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Genetic Crosses

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NF54xNHP4026
Mal31xKH004
NF54xNHP1337
MKK2835xNHP1337

NF54×NHP4026

A world map highlighting two locations. NF54, in West Africa, is circled in blue, and HP4028, in Southeast Asia, is marked with a blue dot.

One of our most notable and well studied crosses, we crossed the CQ-R and slow patient clearing parasite (NHP4026) which was isolated from the Thai-Myanmar with the well characterized drug-sensitive laboratory strain (NF57). This cross generated a total of 270 progeny, of which 109 were unique recombinants. The use of bulk segregant analysis (BSA) on this cross led to the identification of pfaat1 and its epistatic interaction with pfcrt as well as its role in enhancing chloroquine resistance.

Notable phenotypes and genotypes

NF54: drug sensitive; WT pfcrt, pfkelch13, and pfaat1; high fitness

NHP4026: CQ-R (Dd2-like pfcrt and S258L+F313S pfaat1); slow clearing to ACT (WT pfkelch13); high fitness

See sequence repository for all progeny and parents

Publications

Amambua-Ngwa A, Button-Simons KA, Li X, Kumar S, Brenneman KV, Ferrari M, Checkley LA, Haile MT, Shoue DA, McDew-White M, Tindall SM, Reyes A, Delgado E, Dalhoff H, Larbalestier JK, Amato R, Pearson RD, Taylor AB, Nosten FH, D'Alessandro U, Kwiatkowski D, Cheeseman IH, Kappe SHI, Avery SV, Conway DJ, Vaughan AM, Ferdig MT, Anderson TJC. Chloroquine resistance evolution in Plasmodium falciparum is mediated by the putative amino acid transporter AAT1. Nat Microbiol. 2023 Jul;8(7):1213-1226. doi: 10.1038/s41564-023-01377-z. Epub 2023 May 11. PMID: 37169919; PMCID: PMC10322710.

Kumar S, Li X, McDew-White M, Reyes A, Delgado E, Sayeed A, Haile MT, Abatiyow BA, Kennedy SY, Camargo N, Checkley LA, Brenneman KV, Button-Simons KA, Duraisingh MT, Cheeseman IH, Kappe SHI, Nosten F, Ferdig MT, Vaughan AM, Anderson TJC. A Malaria Parasite Cross Reveals Genetic Determinants of Plasmodium falciparum Growth in Different Culture Media. Front Cell Infect Microbiol. 2022 May 30;12:878496. doi: 10.3389/fcimb.2022.878496. PMID: 35711667; PMCID: PMC9197316.

Button-Simons KA, Kumar S, Carmago N, Haile MT, Jett C, Checkley LA, Kennedy SY, Pinapati RS, Shoue DA, McDew-White M, Li X, Nosten FH, Kappe SH, Anderson TJC, Romero-Severson J, Ferdig MT, Emrich SJ, Vaughan AM, Cheeseman IH. The power and promise of genetic mapping from Plasmodium falciparum crosses utilizing human liver-chimeric mice. Commun Biol. 2021 Jun 14;4(1):734. doi: 10.1038/s42003-021-02210-1. PMID: 34127785; PMCID: PMC8203791.

Two-panel figure. Panel A shows a heatmap of allele frequencies across 14 chromosomes, comparing control and CQ-selected groups. Panel B displays line graphs of 3D7 allele frequency across the same chromosomes for both groups.

Mal31×KH004

A world map highlights two locations with blue markers. Mal31 denotes a location in Southern Africa and KH004 signifies a Southeast Asia location.

This cross was generated to understand genetic determinants which preserve parasite fitness in multidrug-resistant parasites. KH004 is a 2016 Cambodian isolate of the KEL1/PLA1 lineage of parasites. It is resistant to CQ, ART, and PPQ while retaining a moderate level of fitness. Mal31 is a 2016 drug-sensitive isolate from Malawi and harbors a pfcrt allele which has been naturally reverted back to WT after extensive lack of CQ pressure. This cross produced a total of 319 clonal recombinant parasites, of which 104 had unique genotypes. A unique feature of this cross is that KH004 carries 4 copies of plasmepsin II/III and progeny inherited a variety of copies ranging from 1 to 5. Additionally, KH004 has a highly novel PPQ-R pfcrt mutation (G367C) which has recently increased in prevalence in Eastern Southeast Asia.

