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.