Abstract
A collaborative international program was initiated to identify and describe the genetic diversity of living germplasm collections of Theobroma cacao genotypes that are maintained in several international collections scattered throughout tropical cacao growing countries of the world. Simple sequence repeat (SSR) DNA analysis was identified as the most appropriate molecular tool for DNA fingerprinting these collections during an international forum representing academic, government and industry scientists in the cacao community. Twenty-five SSR primers, which had been previously described, were evaluated as potential candidates to define an efficient, standardized, molecular fingerprinting protocol for T. cacao accessions. These primers have been evaluated for reliability, widespread distribution across the cacao genome, number of alleles produced by the SSR primers in cacao and their ability to discriminate between cacao accessions. Approximately 690 cacao accessions were used to evaluate the utility of these SSR primers as international molecular standards, and a small number of test samples of T. cacao were sent to two other independent laboratories for verification. DNA fragments were selectively amplified by PCR, using the SSR primers labeled with fluorescent dyes, and separated by capillary electrophoresis. Based on this study, the 15 SSR primers that had the highest reproducibility and consistency within a common genotype, while allowing the differentiation of separate divergent genotypes, were selected as international molecular standards for DNA fingerprinting of T. cacao.
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Acknowledgements
The authors gratefully thank Dr. Ulrike Krauss from CATIE, Costa Rica, Dr. Ricardo Goenaga from the USDA in Puerto Rico, and Dr. David Butler, Dr. Elizabeth Johnson, and Mr. Oliver Sounigo from CRU, Trinidad and Tobago for providing the cacao plant accessions used in this study. They also thank Dr. Claire Lanaud for recommendations on SSR primers and review of the manuscript and Dr. Mark Guiltinan and Dr. Ray Schnell for the independent verification analysis of the DNA primers.
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Communicated by D.B. Neale
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Saunders, J.A., Mischke, S., Leamy, E.A. et al. Selection of international molecular standards for DNA fingerprinting of Theobroma cacao. Theor Appl Genet 110, 41–47 (2004). https://doi.org/10.1007/s00122-004-1762-1
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DOI: https://doi.org/10.1007/s00122-004-1762-1