contain anthocyanins, lower group does not thus GFP emission is much brighter. Right petri dish, seeds from which the GFP transgene has segregated away are barely visible as they are only illuminated in light from transgenic seeds on the left (photo credit, Guiltinan laboratory).

To date, only a single manuscript has been published that has utilised transgenic cacao for the functional analysis of a gene (Maximova et al., 2006). In this study, a gene encoding the antifungal protein chitinase was isolated from cacao and modified for high-level constitutive expression throughout the plant. Chitinase activity levels were measured using an in vitro fluorometric assay. The transgene was expressed at varying levels in the different transgenic lines with up to a sixfold increase of endochitinase activity compared to non-transgenic and transgenic control plants. The in vivo antifungal activity of the transgene against the foliar pathogen Colletotrichum gloeosporioides was evaluated using a cacao leaf disc bioassay. The assay demonstrated that the TcChi1 transgenic cacao leaves significantly inhibited the growth of the fungus and the development of leaf necrosis compared to controls when leaves were wound inoculated with 5000 spores. These results demonstrated the utility of the cacao transformation system as a tool for gene functional analysis and the potential utility of the cacao chitinase gene for increasing fungal pathogen resistance in cacao. This capability will allow the functional testing of candidate genes for various traits identified in QTL mapping and other genomics projects such as genes controlling disease resistance, seed quality traits and other genes of interest. Although this method could also be used to develop plants for commercial production with enhanced resistance and other valuable traits, consumer resistance to genetically modified organisms has precluded the commercialisation of such plants for the time being. For safety considerations, all transgenic plants are maintained in secure greenhouses in a non-producing country. No transgenic cacao plants have been released into any fields.

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B. RNA INTERFERENCE IN CACAO

RNA interference (RNAi) or post-transcriptional gene silencing is used to manipulate gene expression experimentally and to screen gene function on a whole-genome scale. RNAi is a conserved eukaryotic pathway in which double-stranded RNA triggers destruction of homologous target RNA via production of short-interfering RNA (siRNA) (Eamens et al., 2008). In plants, at least some case of RNA silencing can spread systemically (Voinnet, 2008). Because they provide a rapid, versatile and convenient way for achieving a very high level of gene expression in a distinct and defined zone of leaf, Agrobacterium-mediated transient expression systems have been useful for inducing silencing processes and for dissecting systemic silencing signal (Bendahmane et al., 2000; Maimbo et al., 2007). The feasibility of an agroinfiltration approach for inducing RNA silencing systemic signal production in cacao was tested by Andrieu et al. (2006). This approach could be further used as a tool for large-scale discovery and validation of gene function. Based on the Nicotiana benthamiana agroinfiltration method, an efficient and reproducible technique for transient expression of T-DNA vectors was developed in cacao leaves. Using gene specific tag from phytoen desaturase gene (pds) and efflux carrier pin-formed gene (pin1), the feasibility of an agroinfiltration method for inducing gene-silencing processes in a defined zone of leaf was demonstrated (Andrieu et al., 2010). The diagnostic presence of siRNA was monitored by Northern blot. siRNA amplification was demonstrated for the two genes in the cacao plants, indicating that the RNAi mechanism was initiated in planta. However, the RNAi spreading throughout the whole cacao plant still remains under study.

C. MODEL PLANT SYSTEMS FOR ACCELERATION OF CACAO FUNCTIONAL GENOMICS STUDIES

While considerable progress has been made in the field of cacao functional genomics, progress has been limited by a number of contributing factors including: large plant size, long generation time, lack of genomic resources, such as mutant collections and full genome sequence, difficult transformation system and a high degree of heterozygosity in most accessions. Conversely, several plant species have been extensively developed as model plant species such as Arabidopsis, tomato, rice, maize, cotton, medicago and others. These species all have the following common attributes which contribute to their usefulness for genomics research; small size, rapid

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life cycle, relative ease of transformation, genome sequence determined or in progress, large mutant collections and availability of homozygous lines. So far only tomato has been confirmed to be a host of M. perniciosa. In Bahia (Brazil), natural infection of M. perniciosa has been observed on tomato, pepper and egg plants. One strategy to accelerate cacao genomics research is to make use of the model plant systems to provide information and test systems useful for cacao research. This approach is known as translational biology. The mouse model is analogous to this for the translational biology of vertebrates as applied to human biological and medical research. Model systems can be used to: (i) isolate genes in the target species based on sequence similarity with the model species genes; (ii) test gene function by introduction of target genes into model plants; (iii) compare developmental pathways and signal transduction mechanisms which may be conserved between the target species and the model plant and (iv) provide a test system for interactions with plant–pathogens and other biotic and abiotic factors of interest. One example of a translation biology approach with cacao is in the study of the genes involved in proanthocyanidin (ProA) biosynthesis. The flavonoids catechin and epicatechin, and their polymerised oligomers, the ProAs, accumulate to levels of approximately 10% of the total weight of dry cacao seeds. These compounds have been associated with human health benefits and also play important roles in pest and disease defence throughout the plant. To dissect the genetic basis of the flavonoid biosynthetic pathway in cacao, three genes encoding key ProA synthesis enzymes were isolated: anthocyanidin synthase (ANS), anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR) (Liu and Guiltinan, in preparation). This was done by using gene sequence information from Arabidopsis to design PCR primers, which were used to amplify the genes from cacao. To test the function of the putative cacao genes, they were transferred into transgenic Arabidopsis plants and their functions assayed. For example, constitutive over-expression of TcANR in an Arabidopsis mutant lacking ProA synthesis complemented the ProA deficient phenotype in seeds (Fig. 3). Using similar strategies, transgenic over-expression of TcANS in tobacco resulted in increased content of both anthocyanin and ProAs in flower petals. Constitutive over-expression of TcANS in an Arabidopsis ldox mutant complemented its ProA deficient phenotype in seeds. Transgenic tobacco over-expression of TcLAR resulted in decreased amounts of anthocyanins and increased ProAs. Overexpressing TcLAR in Arabidopsis ldox mutant also resulted in elevated synthesis of catechin and epicatechin. The results confirmed the in vivo enzymatic activities of cacao ANS and ANR that were predicted

