The power and promise of genetic mapping from Plasmodium falciparum crosses utilizing human liver-chimeric mice

Abstract

Genetic crosses are most powerful for linkage analysis when progeny numbers are high, parental alleles segregate evenly and numbers of inbred progeny are minimized. We previously developed a novel genetic crossing platform for the human malaria parasite Plasmodium falciparum, an obligately sexual, hermaphroditic protozoan, using mice carrying human hepatocytes (the human liver-chimeric FRG NOD huHep mouse) as the vertebrate host. We report on two genetic crosses—(1) an allopatric cross between a laboratory-adapted parasite (NF54) of African origin and a recently patient-derived Asian parasite, and (2) a sympatric cross between two recently patient-derived Asian parasites. We generated 144 unique recombinant clones from the two crosses, doubling the number of unique recombinant progeny generated in the previous 30 years. The allopatric African/Asian cross has minimal levels of inbreeding and extreme segregation distortion, while in the sympatric Asian cross, inbred progeny predominate and parental alleles segregate evenly. Using simulations, we demonstrate that these progeny provide the power to map small-effect mutations and epistatic interactions. The segregation distortion in the allopatric cross slightly erodes power to detect linkage in several genome regions. We greatly increase the power and the precision to map biomedically important traits with these new large progeny panels.

A Malaria Parasite Cross Reveals Genetic Determinants of Plasmodium falciparum Growth in Different Culture Media

“…We observed three genome regions that showed strong distortions in allele frequency in each independent replicate cross in both human serum and AlbuMAX cultures ( Figure 3 ), consistent with our earlier report ( Li et al., 2019 ; Button-Simons et al., 2021 ): on chr. 7 (named QTL chr.…”

“…Combining all the information, we estimate that there were 28 (average 14 oocysts per mosquito × 4 recombinants per oocyst × 0.5 selfed) recombinant genotypes per infected mosquito, which gave us ~2800 (28 × ~100 infected mosquitoes) unique recombinants per pool for each of the three mice (see Preparation of the Genetic Cross ). In addition to the bulk segregant analyses described below, we cloned and carried out whole genome sequencing (WGS) of progeny from these recombinant pools using published methods ( Button-Simons et al., 2021 ) from a mixture of all pools a week after transferring the transitioned blood stages to in vitro culture. This revealed low numbers of selfed progeny (2/55) in this cross with very little redundancy among recombinants ( Supplementary Table 3 ), as observed in previous crosses ( Button-Simons et al., 2021 ).…”

“…In addition to the bulk segregant analyses described below, we cloned and carried out whole genome sequencing (WGS) of progeny from these recombinant pools using published methods ( Button-Simons et al., 2021 ) from a mixture of all pools a week after transferring the transitioned blood stages to in vitro culture. This revealed low numbers of selfed progeny (2/55) in this cross with very little redundancy among recombinants ( Supplementary Table 3 ), as observed in previous crosses ( Button-Simons et al., 2021 ). Based on bulk segregant analyses of the initial recombinant pools, the allele frequencies of 3D7 emerging from the liver were 0.52 ± 0.001, 0.50 ± 0.002 and 0.55 ± 0.002 for the three mice.…”

“…The 3D7 parasite we used for this study (and for Button-Simons et al. ( Button-Simons et al., 2021 ) here it was referred to as NF54) was further isolated from a volunteer in a malaria clinical trial after controlled human malaria infection by 3D7-infected Anopheles stephensi mosquito bite ( Ockenhouse et al., 1998 ). 3D7 has been maintained in the lab for decades, in a variety of media formulations containing both serum and AlbuMAX.…”

“…Carrying out genetic crosses in Anopheles mosquitoes and human hepatocyte-chimeric mice (FRG huHep mice) ( Vaughan et al., 2015 ) now allows us to routinely generate large pools of recombinant P. falciparum progeny without the need for chimpanzee hosts. We have previously generated four independent P. falciparum genetic crosses with large numbers of unique recombinant progeny using this approach ( Button-Simons et al., 2021 ), reviewed in ( Vendrely et al., 2020 ).…”