Leishmania AFLP: paving the way towards improved molecular assays and markers of diversity.

Diversity, phylogenetic, and population genetic studies of the genus Leishmania, causative agent of leishmaniasis, nowadays generally involve multilocus microsatellite and multilocus sequence typing. Even though these are well established and useful applications, amplified fragment length polymorphisms (AFLP) can provide complementary information. In addition, as the technique essentially probes the entire genome at random, without prior sequence knowledge, it is ideally suited as a screening tool for molecular markers linked with biological and clinical traits. We developed an AFLP protocol adapted to the Leishmania genome, tested its repeatability, and validated it on a panel of samples from the Leishmania donovani complex previously analyzed by multiple molecular tests. The technique proved highly reproducible, and showed that genetic relationships between L. donovani strains generally reflect geographic distance. Four main groups were identified: Leishmania infantum, African L. donovani, Indian L. donovani, and a mixed group consisting of L. donovani from India and Africa. Results were highly congruent with previous analyses on essentially the same sample set, indicating that the developed assay produces trustworthy data. This opens possibilities for application in studies of speciation and population dynamics. Moreover, it allows random screening of the entire Leishmania genome for linkage with biological and clinical parasite properties, such as fitness, drug resistance, and disease profile.