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Mature seeds DMACA stained

Col-O

ban (ANR null)

CaMV35S:TcL ar ::ban

Fig. 3. Functional analysis of the cacao lecuoanthocyanidin reductase gene (TcLar) in transgenic Arabidopsis plants. Left: immature seeds in developing of wild-type (Col-0) Arabidopsis siliques are clear, because they lack anthocyanins. The banyuls (ban) mutant of Arabidopsis lacks a functional ANR gene which is necessary for the production of proanthocyanidins (ProAs). This results in a shift in metabolic flux to the production of anthocyanin, and thus the wine colour of the developing seeds. Introduction of the TcLAR gene restores PS biosynthesis, diverting metabolism away from anthocyanins resulting in the wild-type, clear seed phenotype. Seeds depicted in the right panels demonstrate the presence of ProAs in Col-0 and transgenic ban mutant seeds containing TcLAR. DMACA stains ProAs dark purple, enhancing the differences in appearance of the seeds. These results demonstrate the ability of the cacao LAR protein to function in the ProA biosynthetic pathway as predicted based on its sequence identity to genes from other species (Liu and Guiltinan, in preparation).

based on sequence homology to previously characterised enzymes from other species. The Guiltinan laboratory has used similar approaches with a number of other cacao genes, including genes involved in the defence response pathway. Another use of model plant systems is the use of information about plant development and anatomy to infer new knowledge regarding similar processes in cacao. For example, Swanson et al. (2008) examined the development of cacao flowers and compared this with that of Arabidopsis. Using morphometric and comparative anatomy approaches, this report demonstrated that the stages of development are nearly identical in the two species with the significant difference of speed; the process is much slower in cacao; approximately 30 days as opposed to 14 days in Arabidopsis. This information was used to derive a hypothesis regarding the precise timing and localisation of a MADS box transcription factor LEAFY that is known to regulate flower development in Arabidopsis. Swanson et al. (2006) demonstrated the

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precisely predicted expression pattern of LEAFY in cacao using in situ hybridisation. From this analysis, it appears clear that the main mechanisms and developmental programmes regulating flower development are highly conserved between cacao and Arabidopsis. Thus, it is reasonable to extrapolate the detailed information on these processes from Arabidopsis to cacao. Another application of translational biology is in the study of plant– pathogen interactions. In this example, tomato was used as a model plant species to study interactions with the devastating witches’ broom disease of cacao (Marelli et al., 2009) which is caused by the C-biotype of M. perniciosa. While this disease is of major economic importance, the pathogenicity mechanisms and plant responses underlying the disease are difficult to study given the cacao tree’s long life cycle and the limited availability of genetic and genomic resources for this system. The S-biotype of M. perniciosa infects solanaceous hosts, particularly pepper (Capsicum annuum) and tomato (Solanum lycopersicum). These species are much more amenable for performing studies of mechanisms underpinning host–pathogen interactions as compared to cacao. A comparative analysis demonstrated that disease progression in tomato infected with the S-biotype is similar to that described for cacao infected with the C-biotype. The major symptoms observed in both systems are swelling of the infected shoots and activation and proliferation of axillary meristems. Cellular changes observed in infected tissues correspond to an increase in cell size and numbers of xylem vessels and phloem parenchyma along the infected stem. The tomato model system was further utilised to search for disease resistance genes (Marelli and Guiltinan, in preparation). As an alternative cacao host, the tomato plant (S. lycopersicum var. ‘Microtom’ and ‘Santa Clara’), showed susceptibility to the strains of the S-biotype, from Solanum paniculatum (strain 73-6-01), but not to C-biotype strains of M. perniciosa. Comparatively, the tomato’s wild relative, Solanum habrochaites, was highly resistant to the disease when exposed to the same strain of M. perniciosa. One hundred near isogenic lines of tomato containing chromosomal sections of the genome of S. habrochaites in an Solanum lycopersicum background provided specimens to identify genome regions of S. habrochaites that could be responsible for the observed resistance to M. perniciosa. A strong QTL for resistance on the short arm of chromosome 1 was detected for disease severity (P < 0.05), shoot diameter (P < 0.001), and shoot fresh and dry weight (P < 0.0001). That genomic area is known to contain a cluster of genes for resistance against the pathogen Cladosporium fulvum. In the future, these resistance genes could be identified in tomato and homologous genes isolated from cacao. It is possible that similar resistance genes exist in cacao

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and these could be targeted for breeding programmes or used in biotechnology approaches for enhanced resistance to witches’ broom. D. CHARACTERISATION OF LEAFY COTYLEDON1-LIKE DURING EMBRYOGENESIS IN CACAO

