Malaria is a disease caused by the protozoan parasite Plasmodium where the species that causes the most severe form of malaria in humans is known as Plasmodium falciparum. At least 40% of the global population is at risk of contracting malaria with 627 000 people dying as a result of this disease in 2012. Approximately 90% of all malaria deaths occur in sub-Saharan Africa, where approximately every 30 seconds a young child dies, making malaria the leading cause of death in children under the age of five years old. The malaria parasite has a complex life cycle utilising both invertebrate and vertebrate hosts across sexual and asexual stages. The erythrocyte invasion stage of the life cycle in the human whereby the invasive merozoite form of the parasite enters the erythrocyte is a central and essential step, and it is during this stage that the clinical symptoms of malaria manifest themselves. Merozoites invade erythrocytes utilising multiple, highly specific receptor-ligand interactions in a series of co-ordinated events. The aim of this study was to better understand the interactions occurring between the merozoite and erythrocyte during invasion by using modern, cutting-edge proteomic techniques. This was done in the hope of laying the foundation for the discovery of new key therapeutic targets for antimalarial drug and vaccine-based strategies, as there is currently no commercially available antimalarial vaccine and no drug to which the parasite has not at least started showing resistance. In this study healthy human erythrocytes were treated separately with different protein-altering enzymes and chemicals being trypsin, the potent oxidant sodium periodate (NaIO4), the amine cross-linker tris(2-chloroethyl)amine hydrochloride (TCEA) and the thiol cross-linker 1,11-bis(maleimido)triethylene glycol (BM(PEG)3). The resulting erythrocyte protein alterations were visualised as protein band differences on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE), where treated and untreated control erythrocyte ghost protein fingerprints were visually compared to one another. The protein bands showing differences between treated and control samples were in-gel digested using trypsin then sequenced by liquid chromatography tandem mass spectrometry (LC-MS/MS) and identified using proteomics-based software. In this way, the erythrocyte proteins altered by each enzyme/chemical treatment were identified. Malaria invasion assays were performed where each treatment group of erythrocytes as well as the control erythrocytes were incubated separately with schizont stage malaria parasites for the duration of one complete life cycle. Using fluorescent staining and flow cytometry, the invasion inhibition efficiency for each treatment group was evaluated. By utilising these methods, the identification and the relative importance of the erythrocyte proteins involved in the invasion process were determined. Protein fingerprints of control and treated erythrocyte ghosts were visualised and optimised on SDS PAGE where induced protein band differences were successfully identified by LC-MS/MS. It was found that each treatment altered erythrocyte proteins with changes found in Band 3, actin, phosphoglycerate kinase 1, spectrin alpha, spectrin beta, ankyrin, haemoglobin, Bands 4.1 and 4.2, glycophorin A and stomatin. The invasion assays revealed that TCEA inhibited invasion to the greatest extent as compared to the other treatments, followed by BM(PEG)3 and trypsin. Sodium periodate-treated erythrocytes could not be assessed using the invasion assay due to auto-haemolysis. Band 3, glycophorin A, Band 4.1 and stomatin appear to be of higher relative importance in the invasion process as compared to the other altered erythrocyte proteins. These results confirmed the known roles of spectrin alpha, spectrin beta, glycophorin A, Band 3 and Band 4.1 in invasion, and suggested that ankyrin, Band 4.2 and stomatin may also be involved. This study highlighted the potential that modern, cutting-edge proteomic techniques provide when applied to previous comparative studies found in older literature, as previously unidentified proteins that can be involved in invasion were revealed. These results can be used as a foundation in future studies in order to identify new key targets for the development of new antimalarial drug- and vaccine-based strategies, with the hope of preventing the suffering of the millions of malaria-inflicted people worldwide, and ultimately eradicating this deadly disease. / Dissertation (MSc)--University of Pretoria, 2014. / tm2015 / Pharmacology / MSc / Unrestricted

