J. AMER. SOC. HORT. SCI. 119(5):1073–1082. 1994. Reexamining the Classification of Theobroma cacao L. Using Molecular Markers Antonio Figueira1, Jules Janick2, Morris Levy3, and Peter Goldsbrough Department of Horticulture, West Lafayette, IN 47907-1165 Additional index words. RFLP, RAPD, Herrania, cocoa, taxonomy Abstract. Genetic similarities among eight Theobroma and two Herrania species, including 29 genotypes of T. cacao, were estimated by rDNA polymorphism. A phenogram based on these genetic similarities significantly separated two clusters: one cluster included all Herrania and Theobroma species, except T. cacao, while the second contained 28 of 29 T. cacao genotypes. There was no clear distinction between Herrania and Theobroma species. Separation of 29 T. cacao genotypes, representing all races and various origins, had no congruency with the conventional classification into three horticultural races: Criollo, Forastero, and Trinitario. Genetic similarities in T. cacao, estimated with RAPD markers, indicated continuous variation among the generally similar but heterogeneous genotypes. The wild genotypes formed an outgroup distinct from the cultivated genotypes, a distinction supported by the rDNA data. The phenograms constructed from RAPD and rDNA data were not similar within the wild and cultivated cacao subsets. The genus Theobroma consists of small understory trees of the and have been considered as subspecies (Cuatrecasas, 1964). lowland neotropical rainforest from the Amazon basin through Domesticated in Meso-America, the Criollo race is characterized southern Mexico (Purseglove, 1968). Cuatrecasas (1964) recognized by having pods that are red or yellow when ripe, deeply furrowed, 22 species and divided the genus into six sections, based on mode of warty, and with a pointed end and a thin husk. Criollo seeds are germination, branching pattern, and flower morphology. Interspe- plump with white or pale violet cotyledons, and have superior cific crosses can produce viable hybrids and certain species are flavor. Historically, all nonCriollo cacaos were considered to be graft compatible (Addison and Tavares, 1951). Most species have Forastero, but the latter name is currently used to describe cacao a restricted geographical distribution, with the Andes establishing types that have nonpigmented pods, with thick, hard pod husks, a major separation between species (Baker et al., 1953). and flat, dark purple seeds. Forastero can be subdivided into Upper The genus Herrania has a similar geographic distribution, with Amazonian [wild or semi-wild cacaos as described by Pound only one species occurring in Central America and the other 16 (1938)] and Lower Amazonian, characterized by a rather uniform limited to South America (Schultes, 1958). Herrania species are pod type called Amelonado (nonpigmented, smooth, melon-shaped morphologically similar to Theobroma, being distinguished by the pods with a blunt end). Lower Amazonian cacaos now constitute unbranched orthotropic growth, palmately compound leaves, very the most prevalent cultivated type worldwide, grown notably in long petal laminae, and nonconvoluted cotyledons (Baker et al., West Africa and Brazil. These types grow wild in the Guyanas and 1953). Until the revision of Schultes (1958), Herrania was consid- the eastern Brazilian Amazon, and may have been partially domes- ered a section of Theobroma (Cuatrecasas, 1964). The chromo- ticated by pre-Colombian Amazonian natives for their aromatic some number for both genera is 2n = 20 (Muñoz Ortega, 1948). pulp (Barrau, 1979). Cacaos with red pods have not been found in Theobroma cacao (cacao) is the only species of major eco- the Amazon region, and pod color remains the unique characteris- nomic importance, because its oil-rich seed is the unique source of tic that discriminates Amazonian and other populations (B.G.D. cocoa solids and cocoa butter. Cacao was domesticated in pre- Bartley, unpublished). Colombian times by Meso-American natives (Bergmann, 1969). Trinitario is considered to be either an intermediate type be- Cuatrecasas (1964) postulated that a natural cacao population once tween Criollo and Forastero (Lockwood and Gyamfi, 1979) or a extended from the Guyana-Amazon region through southern group of hybrids that display characteristics that include the total Mexico and ultimately developed into two forms separated by the range of variation (Cheesman, 1944). Trinitario types have not Panama isthmus. Cheesman (1944) proposed the headwaters of the been found under wild conditions. Because Criollos and Forastero Amazon basin region as the center of diversity for cacao. Isozyme types are so heterogeneous, the resulting hybrids might not be studies by Lanaud (1987) have confirmed that populations found