Cacao can be propagated by somatic embryogenesis, and it is possible to produce somatic embryos and plantlets from a large number of genotypes (Maximova et al., 2002). However, the efficiency of the process is not sufficient for a scaling-up step. Moreover, many genotypes remain recalcitrant to somatic embryogenesis (Figueira and Alemanno, 2004). Information has become available about genes involved in the early and late phases of embryogenesis in Arabidopsis. Among them, the leafy cotyledon genes, isolated from loss-of-function mutants in Arabidopsis have a central regulator role in both the early and late phases of embryogenesis, with several functions such as maintaining suspensor cell identity, specification of cotyledon identity, desiccation tolerance, synthesis and accumulation of storage reserves, and inhibition of germination (Harada, 2001). Given the recalcitrance of cacao in somatic embryogenesis, Alemanno et al. (2008) isolated leafy cotyledon gene homologues in cacao (TcL1L) and characterised their structure and function in zygotic and somatic embryos at various stages of development, and in vegetative organs and tissues. TcL1L is a homologue of AtL1L which encodes a 213-amino acid polypeptide with sequence similarity to the HAP3 subunit of the CCAAT binding transcription factor, also called CBF or NF-Y (Lotan et al., 1998). Alemanno et al. (2008) determined the localisation of TcL1L RNAs in zygotic and somatic embryos at different stages by in situ hybridisation. During zygotic embryogenesis, L1L RNA was detected in all tissues and cells of the globular embryos and young cotyledonary stages. During the embryo growth phase, the gene was expressed everywhere, but more in meristematic cells of root and shoot apex. When the embryo had reached its final size, but was still immature, the TcLIL expression drastically decreased and was restricted to the meristematic cells of the shoot and root apex. The expression variation through somatic embryo growth was similar to the one observed in zygotic embryos, even if the intensity was lower in somatic embryos as compared to zygotic ones at equivalent stages. In non-embryogenic primary calli obtained from staminodes, the TcLIL expression was detected in some rare zones of internal meristematic tissue. The results observed by in situ hybridisation were confirmed by RT-qPCR. To assess the functionality of the cacao L1L gene, the Arabidopsis lec1 mutant was transformed by the TcLIL gene. Expression of TcL1L was able to complement most of the morphological defects of lec1At

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embryos suggesting a similarity of function in zygotic embryogenesis. These results strongly support a role for TcL1L in cacao embryogenesis similar to the one of L1L in Arabidopsis. The authors concluded that an over-expression of this gene in cacao would be interesting to look for in order to improve cacao somatic embryogenesis.

IX. EPIGENETICS AND REGULATION OF THE CACAO GENOME The term epigenetics is generally used to describe changes to phenotype or to gene expression that arise from mechanisms other than variation in the underlying DNA code. The collective epigenetic control of gene regulation allows plants to exert plasticity in form and function, and therefore, provides the capacity respond to the changing growing environment. This flexibility is essential for sessile organisms like plants since they must endure rather than avoid stochastic and periodic challenges (such as short periods of water shortage or exposure to disease epidemics) that are overlaid onto regular climatic cycles imposed on daily, seasonal and in the case of perennials, yearly time frames. However, plasticity in the response of breeding lines to variations in the growing environment (so-called Genotype  Environment [G  E] effects) can have a confounding effect on the selection process, and impels breeders of all crops to conduct field trials over several sites and years to mitigate against the effect. Indeed, much of modern agricultural practice, particularly in an arable context, seeks to homogenise growing conditions such that the effects of crop plasticity are minimised. However, this approach is not possible for long-lived perennial crops such as cacao that are predominantly cultivated by small-holder farms and are exposed to a far wider range of growing conditions than their arable counterparts. In these and similar crops, it follows that there is a greater need to develop an understanding of the epigenetic control mechanisms giving rise to phenotypic and physiological plasticity. Several active systems of epigenetic control have been described in plants, most notably histone modifications (Sridhar et al., 2007; Zilberman et al., 2008), the action of small interfering RNAs and DNA methylation (Henderson and Jacobsen, 2007). Of these, it is only DNA methylation that can be heritable between seminal generations and where greatest interest has resided for crop application. There are several ways in which the study of epigenetics can yield tangible benefits for the productivity of a crop. Perhaps, the simplest and most direct of these relate to the characterisation of unwanted epigenetic changes arising from in vitro culture. Such changes commonly

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correlate with somaclonal variants arising from in vitro culture (Kaeppler et al., 2000). Perhaps, the most celebrated example of this in a tropical perennial crop comes from oil palm (Elaeis guineensis Jacq.). Development of a high-throughput system of somatic embryogenesis in this crop led to commercial-scale application of the protocol in Malaysia for the vegetative multiplication of elite clones and their transfer into plantations (Corley et al., 1986). Whilst vegetative growth characteristics of the regenerants from in vitro culture appear normal, around 5% of plants developed abnormal flowers in which stamen primordia were converted into carpel-like tissues (Jaligot et al., 2004). This condition, known as mantled fruit, only becomes apparent during flowering (2–3 years after germination) when it prevents harvestable yield in all affected individuals. Matthes et al. (2001) investigated the mechanisms giving rise to the phenomenon using conventional Amplified Fragment-Length Polymorphism (AFLP) (using methylation-insensitive restriction enzymes), but also with a technique known as methylation sensitive amplified polymorphism or MSAP analysis, that uses methylation sensitive restriction enzymes that target the same DNA sequence recognition motif (isochizomers). They found no consistent differences in AFLP profiles generated with insensitive restriction enzymes, implying a lack of genetic differentiation between phenotypically normal and abnormal palms. In contrast, there was genome-wide demethylation in the mantelled forms from the MSAP results, with 0.3% of the amplicons generated by the methylation sensitive HpaIII proving to be polymorphic between forms. Isolation of nine of these markers and their use as Southern probes indicated that all derived single-copy sequences. Later, Jaligot and colleagues (2004) made a concerted effort to identify a universal epigenetic marker associated with the mantelled phenotype by isolating discriminatory products between ‘mantelled regenerants’ and the source ortet plants using 64 MSAP primer combinations. The authors noted that none of the 23 candidate epigenetic marks isolated could discriminate between phenotypes in genotypes other than the ortet-regenerant pairing from which they were isolated. They also reported that CCGG methylation contexts were notably less affected by the genome-wide demethylation associated with in vitro culture. This finding has resonance with more fundamental studies on the mechanisms creating and maintaining DNA methylation. Cytosine methylation in the CpG context appears to be the only form of methylation that can be inherited between generations in plants (Mathieu et al., 2007; Saze et al., 2003) and is maintained by a collection of DNA methyltransferases (DNMTs). Changes to the methylation status of specific CpG sites can be associated with changed expression and can result in disrupted phenotype such as a change of floral symmetry in Linaria vulgaris and with increased stamen number in Arabidopsis (Cubas et al., 1999;

