Example Student Project

by J-D Swanson, A.C. Lee and M. J. Guiltinan
Penn State University

Introduction

In fall of 2000, at the INGENIC meeting in Malaysia, a presentation was made by Dr. James Saunders of the USDA reporting on the development of a program to fingerprint most if not all cacao germplasm in the international collections using microsatellite markers. Microsatellite markers are small repetitive DNA sequences dispersed in genomes (Tautz, 1989). The lengths of these elements are hyper-variable and thus are highly polymorphic, making them ideal in genomic fingerprinting applications (Devey et al., 2002; Rahman and Rajora, 2002; Testolin et al., 2000). It was generally agreed that a cacao fingerprint database would be useful in establishing the genetic diversity of the collections, in understanding the relatedness between clones, in evaluating labeling consistency and mistakes, and in validation of the identities of clones that have been transferred between germplasm collections.

A discussion followed as to the adaptability of the method to different labs, its reproducibility and the ability to share data across platforms. An agreement was made to test the established protocols in several participating laboratories to validate the reproducibility of the method and establish agreed upon, international standards for cacao genomic fingerprinting. In November of 2000 , Dr. David Butler distributed leaf samples from eight cacao accessions and in January of 2001, Dr. Saunders distributed sequences of 15 microsatellite primers chosen from the CIRAD collection as optimal for the test (Lanaud et al., 1999). We present here, the results of the testing done at Penn State University with these materials. It is hoped that other participating laboratories can use this data to compare with their own, and eventually all the data will be combined into one summary document.

Cacao Plant Materials and DNA Extraction

The Trinidad Cocoa Research Unit provided all plant materials. The genotypes tested were PA30 T1, LX31, PA30 T10, GU114P, PA30 T5, GS4/4A, LCTEEN 68-1, and IMC47. DNA was extracted using the Qiagen DNeasy DNA extraction kit and the recommended manufactures protocol. Once quantified, the DNA samples were stored at a concentration of 10ng/mL at 4°C.

Primers

Fifteen fluorescent microsatellite primers were obtained from the US Department of Agriculture: mTcCIR7, mTcCir18, mTcCIR40, mTcCIR33, mTcCIR1, mTcCIR60, mTcCIR22, mTcCIR24, mTcCIR15, mTcCIR11, mTcCIR12, mTcCIR26, mTcCIR37, mTcCIR6, mTcCIR8. The first six primers listed had annealing temperatures of 51°C, while the remaining nine had annealing temperatures of 46°C. The sequences of individual primers may be found in (Lanaud et al., 1999).

PCR and Electrophoresis

PCR was carried out in 25mL total volume containing the following final concentrations: 1x GeneChoice Reaction Buffer (PGC Scientifics; 100 mM Tris-HCl pH 8.5, 500 mM KCl, 15 mM MgCl₂, 1% Triton X-100), 1 mM dNTP, 300 nM of both forward and reverse primer, 0.5u Taq DNA polymerase, 30 ng of DNA. The reactions were incubated in a Perkin-Elmer GeneAmp 9700 thermocycler for an initial melting step of 94°C for 3 min, followed by 30 cycles of a melting step of 94°C, and annealing step of 46°C or 51°C dependant on the primers used, and an elongation step of 51°C. Once the thirty cycles were complete the reactions were incubated at 72°C for 7 minutes and then stored for electrophoresis at 4°C. Each PCR was replicated a total of three times.

PCR reactions were separated on an ABI 3100 automated DNA sequencing apparatus. Electrophoresis was carried out in 36 cm capillaries with the POP4 polymer at 60°C at 15 kV for 1350 sec with an injection time of 22 sec. Individual PCRs were separated with 0.5 mL of an internal size standard (X-Rhodamine MapMarker, Bio Ventures Inc.). This allowed accurate sizing of microsatellite bands by the Perkin Elmer Genotyper software.

Data Collection

The resulting microsatellite fragment sizes were recorded using the P-E Applied Biosystems Genotyper software. In most cases, more than a single or pair of band(s) were produced, as would be expected by co-dominant markers such as microsatellites. In a co-dominant case it would be expected that any one genotype would have two bands present if it was heterozygous for that marker, or a single band if it was homozygous for that marker. Since we often scored more than two bands per primer pair and we did not have access to parental genotypes the data was treated as being dominant in nature and scored in a binary fashion.