MSc (Biochemistry) / Department of Biochemistry / In 2017 malaria cases were estimated at 219 million and of these 435 000 resulted in death. Malaria is transmitted by female Anopheles mosquitoes which thrive in tropical and sub-tropical areas. Malaria is caused by five species from the genus Plasmodium, namely P. falciparum, P. vivax, P. ovale, P. malariae and P. knowlesi. P. falciparum causes the most severe form of the disease. P. falciparum has a complex life cycle in the human and mosquito hosts exposing the parasite to environmental changes, resulting in upregulation of heat shock proteins (Hsps). These Hsps facilitate protein folding and protein disaggregation. Hsp70 is a molecular chaperone whose function is to facilitate protein folding. P. falciparum Hsp70-x is the only member of this family of proteins that is exported to the erythrocyte cytosol by the parasite. PfHsp70-x has been implicated in the development of malaria pathogenesis. This is largely due to its association with P. falciparum erythrocyte membrane protein 1 (PfEMP1), an important virulent factor that is exposed to the exterior of the infected erythrocyte. In tumour cells, cell surface- bound Hsp70 is known to sensitize the tumour cells to cytolytic attack that is mediated by NK cells. Cell surface bound Hsp70 is thought to recruit NK cells and Granzyme B (GrB) via its 14 amino acid sequence, TKDNNLLGRFELSG, known as the TKD motif. Both PfHsp70-x and human Hsp70 (hHsp70) contain the TKD motif. Thus, this study sought to investigate the role of Hsp70 in facilitating the selective targeting of malaria parasite-infected erythrocytes by GrB. To this end, recombinant hHsp70 and PfHsp70-x were successfully expressed in E. coli and purified. Using slot blot and ELISA, it was observed that both PfHsp70-x and hHsp70 directly interact with GrB. PfHsp70-x showed greater affinity for GrB than hHsp70. In addition, using parasites cultured at the erythrocyte stage it was noted that GrB exhibits potent antiplasmodial activity (IC50 of 0.5μM). In addition, the findings suggest that GrB interacts with both Hsp70s (of parasite and human origin) resident in the infected erythrocyte. This makes GrB a promising antimalarial agent. / NRF

Magister Pharmaceuticae - MPharm / Malaria disease poses substantial health risks to many nations, especially in Africa, where it primarily affects pregnant women, children, and immunocompromised patients. However, current antimalarial drugs have limitations such as low safety profile and particularly widespread treatment failure due to the increasing resistance of Plasmodium falciparum, the major causative organism to artemisinin-based therapy (ACT) and other chemotherapeutics. In the light of this, there is a pressing need for new antimalarial drugs with novel mechanisms of action and satisfactory pharmacokinetic properties, which has led to the current study. Furthermore, current antimalarial drugs target specific stages of the Plasmodium life cycle. For instance, chloroquine targets the erythrocytic stage while primaquine targets the liver stage. However, these therapies cannot achieve complete elimination of the parasite once the life cycle has been established in the body. Hence, the goal of this study is to combat resistance by finding novel compounds that can bind to a multiple-staged protein in Plasmodium falciparum. Based on this consideration, falstatin was chosen as the protein target for this study because it was observed to play a crucial role in the degradation of haemoglobin, rupture of erythrocytes by mature schizonts, and subsequent invasion of erythrocytes by free merozoites. Hence, the protein, falstatin can be targeted to inhibit cell growth and cause plasmodial cell death in merozoites as well as schizonts of Plasmodium falciparum. Therefore, it is intended that compounds that bind to falstatin could serve as novel antimalarials that target multiple stages of the Plasmodium life cycle. Consequently, this study explored the structure-based virtual screening approach to identify compounds that could bind to the protein target, falstatin in Plasmodium falciparum. An extensive literature review identified falstatin as the multi-staged drug target for this study, while homology modelling was used to generate the three-dimensional structure of falstatin. Molecular docking was conducted to predict the binding energy of compiled antiplasmodial compounds to falstatin while druglikeness analysis was used to prioritize compounds according to their ADMET (absorption, distribution, metabolism, excretion and toxicity) properties. The top-ranked compound, based on a novel ligand scoring function, was then