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Jacobsen and Meyerowitz, 1997). It is perhaps for this reason that much of the efforts to relate methylation changes to phenotype have focussed on changes to the methylation status of crops in this context. In many ways, the story of somatic embryogenesis in cacao has mirrored that seen in oil palm. Cacao is a recalcitrant species in tissue culture and somatic embryogenesis systems initially proved to be highly problematic. However, the development of systems based on the use of immature floral tissues as explants (Alemanno et al., 1996; Li et al., 1998; Lopezbaez et al., 1993; Maximova et al., 2002) eventually allowed the creation of efficient transformation protocols (Antunez de Mayolo et al., 2003; Maximova et al., 2003; Perry et al., 2000; Silva et al., 2009), new methods for cryopreserving cacao genotypes (Fang et al., 2004) and even a novel system for the removal of viral infection (Quainoo et al., 2008). In all cases, the economic utility of the systems created could be compromised by excessive genetic or epigenetic change occurring prior to plant regeneration. Maximova et al. (2008) surveyed for phenotypic abnormalities arising from such perturbations by studying six agronomic traits in somatic embryogenesis regenerants from nine ortets grown under field conditions. They found no significant differences between the somatic embryo-derived trees as a whole and isogenic cuttings derived from the same trees, implying that obvious epigenetic abnormalities such as the mantelled inflorescences of oil palm do not appear in cacao following somatic embryogenesis. This important work highlighted the commercial potential of the technique for large-scale multiplication of stocks and possibly for germ plasm storage. The work further served to limit concerns about the appearance of aberrant cacao plants from somatic embryogenesis to cryptic changes, at least for the protocol employed in the study. There are nevertheless some grounds for questioning whether there are always such high levels of genetic and epigenetic fidelity among regenerants. For example, Rodriguez-Lopez et al. (2004) surveyed for slippage mutations among 233 cacao regenerants of somatic embryogenesis using mapped microsatellites (Lanaud et al., 1999b; Risterucci et al., 2000) and found evidence of fixed or chimeric de novo mutations in 33% of the regenerants studied. Similar rates of substitution mutations (2.8  10 3 substitutions/ base screened) were similarly reported in 26% (of 114) cacao regenerants studied by Rodriguez-Lopez et al. (2010a). Furthermore, the first direct comparison of genetic and epigenetic change arising from somatic embryogenesis produced some rather surprising results (Rodrı´guez-Lopez et al., 2010b). As expected, the 15 microsatellite markers used proved to be highly conserved among leaves taken from source trees, with only a single slippage mutation being noted in one marker from one leaf of one tree. In common with previous studies, genetic variation among somatic embryogenesis

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regenerants was again high, with at least one slippage being detected in 35% of regenerants. Interestingly, the frequency of genetic variation initially seemed to increase in frequency with time in callus culture but then to decline. Epigenetic profiling by multivariate analysis of MSAP results revealed clear differences between leaf and staminode tissues (used as an explant source material) on the source trees. The study reported that leaves taken from regenerant plants occupied an intermediate position between those occupied between the explant tissue (staminodes) and the leaves of the source tree. Curiously, various statistical analyses applied to these profiles revealed that leaf profiles of late-emerging regenerants were far closer to those of the parental leaves than those from the earlier regenerants. Thus, the authors appeared to have shown that, counter-intuitively, the late regenerants are both genetically and epigenetically closer to the original source trees than those arising early from culture. By implication, this finding suggested that mutation and epigenetic divergence do not simply accumulate during callus culture. The authors hypothesised that the results could be most plausibly explained if, after an initial period when mutations increase with time in culture, increasing metabolic loads in mutant and epimutant cell lineages meant that they progressively lose their totipotency. In this way, only those lineages that remain free of mutations and epimutations retain the capacity to produce late somatic embryos. As a consequence, late-forming regenerants tended to contain fewer genetic and epigenetic abnormalities. If confirmed, these results would seem to imply scope for subtle changes in culture protocols could have a profound effect on the genetic and epigenetic integrity of the regenerated plants they produce. Moreover, it may ultimately prove possible to use a combination of genetic and epigenetic profiling in this way to optimise in vitro protocols to minimise the incidence of somaclonal variation. A key limitation of MSAP-based epigenetic profiling such as those described above arises from the anonymous nature of the amplicons generated by the technique. This means that it can be difficult to uncover causal relationships between epigenetic change and associated changes to gene expression or to phenotype. One way to circumvent this difficulty is to seek to exploit existing knowledge of gene pathways that control traits of general agronomic importance using the model plant Arabidopsis. Tricker et al. (2008) used this strategy to exploit the apparently conserved nature of genetic control of stomatal density to investigate epigenetic control of plasticity in Arabidopsis in their response to changes in relative humidity. Water use efficiency (WUE) denotes the physiological balance between the photosynthetic assimilation of carbon from carbon dioxide and the loss of water through transpiration, largely through the stomatal pores. As a feature, WUE has huge importance since it can restrict crop productivity and