distinct from the parental populations, making Trinitarios impos- in this region exhibit the greatest genetic diversity, and contain the sible to define except by geographical origin (B.G.D. Bartley, majority of known isozyme alleles. unpublished). Consequently, the race classification system based Various attempts have been made to categorize cacao geno- on pod and seed characters is, at best, imprecise. types into three horticultural races (i.e., Criollo, Forastero, and Isozyme markers have been used in an attempt to validate the Trinitario) based on morphological descriptors and geographic conventional classification, but limited polymorphism does not origin (Engels, 1981; Enriquez and Soria, 1967). Criollo and allow discrimination between cacao types (Lanaud, 1987). The Forastero races have distinct historical and commercial features, development of molecular genetic markers such as restriction fragment length polymorphisms (RFLPs) and random amplified polymorphic DNAs (RAPDs) has made it possible to evaluate Accepted for publication 3 Mar. 1994. Received for publication 12 Nov. 1993. Journal paper no. 13,791 of the Purdue Univ. Agricultural Experiment Station. The phylogenetic relationships more efficiently (Figueira et al., 1992; cost of publishing this paper was defrayed in part by the payment of page charges. Lerceteau et al., 1992; Wilde et al., 1992). Under postal regulations, this paper therefore must be hereby marked advertise- The objectives of this study were 1) to use molecular markers ment solely to indicate this fact. to examine phylogenetic relationships among various Theobroma 1Current address: Alimirante Cacau, Caixa Postal 55, Itajuipa, Bahia, Brazil. 2To whom reprint requests should be addressed. and Herrania species based on RFLPs of ribosomal RNA genes 3Dept. of Biological Sciences. (rDNAs) and 2) to determine if the organization of genetic diver- J. AMER. SOC. HORT. SCI. 119(5):1073–1082. 1994. 1073 sity among a broad geographic sample of Theobroma cacao 1981), and was labelled with 32P-dCTP by random priming using genotypes examined with rDNA polymorphisms and RAPDs was a Decaprime kit (Ambion, Austin, Texas). The hybridization congruent with the traditional separation of T. cacao into the conditions were as described by Goldsbrough et al. (1990). horticultural races Criollo, Forastero, and Trinitario. Random amplified polymorphic DNAs. Amplification reactions were performed as described by Williams et al. (1990) with minor modifica- Materials and Methods tions. The total reaction volume was 25 µliters, containing 10 mM Tris- M M µM HCl (pH 8.8), 50 m KCl, 1.5 m MgCl2, 0.1% Triton X-100, 100 Plant material. Thirty-eight accessions of Theobroma and dATP, dCTP, dGTP, TTP, 0.2 µM primers, 25 ng genomic DNA, and 1.5 Herrania, including 30 genotypes of T. cacao representing various units Taq DNA polymerase (Promega Corp., Madison, Wis.). The populations and origins, were collected at the International Cocoa primers used in this study were from Operon (Alameda, Calif.), Kits B and Germplasm repository, Centro Agronomico Tropical de C, with the following sequences: B01, GTTTCGCTCC; B03, Investigacion y Ensenanza, Turrialba, Costa Rica. The origin and CATCCCCCTG; B04, GGACTGGAGT; B05, TGCGCCCTTC; B07, description of genotypes are given in Tables 1 and 2 and Fig. 1. GGTGACGCAG; B08, GTCCACACGG; B10, CTGCTGGGAC; B11, DNA extraction method. Young leaves from each accession GTAGACCCGT; B12, CCTTGACGCA; B13, TTCCCCCCGCT; B15, were brought into the laboratory, washed in distilled water, blotted GGAGGGTGTT; B16, TTTGCCCGGA; B17, AGGGAACGAG; B18, dry, and frozen in liquid nitrogen. The frozen leaves were kept in CCACAGCAGT; C02, GTGAGGCGTC; C04, CCGCATCTAC; C05, dry ice, transported to the United States, and stored at –70C for up GATGACCGCC; C06, GAACGGACTC; C08, TGGACCGGTG; C09, to 6 months. DNA was extracted from frozen leaves using the CTCACCGTCC; and C13, AAGCCTCGTC. Amplification was con- method described by Figueira et al. (1992). Final DNA concentra- ducted in a Cetus DNA thermalcycler (Perkin-Elmer, Norwalk, Conn.) tions were measured spectrophotometrically. for 45 cycles of 1 min at 94C, 1 min at 37C, and 1 min at 72C, followed DNA hybridization experiments. DNA samples (0.5 µg) were by a final incubation at 72C for 7 min, using the most rapid transition time digested for 4 h with 20 units of DraI, EcoRV, EcoRI, (Promega between temperatures. Products were analyzed on 1.4% agarose gels run Corp., Madison, Wis.), or BamHI (New England Biolabs, Beverly, at 6.4 V/cm in TAE buffer. Mass.). The digested DNAs were separated on a 0.7% agarose gel Statistical analysis. Ribosomal DNA restriction fragments and using a voltage gradient of 2 V/cm in TAE buffer (40 mM Tris-base, RAPD products were scored visually