Numerous, robust genetic markers for Plasmodium chabaudi by the method of amplified fragment length polymorphism

We have used the method of amplified fragment length polymorphism (AFLP) to identify genetic polymorphisms between two cloned isolates of the rodent malaria parasite Plasmodium chabaudi chabaudi. The method employs polymerase chain reaction (PCR)-amplification of genomic DNA fragments cut with specific combinations of restriction endonucleases; we used EcoRI and Tru1I (isoschizomer of MseI). We have identified 819 parasite clone-specific AFLPs between P. c. chabaudi clones AS and AJ. Of these, 403 fragments were specific to AS and 416 to AJ. In preparing blood stage parasites for DNA, nucleated host cells were removed by successive filtration of infected blood through powdered cellulose and Plasmodipur filters. This reduced nucleated host cell contamination to around 1-10 per million parasite nuclei and reduced host DNA to below the limit of detection by the AFLP method. Analysis of our results showed that the total number of PCR-amplified fragments of parasite DNA was consistent with the predicted number of EcoRI sites in the parasite genome. 19.4% of all amplified fragments were P. c. chabaudi clone-specific. From this figure we estimated that the diversity between clones AS and AJ, measured as the probability of a sequence difference, was between about 8 x 10(-3) and 4.6 x 10(-4) per base pair. This is consistent with the sequence diversity found between alleles of candidate drug resistance genes from P. c. chabaudi clones AS and AJ identified and sequenced in this laboratory.