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influence the distribution, fitness and ecological range of both wild and cultivated species. The development of stomata is under control of a wellcharacterised gene pathway (Pilliterri et al., 2007) and is itself determined by WUE (Lake and Woodward, 2008). Thus, plants appear to be able to maintain plasticity in their capacity to moderate stomatal density during subsequent leaf growth in response to changes in water availability. Tricker and colleagues (2008) first sought to investigate whether this plasticity was mediated by epigenetic control in Arabidopsis, with an ultimate aim of studying the phenomenon in cacao. The team focussed their efforts on screening for the impact of environmental change in the methylation status and expression of genes implicated in the stomatal development pathway. Change in stomatal density was induced in four replicated experiments of cv. ‘Landsberg erecta’ by lowering the relative humidity from 65% in the control sets to 45% in the treated samples. As expected, stomatal density fell under the low relative humidity conditions, as did dry weight and seed set. Methyl capture of genomic DNA followed by qPCR using primers targeting all genes in the stomatal development pathway revealed that methylation status of two genes in the pathway (SPEECHLESS and FAMA) was markedly more methylated under low relative humidity. High resolution melting analysis of the same loci following bisulphite treatment appeared to confirm the finding. Thus, it appeared that the reduction in stomatal frequency was negatively correlated with the increased methylation of the two genes marking the start (SPEECHLESS) and end (FAMA) of the gene pathway controlling stomatal development. The group is currently exploring expression of these genes, with the expectation that, in common with many other systems, expression of both SPEECHLESS and FAMA will be markedly reduced by the induction of de novo methylation by low relative humidity conditions. They are also studying the inheritance of methylation patterns and phenotypic response to the same stress in the seminal generations. In the longer term, there is ambition to transfer the results to cacao. As part of this effort, they have now isolated putative homologues of both SPEECHLESS and FAMA, along with genes implicated in the initiation and maintenance of methylation (sequence homologues of the methyl S-transferase genes MET1 and DRM1/ 2 responsible for maintenance and de novo methylation of CpG sites in Arabidopsis). They have also confirmed that at least some cacao plants respond in a similar manner to reduced relative humidity as Arabidopsis, implying similar mechanisms may be in operation. In the longer term, the planned provision of a complete genome sequence for cacao will facilitate comprehensive screening of the genome for base-pair resolution of methylation in the face of various environmental stresses that evoke plastic responses such as that described above. This provision will undoubtedly open the way

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for developing a fuller understanding of plasticity and will ultimately help optimise agronomic practices to minimise adverse plastic responses that impinge on harvestable yield. As yet, however, agricultural epigenetics should be regarded as an emergent field but one likely to yield greatest benefits for perennial crops such as cacao.

X. MOLECULAR GENETIC STUDIES OF IMPORTANT CACAO TRAITS AS SUPPORT FOR GENOMIC STUDIES Selection of improved cacao clones for resistance and/or bean quality is the main goal of all genomic and genetic studies presently conducted. The combination of functional genomics and genetic approaches will facilitate the identification of candidate genes underlying QTLs, and the further exploitation of genetic resources for cacao breeding. Over the last 20 years, a large number of molecular genetic markers were developed and used for genetic studies of cacao useful traits. These studies will be the basis to discover the genes underlying trait variation. A. PLANT GENETIC RESOURCES

Each national institution has mostly its own collection of genetic resources used for breeding programmes. However, two international collections, with plant material available for all research community, exist. The largest one, the International Cocoa Genebank, is located at Cocoa Research Unit (CRU, University of West Indies, Trinidad and Tobago). This collection comprises more than 2000 accessions from all genetic groups, and particularly Forastero accessions collected in the Upper-Amazon regions of Peru and Ecuador. The second international collection is located in the Centro Agronmico Tropical de Investigacin y Ensen˜anza (CATIE, Costa Rica) and comprises 1107 accessions from different genetic origins, but is richer in Criollo and Trinitario types. Other collections are available in Brazil. The Comissa˜o Executiva do Plano da Lavoura Cacaueira (CEPLAC) has two important cacao germ plasm collections, one established at the Estac¸a˜o de Recursos Gene´ticos Jose´ Haroldo (ERJOH), at Marituba, Para´ state, and the other established at the Cocoa Research Center—CEPEC in Itabuna, Bahia. The former, currently, holds 1800 Forastero accessions (denominated Cacao of the Brazilian Amazon—CAB), of which 940 were of clonal origin and 877 are families derived from open-pollinated seedlings, representing 36 river basins of the 186 Brazilian Amazon basins (Almeida et al., 1995; Bartley, 2005). The second one holds 1300 accessions of several genetic groups

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including accessions of more than 20 countries, origin centre, farms and breeding selection. Others important collections are located in Ecuador and Peru. Ecuador’s collection is considered as a very important one because the amount of the genetic diversity it contains; it holds mainly Forastero and ‘Nacional’ populations.