subjected to molecular dynamics (MD). Following this step, rescoring analysis was performed on the top 5 compounds using the Molecular Mechanics-Generalized Born Surface Area (MM-GBSA) scoring function to gain insight into their component binding energies. Thereafter, a pharmacophore hypothesis was developed based on the 5 top-ranking compounds in order to screen other compound libraries in the future. From the results, TCMDC 131646, TCMDC-124274, TCMDC-138266, TCMDC 123844 and TCMDC 131234 possessed good binding energies and satisfactory ADMET properties showing high ligand scores of 77.1, 75.4, 75.4, 75.4 and 73.1 respectively (on a total scale of 100). Also, the study revealed that the top-ranked compound, TCMDC 131646 had a binding energy of -6.15 KJ/mol, contained no toxicophore and conformed to Lipinski, Egan and Muegge rules of druglikeness. Findings from the MD simulation demonstrated that TCMDC 131646 strongly interacted with the protein, falstatin. Morealso, the study revealed that TCMDC 131646 is structurally diverse from chloroquine, artemisinin, artemether and lumefantrine, indicating that it may possess a distinct mechanism of action. The rescoring analysis of TCMDC-131646, TCMDC 124274, TCMDC-138266, TCMDC 123844 and TCMDC 131234 predicted negative binding energies ≤ -4.662 KJ/mol for the top compounds, further indicating that these compounds are likely to bind strongly with falstatin. Additionally, the developed pharmacophore hypothesis contained -H-N-C=O and N-H moieties which strongly suggested that the presence of electron-withdrawing groups could be vital for the inhibition of falstatin at the active site. Overall, TCMDC 131646 was predicted to be a drug-like and safe compound that could inhibit falstatin in Plasmodium falciparum. Chemical-disease co-occurrence analysis in literature revealed that this compound showed in-vitro antiplasmodial activity at an IC50 of 0.226μM and has also shown in vitro activity for neuralgia, hyperalgesia and arthritis. The research recommends TCMDC 131646 as a potential antimalarial hit compound that could yield novel analogues by hit expansion. However, confirmatory in-vitro and in-vivo studies are required to substantiate these predictions

With the increase in recent years in the prevalence of malaria, and in drug resistance of Plasmodium falciparum, there has been much interest in natural plant products for new antimalarials with novel modes of action against Plasmodium. Artemisinin or Qinghaosu is one such antimalarial isolated from a Chinese herb, Anemisia annua (Asteraceae) and it is currently undergoing phase I and II clinical trials. The Southern African species, Artemisia afra (African wormwood, wildeals, lengana) is commonly used by local traditional healers for symptoms of malaria, in particular fever. Thus it seemed appropriate to investigate this species for antimalarial activity. Crude petroleum ether soxhlet extracts of Anemisia afra had demonstrated antimalarial activity against Plasmodium falciparum, FCR-3, cultured in vitro. The IC₅₀ values ranged from 5-13μg/ml. The extract from leaves and flowers was then screened against D10 (chloroquine-sensitive) and FAC8 (chloroquineresistant) P. falciparum, in vitro, with IC₅₀ values of 1.03μg/ml and l.5μg/ml respectively. This extract was fractionated by column chromatography using silica gel-60 and the fractions obtained were screened for antimalarial activity. The most active fraction had an IC₅₀ of 0.5μg/ml against D10 and FAC8. Using TLC and HPLC-UV analysis with pure artemisinin as a standard, no artemisinin could be detected in this fraction. This result was confirmed by thermospray LC-MS analyses. Purification of this fraction yielded ultimately a single pure compound; a clear colourless oil identified by MS and NMR analyses as hydroxydavanone. The compound was screened against a variety of P. falciparum strains with varying degrees of sensitivity and resistance to both chloroquine and mefloquine. Their sensitivity against artemisinin was also established. IC₅₀ values obtained for the isolated pure compound against P. falciparum ranged from 0.87 to 2.54μg/ml. The IC₅₀ values obtained for general cytotoxicity of the crude extract and isolated pure compound against RAT-I fibroblast cells were 34.78 ± 8.23 and 6.29 ± 0.95 μg/ml (n=4) respectively. Thus the crude extract and isolated pure compound exhibited a greater antimalarial than cytotoxic effect. Hence, there are implications for A. afra to be used as a phytomedicine for the treatment of malaria. In vivo studies are recommended for hydroxydavanone in order to fully assess its potential for clinical use.

