Typing Plasmodium yoelii microsatellites using a simple and affordable fluorescent labeling method.

The rodent malaria parasite Plasmodium yoelii has been an important animal model for studying malaria pathology and host-parasite interactions. Compared with other rodent malaria parasites such as Plasmodium chabaudi, however, genetic mapping studies on P. yoelii have been limited, partly due to the absence of genetic markers and the lack of well characterized phenotypes. Taking advantage of the available genome sequence, we initiated a project to develop a high-resolution microsatellite (MS) map for P. yoelii to study malaria disease phenotypes. Here we report screening the P. yoelii genome for simple sequence repeats and development of an inexpensive method (modified from a previously reported procedure) for typing malaria parasite MS: instead of labeling individual polymerase chain reaction primers, a single fluorescently labeled primer was used to type the MS markers. We evaluated various polymerase chain reaction cycling conditions and M13-tailed/labeled M13 primer ratios to establish a simple and robust procedure for typing P. yoelii MS markers. We also compared typing efficiencies between individually labeled primers and the M13-tailed single labeled primer method and found that the two approaches were comparable. Preliminary analyses of seven P. yoelii isolates deposited at MR4 with 77 MS showed that the markers were highly polymorphic and that the isolates belonged to two groups, suggesting potential common ancestry or laboratory contaminations among the isolates. The MS markers and the typing method provide important tools for genetic studies of P. yoelii. There is a good possibility that this method can be applied to type MS from other malaria parasites including important human pathogens Plasmodium falciparum and Plasmodium vivax.