Entry: USDA/ARS Colleagues and Bill Guyton, World Cocoa Foundation

Through the Cocoa Borlaug Fellowship Program, the U.S. Department of Agriculture/Foreign Agricultural Service and the World Cocoa Foundation have supported the Cocoa Research Institute of Ghana’s breeding program. In 2008, the program supported cocoa breeder Abu Dadzie to complete a fellowship with the U.S. Department of Agriculture/Agricultural Research Service (USDA/ARS) in Miami. The Cocoa Research Institute of Ghana (CRIG) then supported him to continue his stay into mid-2009. In August/September 2009, USDA/ARS Miami supported Kathleen Kelley to travel to Ghana to provide training to CRIG’s laboratory technicians. Below is an excerpt from the report on her work.

A problem facing cacao breeders is the potential for misidentification of the clones used to produce hybrid seedlings through mislabeling or errors in clonal propagation. A recently developed genetic test, the single nucleotide polymorphism (SNP) assay, can identify such mislabeling. Two significant advantages of the SNP assay over the currently more commonly used microsatellite genetic marker assay are that SNPs can be reliably assayed without expensive electrophoretic equipment and that the SNP assay results in an unambiguous genotype requiring no further post-assay analysis as is the case with microsatellites. The SNP assay is a small first step to making reliable genotyping available in cocoa producing countries.

In August 2009, the Cocoa Research Institute of Ghana (CRIG) acquired a fluorescent plate reader and the probes and primers necessary to do SNP assays of cocoa plants. Performing the assays, however, requires some technical expertise. The training provided at the installation of the plate reader consisted of a PowerPoint presentation by the installer on the potential uses of the equipment. As the technicians in the laboratory were unfamiliar with both the equipment and the specialized SNP assay that the equipment was for, additional training was necessary. Dr. David Kuhn, one of the molecular biologists at the USDA-ARS Subtropical Horticulture Research Station (SHRS) lab involved in the cocoa genotyping project, suggested that someone familiar with both the techniques and with the Ghanaian work environment be sent to Ghana to train the laboratory technicians. Kathleen Kelley was chosen for this job. Kathleen is currently a graduate student in Chemistry at the University of Illinois at Urbana-Champaign and recently returned from two years (2006-8) teaching secondary school science in Ghana in the Peace Corps. After being trained in the SNP assay at the USDA station in Miami, FL, Kathleen returned to Ghana for a month to teach the CRIG staff the procedures to quantify the amount of DNA and subsequently run SNP assays on their cocoa samples. The extended stay was designed to assure that the operators became completely comfortable with the theory and operation of the plate reader and the assays that they would be performing, as well as to allow for any delays from equipment malfunction or difficulty in shipping.

DNA Quantification
Over the course of the month, Kathleen trained the permanent technicians at the CRIG Molecular Biology laboratory in two assays. The first was a simple quantification protocol that measured the amount of DNA in an extracted sample by binding a fluorescent SYBRGreen dye to the DNA and comparing the fluorescence to a standard curve. After the practice gained in making a satisfactory standard curve, however, the group had few problems getting decent results in the SYBRGreen assay and figuring the volumes of sample needed to do the polymerase chain reactions (PCR) that are part of the SNP procedure. The CRIG lab anticipates being able to quantify DNA for cocoa and all the other plant species involved in their research.

SNP Assays
The standard laboratory procedure for genetic analysis is a polymerase chain reaction (PCR) of DNA fragments isolated from individuals of interest, followed by agarose gel electrophoresis of the PCR products and comparison of electrophoretic banding patterns among individuals. This procedure can take upwards of an hour and frequently gives uncertain results. The SNP assay in which Kathleen was training the laboratory technicians completely eliminated the agarose gel stage and substituted, instead, a two-minute fluorescence reading on the plate reader before and after PCR. Because the PCR reaction was quite robust and the samples were classified qualitatively according to the differences in their pre-and post-reads, careful mixing and pipetting were not as much of a challenge as they were in the DNA quantification, where the samples were compared to an external standard curve. Problems were encountered when samples failed to amplify enough in the PCR to give a fluorescence signal that allowed the technicians to definitively say whether the sample was homozygous or heterozygous at that allele. However, because the fluorescence read took only two minutes and was completely non-destructive, the solution to this problem was that if a sample failed to amplify, it was simply put through another round of PCR, and the readings on the sample were retaken. This process allowed the technicians to genotype samples that they were not able to assign the first time. This solution would not have been possible with an agarose gel procedure; in addition to the time cost, agarose gels would render the sample useless for any further analysis.

Equipment in DNA analysis lab

Uses for the SNP Assay
The SNP assays are very powerful tools for genotyping individuals. An example of the data that the new assays can now provide CRIG researchers and plant breeders came from samples that the technicians were using for training. They were given five samples of the PA 150 clone included in the CRIG clonal stock. The genotypes for four of the samples of that clone were the same at each SNP tested. One sample, however, was consistently different from the other four. Either this sample had been mislabeled in the collection, or, more seriously, a tree that CRIG was using in its breeding program was not what the breeders believed it to be. Kathleen and the technicians were able to draw this conclusion just by looking at the results of the first test; running subsequent SNP assays that used other probes and primers simply confirmed what they already suspected. In this instance, the assay showed that something was wrong because the unusual sample was compared to other samples in the same sample set that were theoretically genetically identical. As time goes by, however, CRIG will build up a database of SNP genotypes for all of their clones, so they will be able to compare a sample with their records to see if the sample is what they expect it to be. The assay will also be used to identify clones in the seed gardens, and to make sure that crosses have genotypes consistent with the DNA of the parents.