B. MOLECULAR MARKERS

Genomic markers were the first ones produced and among them Restriction Fragment Length Polymorphism (RFLP) was first used to establish genetic maps and detect QTLs in cacao (Crouzillat et al., 1996; Lanaud et al., 1995). However, with the development of the polymerase chain reaction (PCR) technique, other types of markers, such as microsatellites or SSRs, have been produced and extensively used in further cacao genetic studies (Brown et al., 2005; Lanaud et al., 1999a,b; Pugh et al., 2004; Risterucci et al., 2000). The high level of polymorphism of SSR markers allowed their mapping in most of the studied progenies, and consequently enabled map and QTL comparisons between the many genetic maps produced. In the last years, most of the markers developed for cacao were defined in genes to facilitate the identification of candidate genes involved in trait elaboration. Two main strategies were adopted for this goal: production of markers (i) from defence gene analogues isolated using degenerated primers and (ii) from EST libraries. In the first strategy, resistance and defence gene analogue (RGA/ DGA) sequences were isolated in cacao using a PCR approach with degenerate primers designed from conserved domains of plant resistance and defence genes identified in other species. Such degenerate primers were defined in the nucleotide-binding site (NBS) motif present in a number of resistance genes such as in: (i) the tobacco N gene (Kuhn et al., 2003, 2006; Lanaud et al., 2004); (ii) the subdomains of plant serine–threonine kinases like Pto tomato gene and (iii) conserved domains of two defence gene families: class 2 and five PR proteins (
1-3 glucanase and thaumatin genes; Lanaud et al., 2004). In all, 6 and 16 RGA/ DGA were mapped by Kuhn et al. (2003) and Lanaud et al. (2004), respectively. Several co-localisations were observed between RGAs, DGAs and QTLs for resistance to Phytophthora detected in several progenies, particularly on chromosome 4, where a cluster of Pto-like sequences and four QTLs for resistance to Phytophthora have been identified. WRKY genes—which are responsible for the regulation of plant responses to abiotic and biotic stresses—were also isolated by Borrone et al. (2004) using degenerate PCR primers designed in the conserved DNA-binding domain and other conserved motifs of the studied genes. Four individual WRKY fragments have been mapped by these authors.

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From 3487 ESTs developed by Gesteira et al. (2007), 505 EST-SSRs were identified, with three types of motifs: dinucleotides (72.1%), trinucleotides (27.3%) and tetranucleotides (0.6%) (Lima et al., 2008, 2010). A high frequency of SSRs was found at the 50 untranslated region (UTR) and in the Open Reading Frame (ORF) (about 27%), while a low frequency (about 8%) was observed at the 30 UTR. The difference of SSR numbers between the two UTR regions may be related to a longer 50 UTR sequence due to the directional sequencing made from the 50 end for all the ESTs in order to avoid the polyA tail sequencing (Gesteira et al., 2007). In these conditions, the 50 UTR was systematically sequenced, while the 30 UTR was either partially or not sequenced, depending of the ORF length and sequencing capacities. Fortynine EST-SSR primers were designed and evaluated in 21 cacao accessions presenting different resistance levels to witches’ broom disease, with 12 revealing polymorphism with a total of 47 alleles, and an average of 3.9 alleles per locus. Among the 12 polymorphic EST-SSR markers, two were mapped on the Sca6  ICS1 F2 population, reference for witches’ broom disease resistance (Lima et al., 2010). In parallel, the EST collection produced by Argout et al. (2008) provided a larger number of genetic markers found in genes. A total of 2252 SSRs were identified in 2164 unigenes. Dimers and trimers were the most common types and represented 94.2% of SSRs found in unigenes. The poly(AG)n and poly(AAG)n groups were the most abundant motifs in cacao unigenes. A first subset of 314 EST-SSR displaying a similarity with known function genes was screened for its polymorphism on 8 contrasting genotypes, revealing 174 polymorphic SSR, and among them, 115 which could be mapped in the reference map (Lanaud et al., 2006; Fouet et al., in preparation). Dinucleotide repeat loci revealed more polymorphisms (78%) among cacao genotypes compared to those revealed by trinucleotide repeat loci (58%). The polymorphism of SSRs loci differs according to the different gene regions: the protein-coding sequences (CDS) region is the least polymorphic, with 54% of polymorphic loci, compared with non-coding untranslated 50 UTR regions (69%) and 30 UTR regions (82%) (Fouet et al., in preparation). In this study, the 50 UTR region is less polymorphic than the 30 UTR region. This result is in agreement with a better conservation of the coding sequences and the presence of important regions involved in the regulation of gene expression in the 50 UTR that needs to be more conserved. These results differ from Lima et al. (2008) due to the method used for sequencing. Diversity in these EST-SSRs sequences is thought to have significant impact on gene function and regulation (Young et al., 2000). Variation in the length of SSR motifs in non-coding sequences of genes (i.e. promoters, UTRs and introns) may affect the process of transcription and translation through slippage, gene silencing and pre-mRNA splicing as has been

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observed for many human diseases disorders (Kim et al., 2001). EST-SSRs cacao markers, particularly those based on such non-coding untranslated 50 UTR regions could be useful as ‘functional genetic markers’ for various diversity and association mapping studies. SNP markers were more recently defined in cacao. Using 153 resistancerelated sequences from ESTs libraries related to witches’ broom disease resistance (Gesteira et al., 2007), Lima et al. (2009) detected, by in silico analysis, 71 putative SNPs, which remain to be validated. Forty-four per cent of the putative SNPs were found in ORFs, and 32% at the 50 UTR; 42% and 34% of these SNPs were synonymous and non-synonymous, respectively. A large number of SNPs was also identified in the Argout et al. (2008) EST collection. In order to avoid contigs containing paralogues, contigs including 4–100 sequences were selected and allowed to define 5246 SNP in 2012 contigs. Transitions (A/T–G/C) represented 54.2% of the SNPs found, transversions 32.1% and InDels 13.7% (Argout et al., 2008). A panel of 1536 SNP was selected in genes having similarity with known function genes. Based on the Illumina Golden Gate technology, they were used to genotype several mapping and diversity populations (Allegre et al., in preparation). Kuhn et al. (2009) identified SNPs from leaf transcriptome isolated from 20 cacao genotypes from the various genetic groups defined by Motamayor et al. (2008). cDNAs were sequenced using the 454 roche and Illumina GAII (Solexa) technologies. A pool of 285,000 putative SNP was identified with the goal to establish a 30K Illumina Infinium chip. C. GENETIC MAPS FOR QTLS AND ASSOCIATION STUDIES

