Probing the genetic population structure of Trypanosoma cruzi with polymorphic microsatellites

Abstract

We describe here the identification of eight polymorphic microsatellite loci with (CA)n repeats in the Trypanosoma cruzi genome based on the affinity capture of fragments using biotinylated (CA)12 attached to streptavidin-coated magnetic beads. The presence of two peaks in PCR amplification products from individual clones confirmed that T. cruzi is diploid. Hardy-Weinberg and linkage disequilibrium analyses suggested that sexual reproduction is rare or absent and that the population structure is clonal. Several strains, especially those isolated from nonhuman sources, showed more than two alleles in many loci demonstrating that they were multiclonal. The phylogenetic analysis of T. cruzi based on microsatellites revealed a great genetic distance among strains, although the strain dispersion profile in the Wagner network was in general agreement with the species dimorphism found by PCR amplification of the divergent region of the rRNA 24Salpha gene.

Figure 1

Evaluation of enrichment for (CA)_n-containing fragments after affinity capture. We show an ethidium bromide-stained agarose gel (A) and a Southen blot hybridized with an alkaline phosphatase-labeled (CA)₈ probe (B). Lanes: 1, T. cruzi DNA digested with Sau3AI; 2, T. cruzi DNA digested and ligated with Sau3AI adapters; and 3, T. cruzi DNA digested with Sau3AI with adapters after capturing the (CA)_n-containing fragments. Molecular size markers were from a 1-Kb ladder (Life Technologies, Gaithersburg, MD) are indicated.

Figure 2

(A) Tracing of electrophoresis of alleles of the MCLE03 microsatellite locus of the Y strain and three of its clones. (B) Tracing of electrophoresis of alleles of the SCLE10 locus of different T. cruzi strains. Strain 1023 shows four different alleles, indicating multiclonality.

Figure 3

Unrooted Wagner network of 20 T. cruzi strains based on the amplification of eight polymorphic loci with (CA)_n repeats. Considering each microsatellite allele as one state from a multistate character, the genetic distance between any two strains was estimated as the number of mutational steps necessary to transform one into the other. The figures in parenthesis indicate the number of times that the branch was observed in 1,000 bootstraps. Bootstrap values inside the lower cluster (below strain 138) were all low (below 300) and are not given individually. The classification of each strain indicated in italics (1, 2, or 1/2) was based on the rRNA 24Sα amplification.