Evalúa la relación entre parasitemia, respuesta de la médula ósea y anemia por infección de P. falciparum en el modelo experimental Aotus nancymaee de estudios de vacunas conducidos en NAMRU-6 entre el 2012 - 2016. De los resultados se desprende que de los 74 monos, el 35.14% desarrolla alta parasitemia, de los cuales este grupo representa el 38.89% de los casos de anemia, con un p=0.266. Sin embargo, en este grupo no se observa casos de anemia severa (p=0.001). Con respecto a la parasitemia acumulada se halla que es un factor importante para el desarrollo de anemia con un OR=2.27 (IC 95%,1.18 – 4.64,p=0.014), mientras que para la severidad de la anemia días de parasitemia patente es un factor con mayor impacto OR=2.1(IC 95% ,1.4-3.2,p=0.001). Al evaluar parasitemia y respuesta de la médula ósea se encuentra diferencias significativas, se aprecia un clearance total en casos con respuesta de la médula ósea (p=0.02) en el día 25 de seguimiento. Asimismo, se observa diferencias significativas con respecto al números de días de clearance entre el grupo con respuesta y no respuesta (medianas respectivas:5 días, 0 días (p=0.002)). No se evidencia respuesta de la médula ósea frente a un estado de anemia, no se muestra diferencias significativas entre los grados de anemia (p<0.05). Para el desarrollo de anemia en un estado de infección por P. falciparum no importa el nivel de parasitemia, sin embargo, el tiempo de la parasitemia “cronicidad” parece ser un factor a tomar en cuenta. Y con respecto a la respuesta de la médula ósea en un estado de anemia, la médula ósea no responde si no hay un previo clearance total de la parasitemia y también es necesario al menos 5 días de clearance. Se recomienda hacer otros estudios enfocados en la “cronicidad” de la enfermedad. Asimismo, estudios directos de médula ósea antes, durante y después del pico de parasitemia y durante el clearance. / Tesis

Malaria is a life-threatening parasitic disease that causes at least 300 million acute illnesses annually, of which at least one million infected humans die, mainly children under the age of 5 years. This overwhelming burden is due to the most virulent causative agent, Plasmodium falciparum, as a result of its prevalence in sub-Saharan Africa, as well as its resistance to nearly all anti-malarials in use. There is thus an urgent need to discover and characterise new novel parasitic targets for chemotherapeutic intervention. Folate metabolism is the target of several anti-malarials such as pyrimethamine and sulfadoxine. These drugs cause a decrease in parasite growth since Plasmodia have a high rate of replication and demand for nucleotides during DNA synthesis. The parasite is almost totally resistant to the current antifolates. Further insights into the folate pathway and its drugs are essential for the understanding of the resistance mechanism and to identify/characterise new drug targets for inhibition. Three possible new drug targets were identified and characterised in the folate pathway (Lee C.S. et al., 2001). One of these targets is the bifunctional enzyme, dihydrofolate-synthase folylpolyglutamate synthase (DHFS-FPGS). The bifunctionality and activity of the -dhfs-fpgs gene in Plasmodium was confirmed by functional complementation in yeast and bacteria and shown to be unique to Plasmodia and bacteria. This gene indicated only a 15-17% homology to other organisms; DHFS activity is usually only present in prokaryotes but not in humans or other eukaryotes (Salcedo E. et al., 2001). Although part of a bifunctional protein and having closely related catalytic functions, the DHFS and FPGS activities have distinct roles to play in both the de novo and salvage pathways of folate metabolism. These characteristics indicate DHFS-FPGS as a potentially good drug target since a single inhibitor is likely to have a drastic effect on both routes and consequently arrest DNA synthesis in the malaria parasite. This could prove to be a very effective and novel antimalarial strategy. Comparative expression studies of synthetic and native DHFS-FPGS presented here indicated that the highest quantity of protein is expressed from the synthetic gene. However, results indicated that most of the recombinant protein expressed in various E. coli cell lines, produced insoluble protein aggregates. Various strategies were employed in an attempt to improve recombinant soluble expression including auto-induction of T7 promoter activity. However, this did not result in an increased percentage of soluble protein expression even though improved total protein expression was observed. The inclusion of chaperone proteins resulted in a minor change in soluble expression. Activity assays of the DHFS-FPGS from these two methods indicated that active protein was produced in a correctly folded manner. Due to the high amount of recombinant protein present in the inclusion bodies, various methods were investigated to isolate and refold the DHFS-FPGS protein. The use of a non-ionic and ionic detergent for refolding resulted in pure, solubilised, active protein. Activity assays of the refolded, soluble protein indicated that the protein is active. Preliminary kinetic analysis was unsuccessful and requires further investigations. / Dissertation (MSc (Biochemistry))--University of Pretoria, 2007. / Biochemistry / unrestricted

No description available.

