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

^a USDA -ARS, BARC, PSI, SPCL, 10300 Baltimore Ave. Bldg. 50 BARC-W, Beltsville, MD 20705, USA
^b USDA-ARS, Tropical Agric. Research Station, P.O. Box 70, Mayaguez, PR 00681, Puerto Rico
^c Genetic Engineering & Biotechnology Research Institute (GEBRI), Sadat City, Minufiya Univ., Egypt
^d Molecular Biology, Biochemistry and Bioinformatics (MB3), 360 Smith Hall, 8000 York Road, Towson Univ., Towson, MD 21252, USA

View larger version (11K): [in a new window]   Fig. 1. This electropherogram shows the DNA fragment profiles of SSR primers for three different loci, amplified separately using three different dyes, multiplexed and separated by capillary electrophoresis. The sample was the T. cacao accession PA44, an Upper Amazon Forastero type that is part of the USDA germplasm collection maintained in Puerto Rico. Alleles from three loci were amplified as shown in the electropherogram sample: The heterozygous locus Y16980 (mTcCIR6) is located on chromosome 6 and shows alleles sizes of 228 and 232 base pairs. The two homozygous loci, Y16996 (mTcCIR24) located on chromosome 9 and the Y16995 (mTcCIR22) locus on chromosome 1, each show a single allele at 187 and 290 bp, respectively. Internal standards, labeled with red, are run concurrently with all samples for base pair determination during the capillary electrophoresis DNA fragment separations.

View larger version (10K): [in a new window]   Fig. 2. Sibling probabilities of identity (PID-sib) from 141 cacao accessions in the USDA cacao collection maintained at Mayaguez, Puerto Rico. The probability that two sibling individuals drawn at random from this collection have the same mutilocus genotype became close to zero after seven SSR loci with highest PID-sib were applied.

View larger version (16K): [in a new window]   Fig. 3. Mismatch distribution of 141 cacao accessions in the USDA cacao collection maintained at Mayaguez, Puerto Rico. Computer program MM-DIST (Kalinowski et al., 2006) was used for computation of mismatch distribution. The data set from the single genotyping was used to compute the probability that two individuals differ at k loci. Both empirical and expected distributions showed that the full siblings will likely differ by at least three out of 15 loci in their multilocus SSR profiles. The unrelated individuals will likely differ by at least five out of 15 loci. The error mismatch distributions are therefore unlikely to overlap with distributions of true genotypic difference (McKelvey and Schwartz, 2004; Kalinowski et al., 2006).

View larger version (12K): [in a new window]   Fig. 4. The probability of obtaining a consensus genotype (at least two correct microsatellite genotypes) for a given number of repeated genotypings (independent PCRs). The simulation was based on the observed error rate in an independently repeated genotyping experiment as described in this paper. The simulation result shows that 98.7% of the consensus genotype can be identified with a minimum of three PCR repetitions, and 99.7% of the consensus genotypes can be identified with four PCR repetitions. Further increase of PCR repetitions offers little further improvement in terms of genotyping accuracy.