Accuracy and Reliability of High-Throughput Microsatellite Genotyping for Cacao Clone Identification

Microsatellite-based DNA fingerprinting has been increasingly applied to cacao (Theobroma cacao L.) genotype identification. However, the accuracy and reliability of using high throughput microsatellite analysis for cacao clone identification have not yet been rigorously assessed. Despite the use of highly robust fingerprinting protocols, cacao genotype identification has been affected by genotyping errors, which potentially mislead the result of clone identification. In this paper, we calculated the probability of identity for 15 selected microsatellite loci. We then quantified the genotyping error rate through repeated genotyping and simulated the impact of the genotyping error on cacao clone identification. Allelic dropout (ADO), or failure to amplify one allele for a heterozygous locus, and false allele (FA), or an amplicon size error by the polymerase, accounted for 48 and 52% of the genotyping inconsistencies, respectively. The result of simulation showed that 99% of the consensus genotype can be generated for the ambiguous loci through a minimum of three polymerase chain reaction (PCR) repetitions. On the basis of the error rate and probability of identity (PID), we designed a genotyping scheme and applied it to the cacao germplasm held in the USDA cacao collection at Mayaguez, Puerto Rico. Out of the 141 samples, we unambiguously identified nine duplicated groups consisting of 34 cacao accessions. This genotyping scheme is being implemented in large scale fingerprinting of cacao germplasm. CACAO is an important tropical crop, native to the South American rainforest (Cuatrecasas, 1964; Coe and Coe, 1996; Young, 1994). The species comprises a large number of highly morphologically variable and mutually interfertile populations. A cacao pod may contain numerous seeds, but the seeds do not remain viable for much longer than a week once the pod has been harvested (Coe and Coe, 1996). As a result, cacao germplasm collections must be maintained as clonally propagated, living trees. A dozen major cacao germplasm collections in tropical regions of the world serve as germplasm repositories. The genetic diversity is not fully characterized. A number of molecular tools have been used to examine cacao populations by DNA fingerprinting procedures and a large number of cacao accessions in these collections have been reported to be misidentified, duplicated, or to have no identification at all (Kennedy and Mooleedhar, 1993; Lockwood and End, 1993). The incorrect labeling of accessions is a major limitation to efficient conservation of cacao germplasm. Further, this constraint impedes the progress in genetic improvement of cacao throughout the world. The development of simple sequence repeats (SSR) markers in cacao (Lanaud et al., 1999) has significantly enhanced the capacity of molecular characterization of cacao germplasm. This technique has been applied for cacao clone identification (Cryer et al., 2006; Saunders et al., 2004), parentage analysis (Schnell et al., 2005), diversity assessment (Lanaud et al., 1999, 2001), and investigation of the origin and dispersal of cacao (Motamayor et al., 2002, 2003). The USDA, together with its collaborating institutions, has undertaken a program to identify cacao genotypes and describe the genetic diversity of living plant germplasm collections that are maintained in 10 to 12 national and international collections located within tropical cacao growing countries of Central and South America. During international forums held in England and in France in 2001, a consortium of scientists and representatives from the cacao industry, academic centers involved in cacao research, and representatives from multiple international, government-sponsored laboratories reached an agreement that a set of standardized SSR primers would be used to characterize all T. cacao germplasm collections (Saunders et al., 2001, 2004). The strategy for the identification of mislabeling is to develop a reference SSR profile for each original accession. Then the reference SSR profiles will be compared with the putatively mislabeled accessions. Corrections will then be made as necessary (Turnbull et al., 2004). However, the accuracy and reliability of using high throughput microsatellite analysis for cacao clone identification have not yet been rigorously assessed. Several questions must be addressed if this tool is to be applied for large-scale genotyping of cacao germplasm. First, we need to know if the set of proposed SSR loci have enough discriminating power to establish unique multilocus profiles. If too few loci are examined, multilocus genotypes in a population may not be unique; thus, closely related clones will be indistinguishable. The problem of finding different clones with the same SSR profile can be solved by increasing the number of loci examined, so that the probability that two different clones have the same multilocus genotype is small. This probability, often called the “probability of identity” (PID) in forensic science, can be estimated from the allele frequencies in a population (Waits et al., 2001). Dapeng Zhang and Sue Mischke, USDA-ARS, BARC, PSI, SPCL, 10300 Baltimore Ave. Bldg. 50 BARC-W, Beltsville, MD 20705, USA; Ricardo Goenaga, USDA-ARS, Tropical Agric. Research Station, P.O. Box 70, Mayaguez, PR 00681, Puerto Rico; Alaa A. Hemeida, Genetic Engineering & Biotechnology Research Institute (GEBRI), Sadat City, Minufiya Univ., Egypt; James A. Saunders, Molecular Biology, Biochemistry and Bioinformatics (MB3), 360 Smith Hall, 8000 York Road, Towson Univ., Towson, MD 21252, USA. Received 2 Jan. 2006. *Corresponding author ([email protected]). Published in Crop Sci. 46:2084–2092 (2006). Plant Genetic Resources doi:10.2135/cropsci2006.01.0004 a Crop Science Society of America 677 S. Segoe Rd., Madison, WI 53711 USA Abbreviations: ADO, Allele dropout; FA, false allele; PID, probability of identity; SSR, single sequence repeats. R e p ro d u c e d fr o m C ro p S c ie n c e . P u b lis h e d b y C ro p S c ie n c e S o c ie ty o f A m e ri c a . A ll c o p y ri g h ts re s e rv e d . 2084 Published online September 8, 2006