Plasmodium falciparum is one of four species known to cause malaria in humans and is the species that is associated with the most virulent form of the disease. Malaria causes nearly two million deaths each year, many of these occurring among children in under-developed countries of the world. One reason for this is the prevalence of drug resistant strains of malaria that mitigate the efficacy of existing drugs. Hence, the identification of a new generation of pharmacological agents for malaria is extremely urgent. The recent identification of a group of novel protein kinases within the Plasmodium falciparum genome has provided researchers with a basis for what many hope to be new potential drug targets for malaria. Identified within the Plasmodium genome and a few select apicomplexans, these novel proteins have been predicted to be protein kinases based solely on certain sequence features shared with other eukaryotic protein kinases (ePKs). However, to date, no significant studies to determine the function of these novel kinases have been performed. Termed FIKKs, these proteins all possess a non-conserved N-terminal sequence that contains a Plasmodium export element (Pexel) which may target the proteins for export from the parasite and a conserved C-terminal catalytic domain containing a FIKK sequence common to all twenty members of this family. We analyzed the localization of one of the FIKK proteins, FIKK11, encoded by the PF11_0510 locus, during intraerythrocyte differentiation of P. falciparum by Western blot analysis and indirect immunofluorescence assay. Western blot analysis demonstrated that FIKK 11 is expressed within the parasite at all stages of its erythrocytic life cycle with its highest expression occurring during the schizont stage. Immunofluorescence assays showed that this protein is exported from the Plasmodium parasite into the host erythrocyte cytosol which is consistent with studies on other Plasmodium proteins that also have the Pexel motif. To determine the enzymatic activity of FIKK11, we overexpressed the recombinant protein in E. coli and then purified it. However, no protein kinase activity was detected using several commonly used protein kinase substrates including histone H1, myelin basic protein, or dephosphorylated casein. We also did not detect any kinase activity of the native enzyme using pull-down assays of the Plasmodium falciparum cell extract against those same substrates. In addition, kinase substrate peptide array analysis of FIKK11 showed no evidence of protein kinase activity either for FIKK11. Interestingly, however, we were able to detect some kinase activity using the recombinant protein alone with no substrate. The lack of the glycine triad within subdomain I of these FIKK kinases as compared with most traditional eukaryotic protein kinases may explain why we were unable to find any interactions between FIKK11 and other commonly protein kinase substrates. Of interest was the observation that the protein reproducibly exhibited what appeared to be an autophosphorylation activity when using the standard protein kinase assay. Further analyses, however, showed that FIKK11 actually possesses diacylglycerol kinase activity utilizing 1-Stearoyl-2-arachidonoyl-sn-glycerol as a substrate. This is the first evidence of diacylglycerol kinase activity in Plasmodium falciparum. Because FIKK11 is exported into the host cell and is localized on the erythrocyte membrane, its enzymatic activity may potentially have relevance in the pathophysiology of the disease.

Malaria affects hundreds of millions of people worldwide and equally claims hundreds of thousands of lives each year. With the current spread of drug resistance to standard antimalarial drugs like chloroquine and the emergence of artemisinin-resistant parasites, new antimalarial drugs and formulations are urgently needed. An ethnobotanical survey was carried out in this study in search of novel compounds with promising antiplasmodial activity. Using the ethnobotanical approach, a total of 61 plant species from 59 genera distributed in 34 plant families were found to be used traditionally for the treatment of malaria in Nigeria. Biological evaluation of the plant¿s methanolic extracts was assessed using the parasite lactate dehydrogenase (pLDH) assay against the chloroquine-sensitive (3D7) and chloroquine-resistant (K1) strains of Plasmodium falciparum. A total of five (5) plant species showed more potent antiplasmodial activities against the malaria parasites. These are Acanthospermum hispidum, Cassia occidentalis, Kaempferia aethiopica Prosopis africana and Physalis angulata with MIC values ranging between 7.815µg/ml to 31.25µg/ml (3D7 strain) and 15.63µg/ml to 62.50µg/ml (K1 strain) against the malaria parasites, respectively. Two plants, Prosopis africana (Leguminosae-mimosoideae) and Physalis angulata (Solanaceae) were selected for further study. The phytochemical investigation of the active chloroform extracts of P. africana and P. angulata yielded several compounds with three known alkaloids, namely, prosopinine (I), prosopine (II) and acetamide (III). Their structures were confirmed by MS, 1D and 2D NMR spectroscopy. Compounds I, II and III have moderate in vitro antiplasmodial activity against the malaria parasites. Both chloroquine and artemether were used as standard control. / Association of Commonwealth Universities and the Commonwealth Scholarship Commission in the UK (Commonwealth Scholarship Reference Number: NGCS-2005-259).

