Role of recombination in genetic linkage and genome variation in the human malaria parasite, Plasmodium falciparum

Malaria is a devastating infectious disease that has caused death and debilitation for centuries. It remains a globally significant disease despite relentless efforts to control it over the last century. Infections resulting from the malaria parasite, Plasmodium falciparum, cause approximately 300- 500 million clinical cases and nearly a million deaths each year. Disease control has been confounded by the emergence of multidrug resistant (MDR) parasites. The success of the parasite in defeating eradication and control is largely attributable to its genetic variability, which allows the parasite to overcome human host immunity, and drug treatment regimens. Recombination is a fundamental mechanism by which genetic variation is introduced from the parent to subsequent generations by reshuffling parent genome material and breaks up linkage between loci, allowing for independent evolution of sites. Knowledge of the extent and distribution of genetic variation, as well as the mechanisms that generate variation is fundamental to understanding the evolution of parasite phenotypes, including drug resistance, prevalence and severity of the disease in humans. This dissertation focused on revealing the role of recombination on the genetic variability of the malaria parasite. We used an experimental system based on a well-known genetic cross, HB3ÌÄ' Dd2, to directly observe the products of recombination. In the context of the genetic cross, we carried out linkage analysis, detected the fine structure of recombinant products, and revealed the dynamics and mechanisms of copy number variation (CNV) inheritance in the parasite. This approach allowed for a unique and previously unappreciated glimpse in to the repertoire of genetic variability that is generated within a life cycle of the parasite via recombination.

We demonstrated that combining linkage maps from different mapping populations of P. falciparum strains with genome sequence information can refine existing maps, increase map accuracy, inform genome assemblies, and highlight key aspects of the biology of recombination in the parasite. These observations propelled subsequent work to evaluate the genome-wide distribution of recombination at an even higher marker density. We built on the information on linkage analysis and used two sibling parasite clones from the HB3 ÌÄ' Dd2 that had a high number of crossovers (COs) to describe a method to detect genomewide distribution of COs, non-crossovers(NCOs) and gene conversions (GCs). This study was the first to sequence progeny clones to capture high resolution information on COs, NCOs and genetic variation in malaria parasites. The work emphasized the importance of NCO/GCs in the linkage and haplotype analysis of malaria parasites and the need for the description of a genome-wide rate of GCs for the species to advance ongoing efforts in haplotype analysis and to improve the accuracy of genome-wide association studies (GWAS). We also argued that it is essential to combine linkage analysis and genome-wide distributions of CNVs to determine CNV behavior, distributions and frequencies across genomes to understand the origins of CNV in both evolutionary and generational time frames. We revealed that CNVs are a significant source of segregating and de novo genome variation. The study highlighted that the rate of de novo CNV may be higher than expected in P. falciparum and that both by-products of recombination, COs and NCO/GCs, can not only produce de novo CNVs, but also lead to loss or gain of copies at an already existing CNV locus.

In conclusion, this doctoral thesis emphasized the significance of recombination as a central mechanism generating several distinct classes of genetic variants in P. falciparum, ranging from reshuffling of parent genomes to GCs to genome structural variants. These studies are fundamental contributions to establishing a framework for ongoing studies of recombination and the mechanisms of genome structural variation in malaria parasites.