The first genetic maps were established by Lanaud et al. (1995) and Crouzillat et al. (1996). The map established by Lanaud et al. (1995) was based on an F1 progeny resulting from a cross between two heterozygous cacao genotypes: an Upper-Amazon Forastero (‘UPA 402’) and a Trinitario (‘UF 676’): this ‘reference’ map was successively enriched by all new markers produced (Pugh et al., 2004; Risterucci et al., 2000). More recently, 450 SNP markers were added to this reference map (Allegre et al., (in preparation)). Several other maps dedicated to QTLs analyses were established from various progenies listed in Lanaud et al. (2009). Some consensus maps have been established by Brown et al. (2007) and Lanaud et al. (2008) to be able to carry out meta-QTL analyses. The number of markers and size of these consensus maps were, respectively, 291 markers for 782.6 cM and 676 markers for 807.3 cM. QTLs and association studies are an anchoring step to carry out MAS, as well as to identify the genes underlying the trait variation, to clone them and

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to study their allelic diversity in genetic resources. In cacao, numerous QTLs or association studies have been carried out to study the genetic bases of useful traits. A large number of them were related to disease resistance, particularly resistance to: (i) black pod (Flament et al., 2000; Lanaud et al., 2004a; Motilal et al., 2000; Risterucci et al., 2003); (ii) witches’ broom disease (Albuquerque and Figueira, 2004; Brown et al., 2005; Faleiro et al., 2006; Figueira et al., 2006; Queiroz et al., 2003) and (iii) frosty pod (Brown et al., 2007). QTL analyses were also carried out to study yield factors, seed and fruit traits, quality traits and other morphological or biochemical traits (Cle´ment et al, 2003a,b; Crouzillat et al., 1996, 2000a,b, 2003; Lanaud et al., 1999a,b, 2003, 2004b; N’Goran et al., 2000). Several statistical methods have been described to identify QTLs (Jansen, 1996). Most of the QTLs are generally detected from controlled crosses and characterised by their map position, their contribution in trait variation (R2), their LOD score and confidence interval (CI). The detected QTLs depend on the diversity of the two parents at the origin of the progenies. In cacao, numerous QTL studies have been carried out from controlled crosses to investigate the genetic bases of useful traits. More recently, genome-wide association studies (GWAS) are increasingly being used to study complex trait genetic bases. GWAS allow the detection of a wider diversity of QTLs in a same population. First developed in human genetics to identify genes involved in diseases, its efficiency has been proven in plants, and particularly in cacao, in order to detect genome regions involved in trait variations (Marcano et al., 2007; Pugh, 2005; Schnell et al., 2005). Association mapping studies could be carried out in populations with a large genetic base, which could correspond to wild or cultivated populations, or to germ plasm collections. The number of recombinations following the first population ancestors crosses is influencing the power of resolution of marker/trait association and could reduce the CI of QTL detection. This method is particularly useful for cacao studies for which large controlled progenies are not always available; indeed, recent hybrid populations are generally planted for cultivation and can be the basis for such GWAS studies which are a good complement of classic QTL analyses. In total, about 300 QTLs or marker/trait associations were detected for the various traits (Lanaud et al., 2009). However, QTL mapping or association studies experiments are generally totally heterogeneous, involving different types of populations (F1, F2 and back crosses), variable sample sizes, parents of diverse genetic origins, and under various environmental conditions, different methods of trait evaluation and even different markers to establish genetic maps. A comparative QTL mapping is a difficult but a necessary challenge to synthesize all QTL information for

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cacao improvement using MAS strategies as well as to establish links with functional genomics. Until now, bioinformatic tools, integrated in the CocoaGenDB database (Ruiz et al., 2003; Argout et al., 2006; see Section X) allowed to compare part of the QTLs detected in different maps, using CMAP, a web-based tool that allows users to view comparisons of genetic and physical maps (http://gmod. sourceforge.net/cmap/). This comparison was based on the common markers between maps allows for the alignment of the homologous groups and their corresponding QTLs. Several approaches have been developed more recently for the comparison and integration of multiple QTL mapping experiments, after the establishment of a consensus map. A meta-analysis approach, developed by Goffinet and Gerber (2000) and carried out with the ‘Biomercator’ software (Arcade et al., 2004), allows using the existing published QTL information (location, R2, CI) to determine the most probable real number of QTLs, their position and new CI. Such analyses were made recently by Lanaud et al. (2009) to investigate the genetic bases of disease resistance traits for which 76 QTLs were detected in 16 different experiments located in various countries. The advantages of this meta-analysis strategy is not only to localise all markers and QTLs in a single figure representing the linkage groups but also to synthesise all the information related to a cluster of QTL by identifying consensus QTLs. Moreover, there was a twofold reduction in average CI observed when compared with the CI of individual QTLs (Lanaud et al., 2009). For some traits, like Phytophthora resistance, for which a large body of QTL information exists, this meta-analysis has highlighted genome ‘hot spot’ where QTLs detected in different studies are localised in same genome region. At least eight genomic regions appeared clearly involved in Phytophthora resistance. Some hot spots corresponded to QTLs related to resistance to different species of Phytophthora or even different diseases, suggesting common resistance mechanisms. Such meta-analyses were also carried out for other cacao traits of interest as seed and fruit traits, and developmental traits that also highlighted hot spots gathering QTLs for the same trait identified in very different experiments (Lanaud, unpublished data). The QTLs distribution is not homogenous along the genome and between the chromosomes. It varies from 1.1 QTL/10 cM to 8.74 QTL/cM according to the chromosomes. The detection of QTLs depends on the presence of heterozygous loci at the QTL level, allowing the segregation of markers and phenotypic traits in the progeny. The lower density of QTL present in some chromosome regions could be explained by a lower gene heterozygosity in these genome regions or by a difference in gene space distribution along the genome (Lanaud, unpublished data). A comparative QTL and candidate

