Linkage mapping and genetic analysis of Trypanosoma brucei

Trypanosoma brucei is a protozoan parasite of major public health and economic importance in sub-Saharan Africa, where it is the causative agent of sleeping sickness in man and Nagana in cattle. The complete genome sequence of T.brucei is now available and the diploid genetic system has recently been demonstrated to be Mendelian. This opens up the possibility of using a classical genetic approach to identify genetic loci that determine important phenotypic traits in this parasite, such as host specificity, drug resistance, and pathogenicity. A genetic map of the non human-infective subspecies, T.b.brucei, has already been assembled and successfully used in quantitative trait analysis of a number of traits specific to this pathogen. This thesis describes the construction of a separate genetic map for the sub-species responsible for > 90% of human African trypanosomiasis infections, T.b.gambiense, which differs significantly from T.b.brucei in many key phenotypes. The genetic linkage map was constructed from the analysis of 119 polymorphic microsatellite markers in a population of 38 F1 progeny, obtained from the genetic cross of a T.b.gambiense group 2 strain, STIB 386, with a T.b.brucei strain, STIB 247. Eleven major linkage groups were resolved, one for each of the megabase chromosomes, resulting in a total genetic map length of 733 cM, and an average map unit size of 24 Kb/cM. The map provides a 90% probability of a marker being within 268 Kb of any genetic locus. A comparative analysis of the T.b.gambiense and T.b.brucei genetic maps revealed synteny and marker order to be conserved between the two sub-species. However, variation was observed in the location of regions of high and low recombination frequency (hot and cold spots) in the two maps. The genetic linkage map presented here is the first available for T.b.gambiense and can now be utilised to find the location within the genome of genes responsible for phenotypic traits in this clinically important sub-species. These traits include human infectivity, tsetse transmissibility and virulence, in addition to sensitivity to the trypanocidal drug, pentamidine, for which phenotypic variation between the parents was characterised both in vitro and in vivo in this thesis. The ability of the T.brucei genetic maps to pinpoint loci underlying phenotypic variation is limited by the number of recombination events, and therefore progeny, available for analysis. To increase the utility of this approach for future studies, an improved method for progeny isolation from uncloned genetic cross populations was also developed. This in vitro bloodstream cloning procedure is scalable and efficient, and replaces a time consuming and technically demanding in vivo method. Twelve new progeny clones were isolated by this approach during the trial and incorporated into the analysis, representing a step toward a higher resolution second-generation genetic map. Finally, whilst undertaking genotyping analysis with microsatellite markers the development of spontaneous chromosome 10 abnormalities was observed. A detailed investigation identified seven laboratory-adapted T.brucei lines in which loss of heterozygosity appeared to have occurred. These alterations to the karyotype significantly exceeded the well-characterised genomic rearrangements of subtelomeric regions that are frequently associated with antigenic variation in African trypanosomes. Microsatellite analysis, pulsed field gel electrophoresis and Illumina next generation sequencing demonstrated these changes to be the product of mitotic recombination events in the chromosome core, resulting in an extensive loss of heterozygosity of up to 75% of the chromosome and correlated with an improved growth phenotype. Further work is now required to determine the extent and frequency with which these abnormalities might occur, however these findings do highlight the potential instability of the molecular karyotype of T.brucei in prolonged in vitro culture