The repeated emergence of Plasmodium falciparum resistance to first-line antimalarial drugs necessitates understanding the underlying resistance mechanisms to detect and monitor resistance in the field and to inform drug discovery efforts. With the advent of the FRG NOD human liver-chimeric (huHep) mouse model for P. falciparum genetic crosses, interest has renewed in harnessing this forward genetics tool to study traits including drug resistance. The antimalarial quinine (QN) is of particular interest as it has retained efficacy over 400 years as parasite resistance has been slow to develop against the drug, likely due to a multifactorial mechanism of which only several genes have been partially implicated. Chloroquine (CQ) is a former first-line drug for P. falciparum (that is still in use for P. vivax), and it’s phasing out has been associated with the recent emergence of CQ-sensitive P. falciparum parasites. While the CQ resistance transporter (PfCRT) is known to be the primary driver of resistance, studies have provided evidence for secondary modulators of CQ, of which only the multidrug resistance protein 1 (PfMDR1) transporter has been identified. This thesis addresses the hypotheses that additional mediators are involved in the parasite resistance mechanism to QN and that genes other than pfmdr1 modulate parasite resistance to CQ. In chapter 3, we present the P. falciparum genetic cross that we conducted between the QN- and CQ-sensitive African NF54 and QN- and CQ-resistant Cambodian Cam3.II parasites in huHep mice, in collaboration with Dr. Photini Sinnis’s laboratory at Johns Hopkins University. By applying different selective conditions to cross progeny bulk pools prior to cloning these bulks, we were able to recover 120 unique recombinant progeny from this cross. We observed minimal overlap in the progeny genotypes obtained from CQ and QN pressure, suggesting distinct mechanisms for parasite resistance to these drugs. Bulk progeny selection and progeny clone-based QN linkage mapping approaches identified quantitative trait loci (QTLs) on chromosomes 7 and 12, as well as minor QTLs on other chromosomes, consistent with a multifactorial resistance mechanism. We applied the latter approach to investigate parasite response to CQ and its active metabolite monodesethyl-CQ (md-CQ) and identified a novel chromosome 12 QTL in addition to pfcrt. Interestingly, while the chromosome 12 QTLs overlapped, the chromosome 7 QTL for high-grade QN resistance did not contain pfcrt. In chapter 4, we used bioinformatic approaches, whole-genome sequence data from our cross and field isolates, and literature review to identify the drug/metabolite transporter 1 (DMT1) as the top candidate of the chromosome 7 QTL, and S-adenosylmethionine mitochondrial carrier protein (SAMC), hydroxyethylthiazole kinase (ThzK), and ATP-dependent zinc metalloprotease (FtsH1) as the top candidates for the chromosome 12 QTLs. By harnessing Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 gene editing methodologies (SNP-editing, knockout, and tagging), we obtained evidence favoring DMT1 as a marker of QN resistance and localized this transporter to structures associated with vesicular trafficking, PVM, lipid bodies, and the lysosome-like digestive vacuole. We also harnessed SNP-editing and identified FtsH1 as a potential mediator of QN resistance and a modulator of CQ and md-CQ resistance. QN, mefloquine, and lumefantrine belong to the same aryl-amino alcohol class, and we found that QN is structurally more similar to mefloquine than lumefantrine. We also showed that QN can partially inhibit heme detoxification. While conducting the work outlined in chapters 3 and 4, we identified an unmet need for quickly identifying clonal recombinant progeny and validating parasite identity, which inspired the study presented in chapter 5. We developed a genotyping method that can assess drug resistance-conferring SNPs directly from P. falciparum culture or infected blood as well as a multiplexed microsatellite genotyping method with five broadly informative markers. Both methods were applied in chapter 3 to identify clonal recombinant progeny, and the SNP genotyping method was used in chapter 4 to validate gene editing and progeny identity. We also tested the resolution, sensitivity, time, and cost of each method as well as whole-genome sequencing and recommended the ideal application for each genotyping method. Our data demonstrate that DMT1 is a novel marker for QN resistance, and a new chromosome 12 locus associates with CQ response, of which ftsh1 is a potential candidate. In chapter 6, we discuss the potential mechanisms by which DMT1 is involved in QN resistance, the potential impact of our findings, and future experiments that can further characterize the QN and CQ resistance mechanisms and the functional role of these candidate genes.