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The size of the haploid genome of Theobroma cacao L. (cacao), estimated using laser flow cytometry, was 0.43 pg. An improved DNA extraction procedure was developed based on isolation of a crude nuclei preparation from leaf tissue that effectively eliminated contamination of the DNA by polysaccharid es and produced DNA that was, on average, longer than 50 kb. DNA yields ranged from 2 to 10 µg·g -1 fresh weight of leaf tissue. DNA blot hybridization experiments with a flax (Linum usitatissimum L.) ribosomal DNA probe revealed restriction fragment length poly- morphisms between three cacao genotypes. Differences between the DNA of these genotypes were also detected by polymerase chain reaction amplification of polymorphic DNA fragments, using random oligonucleotide primers. Theobroma cacao, the source of cocoa solids and cocoa but- ter, is a major tropical crop. Despite its economic importance, breeding has not had a major impact on the cacao industry, especially in the New World (Kennedy et al., 1987). The per- formance of cacao "hybrid seed" (actually crosses of selected heterozygous clones) has been disappointing (Hunter, 1990), and clonal propagation has not been widely adopted because of dimorphic growth and costs. Although major efforts have been placed on germplasm collection and maintenance (Allen, 1987; Allen and Lass, 1983), characterization and evaluation of these resources have been minimal. Further, the collections at many sites present problems of duplication and/or misidentification of accessions. Some devastating cacao diseases cannot be con- trolled effectively, indicating the need for genetic solutions. These diseases include witches' broom caused by Crinipellis perniciosa (Stahel) Singer in South America, vascular-streak dieback caused by Oncobasidium theobromae (Talbot & Kenea) in Oceania, and swollen shoot virus in West Africa. Clearly, new approaches are needed for genetic improvement of cacao. For a successful long-term breeding program, genetic infor- mation from well-evaluated parents is necessary. The availabil- ity of molecular techniques has opened new ways to characterize germplasm, to determine genetic diversity, and to establish gene linkages of agronomic traits to molecular markers. An efficient DNA extraction method is a prerequisite for such molecular genetic studies. The only published technique for cacao DNA isolation (Couch and Fritz, 1990) is complex, and although it overcomes polyphenol contamination, it does not take into con- sideration the problem of polysaccharide gums that are ubiqui- tous in cacao (Figueira et al., 1991). The objective of this study was to establish suitable molecular techniques for cacao. This included estimation of the size of the cacao genome, development of a reliable method of DNA ex- traction, and evaluation of the degree of polymorphism found within the species through traditional restriction fragment length polymorphism (RFLP) methods and the new method of random amplified polymorphic DNA (RAPD) analysis (Welsh and McClelland, 1990; Williams et al., 1990).

Genome Size and DNA Polymorphism in Theobroma cacao