Eleven of the fifteen primers amplified clear DNA fragments as expected; the remaining four primers (mTcCIR40, mTcCIR33, mTcCIR12, and mTcCIR6) failed to prime amplification despite repeated attempts. For the eleven primers that did give good amplification, three replicate reactions were compared and the resulting fragments were regarded as being reproducible if they appeared in two out of the three replicates. Bands that appeared only once were regarded as being PCR artifacts and were discarded from the analysis. Next we compared the fragments produced by each genotype, and discarded any monomorphic (non-informative bands) that appeared in all DNA samples. We also found that some fragments which were within one base pair in size of one another. These fragments were considered to fall within the accuracy of the ABI 3100 and thus were considered as being the same, and were expressed as a range of sizes for further analysis.

Results and Discussion

The results were then summarized and presented in binary form (Table 1). From this table it can be seen that the eleven primers are more than sufficient to clearly distinguish among the eight genotypes tested. The primer pairs produced from two to twelve individual DNA fragments which were both reproducible and polymorphic with genotypes tested. A total of 61 such polymorphic markers were scored. However, 33 contained markers that were seen in only two of three amplifications, indicating some variability in the reproducible production of these fragments (Table 1, fragments indicated with a *). The lack of amplification with some of the primer pairs and the lack of reproducibility in the amplification of certain individual fragments, highlights the need for development of unified, accepted international standards for genotype mapping. It will be interesting to see if the other participants in this ring test see similar variability in the amplification of the same fragments. Nonetheless, this method was shown to be very informative in our hands, for the molecular discrimination between the genotypes tested.

It is hoped that by publishing these results that other laboratories involved in the cacao ring test will be able to compare their data to ours. If discrepancies are present then discussion should be made to resolve these issues, so that a definitive protocol for microsatellite fingerprinting can be used globally defined. Our data can also be accessed at the Guiltinan Lab Website at The Pennsylvania State University.

Acknowledgements

The authors would like to thank Dr. J. Saunders for kindly providing us with the fluorescent PCR primers, Dr. David Butler of The University of West Indies-Trinidad Cocoa Research Unit for the cacao leaf material, Dr. Debora Grove of The Penn State University Nucleic Acid Facility, John Carlson for his advice and the use of the Genetyper software and the members of the Guiltinan Lab, in particular, Sharon Pishak, Joe Verica, and Siela Maximova for their discussions and advice.

Correspondence

Mark J. Guiltinan
306 Wartik Building
Penn State University
University Park, PA 16802
USA

Tel: 814 863-7957

E-mail: mjg9@psu.edu

References

Devey, M.E., J.C. Bell, T.L. Uren, and G.F. Moran. 2002. A set of microsatellite markers for fingerprinting and breeding applications in Pinus radiata. Genome. 45: 984-989.

Lanaud, C., A.M. Risterucci, I. Pieretti, M. Falque, A. Bouet, and P.J. Lagoda. 1999. Isolation and characterization of microsatellites in Theobroma cacao L. Mol Ecol. 8: 2141-2143.

Rahman, M.H.and O.P. Rajora. 2002. Microsatellite DNA fingerprinting, differentiation, and genetic relationships of clones, cultivars, and varieties of six poplar species from three sections of the genus Populus. Genome. 45: 1083-1094.

Tautz, D. 1989. Hypervaribility of simple sequence as a general source for polymorphic DNA markers. Nucleic Acids Research. 17: 6463-6471.

Testolin, R., T. Marrazzo, G. Cipriani, R. Quarta, I. Verde, M.T. Dettori, M. Pancaldi, and S. Sansavini. 2000. Microsatellite DNA in peach (Prunus persica L. Batsch) and its use in fingerprinting and testing the genetic origin of cultivars. Genome. 43: 512-520.

Accessions

Table 1. Binary tabular fingerprinting data for each of eight cacao genotypes using eleven microsatellite primers. Column 1 indicates the primer pairs used, column 2 is the sizes in base pairs of DNA fragments that were amplified by the respective primer. Cacao accessions 1-8 are as follows: PA30 T1, PA30 T10, LCTEEN 68-1, LX31, IMC47, GU114P, GS4/4A and PA30 T5. 1 represents the presence of a band, while 0 represents the absence of the band. A * indicates a fragment that only amplified two out of three replicate experiments times. An example is that we would expect the genotype PA30-T1 to have markers at 132-133 bp, 134 bp, 153-155 bp, 156-158 bp, 190 bp, and 215-216 bp when amplified with the primer mTcCIR7.