Notable phenotypes and genotypes

Mal31: drug sensitive (WT pfcrt, pfkelch13, single copy plasmepsin II/III, S258L pfaat1); low fitness

KH004: CQ-R and PPQ-R (Dd2-like pfcrt + G367C, 4 copies plasmepsin II/III, S258L+F313S pfaat1); ART-R (C580Y pfkelch13); high fitness

See sequence repository for all progeny and parents

Project Number: PRJNA524855

Publications

Kane J, Li X, Kumar S, Button-Simons KA, Vendrely Brenneman KM, Dahlhoff H, Sievert MAC, Checkley LA, Shoue DA, Singh PP, Abatiyow BA, Haile MT, Nair S, Reyes A, Tripura R, Peto TJ, Lek D, Mukherjee A,Kappe SHI, Dhorda M, Nkhoma SC, Cheeseman IH,Vaughan AM, Anderson TJC, Ferdig MT.2024. A Plasmodium falciparum genetic cross reveals the contributions of pfcrt and plasmepsin II/III to piperaquine drug resistance. mBio15:e00805-24.https://doi.org/10.1128/mbio.00805-24

Brenneman KV, Li X, Kumar S, Delgado E, Checkley LA, Shoue DA, Reyes A, Abatiyow BA, Haile MT, Tripura R, Peto T, Lek D, Button-Simons KA, Kappe SHI, Dhorda M, Nosten F, Nkhoma SC, Cheeseman IH, Vaughan AM, Ferdig MT, Anderson TJC. Optimizing bulk segregant analysis of drug resistance using Plasmodium falciparum genetic crosses conducted in humanized mice. iScience. 2022 Mar 16;25(4):104095. doi: 10.1016/j.isci.2022.104095. PMID: 35372813; PMCID: PMC8971943.

Two-panel figure. Panel A is a line graph depicting KH004 allele frequency across 14 chromosomes. Shaded gray bars highlight regions of interest. Panel B is a heatmap showing allele frequencies for multiple genes (pfcrt, pfkelch13, pm2/3) across the same 14 chromosomes for Mal31 and KH004 isolates. Orange represents the Mal31 allele and blue represents the KH004 allele.

NF54×NHP1337

A world map highlights two locations: NF54, a strain of malaria, is marked in West Africa. HP133, another malaria strain, is marked in Southeast Asia.

Notable phenotypes and genotypes

NF54: drug sensitive; WT pfcrt, pfkelch13, and pfaat1; high fitness

NHP1337: CQ-R (Dd2 pfcrt, S258L+F313S pfaat1); ART-R (C580Y pfkelch13); low-moderate fitness

See to sequence repository for BSA

Project Number: PRJNA524855

Publications

Brenneman KV, Li X, Kumar S, Delgado E, Checkley LA, Shoue DA, Reyes A, Abatiyow BA, Haile MT, Tripura R, Peto T, Lek D, Button-Simons KA, Kappe SHI, Dhorda M, Nosten F, Nkhoma SC, Cheeseman IH, Vaughan AM, Ferdig MT, Anderson TJC. Optimizing bulk segregant analysis of drug resistance using Plasmodium falciparum genetic crosses conducted in humanized mice. iScience. 2022 Mar 16;25(4):104095. doi: 10.1016/j.isci.2022.104095. PMID: 35372813; PMCID: PMC8971943.

This image presents three scientific figures visualizing genotype clusters and cloning rounds. Figure (a) and (b) depict clusters of colored dots representing different genotypes, with legends indicating parental, recombinant within rounds, and recombinant between rounds. Figure (c) is a bar graph comparing genotyped clone counts after filtering, categorized by unique recombinant progeny, repeat genotypes within and between cloning rounds, non-clonal progeny, and selfed progeny. Labels NF54, MKK2835, and NHP1337 are visible.

MKK2835×NHP1337

Map of Southeast Asia with two blue dots indicating the sample locations: NHP1337 in western Thailand and MKK28 in eastern Thailand.

This sympatric cross utilizes two newly cloned clinical isolates (MKK2835 and NHP1337) from the Thai–Myanmar border. These parasites were respectively isolated before (2003) and after (2013) the emergence of artemisinin resistance in the region. This cross yielded 60 clonal unique recombinant progeny.

Notable phenotypes and genotypes

MKK2835: CQ-R (Dd2 pfcrt, F313S+K541N pfaat1); moderate fitness

NHP1337: CQ-R (Dd2 pfcrt, S258L+F313S pfaat1); ART-R (C580Y pfkelch13); low-moderate fitness

Publications

Li X, Kumar S, McDew-White M, Haile M, Cheeseman IH, Emrich S, Button-Simons K, Nosten F, Kappe SHI, Ferdig MT, Anderson TJC, Vaughan AM. Genetic mapping of fitness determinants across the malaria parasite Plasmodium falciparum life cycle. PLoS Genet. 2019 Oct 14;15(10):e1008453. doi: 10.1371/journal.pgen.1008453. PMID: 31609965; PMCID: PMC6821138.