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gene mapping are among the first step towards the discovery of genes underlying trait variations. An increased precision of QTL location, provided by such a meta-analysis, will facilitate the establishment of these links between genetic and genomic approaches. In a second step, a refined QTL localisation provided by association studies (or fine mapping) will be necessary before validating candidate gene involvement in a studied trait. Such a fine mapping approach is presently conducted for the positional cloning of a major QTL involved in witches’ broom resistance (Cle´ment et al., 2006).

XI. BIOINFORMATIC RESOURCES FOR CACAO FUNCTIONAL GENOMICS Cacao molecular and phenotypic data are stored in several open access databases: –

The International Cocoa Germ Plasm Database (ICGD), which was developed by the University of Reading (http://www.icgd.rdg.ac.uk/), provides phenotypic information on cacao germ plasm. It comprises around 14,127 different entries with detailed information on genetic and geographic origins, history of collection expeditions, morphology, pest, disease and stress reactions, quality and agronomic characteristics and anatomical data. – TropGENE-DB, which was developed by Ruiz et al. (2004), is organised on a crop basis with presently ten modules (cacao, banana, coconut, coffee, oil palm, rice, rubber tree, sorghum and sugarcane). TropGENE-DB is based on the RDMS MySQL software. The most common data stored in TropGENE-DB are genetic and physical maps, marker information, QTL, sequence data and molecular data on genetic resources. To display genetic or physical maps, the TropGENE-DB system uses CMAP; a web-based map viewer from the GMOD consortium, which operates on relational databases. A development that allows the use of CMAP to display maps from data in TropGENE-DB has been carried out with the Perl language. It is planned to add a link with Gbrowse, the genome browser of the GMOD consortium. TropGENEDB model flexibility allows adding new types of data such as ESTs, BAC libraries, SNPs as they become available. Presently, the cacao module comprises around 500 clones with their genotypes at various markers (RFLP, AFLP, microsatellites, isozymes, etc.), 7 genetic maps and their corresponding detected QTLs, and information on the markers themselves. – CocoaGenDB, which is a Web portal combining molecular genetic information contained in TropGENE-DB with phenotypic data contained

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in ICGD. It has been developed through a collaborative project involving Cirad (France), University of Reading (School of Plants Sciences, UK) and USDA (United States Department of Agriculture). CocoaGenDB is available through the internet at the URL http://cocoagendb.cirad.fr. A userfriendly and intuitive web consultation interface allows the performance of complex queries combining genetic and phenotypic information and clone genealogy. This new database is specifically designed to allow endusers (breeders or molecular geneticists) to best exploit genetic information available on cacao germ plasm (Argout et al., 2006). – ESTtik (Expressed Sequence Tag Treatment and Investigation Kit), which is a specific tool constructed to manage and store cDNA sequences (Argout et al., 2008). ESTtik is an information system that contains a pipeline for processing, a database and a web site publically available (http://esttik.cirad.fr) for querying data. The ESTtik pipeline program is a set of Perl packages which contain a main program related to nine modules in charge of completing different processing. The pipeline executes a series of programs to assess quality of nucleotides from chromatograms, then edits, and assembles the input DNA sequence information into a non-redundant data set. Then microsatellites are searched for in the unigene. It is used as input for an annotation against public databases including an extraction of Gene Ontology terms. All the results produced by automatic processing are finally stored into XML files. The information collected from individual program modules of the pipeline is stored into a MySQL database. The database model was specially designed using the UML technology to fit data. To visualise Blast results, database records can be accessed using seven query pages combining PerlCGI, HTML, Javascript and Flash technologies.

XII. CACAO GENETICS RESEARCH COMMUNITY The cacao research community is widely dispersed worldwide. However, many cacao researchers collaborate on large number of projects involving genomics and plant breeding. To foster these interactions, the International Group for Genetic Improvement of Cocoa (INGENIC) was created in 1994 to promote the exchange of information and international collaboration on cacao genetics and improvement of cacao planting materials. The membership currently includes approximately 248 members, representing 35 developing and developed countries around the world. Members of Ingenic share research progress through publication of a Newsletter and in workshops held every 3 years. Informal communications are facilitated using a listserve email forum. The

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newsletter, proceedings of the workshops and instructions for joining INGENIC can be found on the group web site (http://ingenic.cas.psu.edu/).

XIII. CONCLUSION T. cacao has been neglected for a long time due to its tropical nature and long generation period, with a few number of research teams involved in its study. Nevertheless, cacao is an important commodity which represents a large source of income for more than 20 million people in Africa, Asia, South America and many developing countries. Approximately 90% of the production, mainly from the Ivory Cost, Ghana and Indonesia, are exported as beans or semi-manufactured cacao products to Europe and the USA. Currently, farmers have to face numerous cacao diseases and potentially plant biotechnology and publically available molecular resources could improve the actual farmer scenario. Particularly, cacao genome sequencing, associated with integrated genomic and genetic studies, could certainly accelerate the understanding of the main cacao useful traits (resistance, quality) and breeding.

ACKNOWLEDGEMENT We thank C. Hamelin and J. C. Breitler for supplementary information provided on bioinformatics databases and RNA interference experiments.

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