Refining Mendelian genetics of malaria parasites

摘要 一个充分知情的实验遗传杂交是一个强大的正向遗传学工具，因为它产生重组 用于观察到的表型的遗传作图的后代。这种方法已被广泛应用于许多模型中 从苍蝇到老鼠的生物体，但是杂交不同的人类疟疾寄生虫恶性疟原虫， 每年造成数十万人死亡，这是一个挑战。寄生虫的一生 循环是复杂的，在蚊媒和人类宿主之间交替，包括专性有性生殖， 生殖为了产生和表型重组寄生虫，完整的生活史从无性 在蚊子到肝脏的阶段，血液阶段到重组体的形成，然后回到无性 血液阶段必须在实验室进行。以前，肝脏阶段到血液阶段的过渡是唯一可能的 在切除脾脏的黑猩猩中，由于黑猩猩研究的伦理和经济障碍， 在国家卫生研究院禁止黑猩猩研究之前，我们研制出了一种人肝 允许恶性疟原虫肝脏阶段发育和向血液阶段转变的嵌合小鼠模型 感染，并已成功地使用该模型从恶性疟原虫遗传杂交中回收后代。的 这种小鼠模型的易用性现在将允许我们创建许多信息充分的交叉。本研究 项目将首先确定变量和因素，使我们能够最大限度地回收独特的重组 寄生虫我们预测，如果有超过60个独特的重组后代，我们将能够精细定位感兴趣的性状， 基因组区域只包含几个候选基因。此外，我们将使用重组后代， 回交实验，以确定基因参与寄生虫的能力，以避免自交和促进 杂交以及在杂交实验中观察到的细胞质基因组不相容性。加速 杂交可以驱动寄生虫菌株之间的重组，从而加速药物的传播。 通过寄生虫种群传播抗性基因。相反，细胞质基因组不相容性可以防止 寄生虫菌株重组。本计画所分析的交配机制具有重要的意义 在P01赠款总额的背景下，该赠款旨在更充分地了解青蒿素耐药性和耐药性 与伙伴药物，包括哌喹，这是目前消除疟疾工作中的一个重大关切。 最后，我们将建立与基因组测序和数量性状位点相结合的批量分离分析 作图以使与表型相关的基因的更有效的作图成为可能，这对于 分析了东南亚现已记录的新出现的哌喹耐药寄生虫。的 该研究项目的成功完成将大大加强恶性疟原虫遗传学研究， 进一步了解寄生虫抗药性的出现、演变和传播。

ABSTRACT A well informed experimental genetic cross is a powerful forward genetics tool because it generates recombinant progeny for genetic mapping of observed phenotypes. This approach has been extensively used in many model organisms from flies to mice but crossing different strains of the human malaria parasite Plasmodium falciparum, responsible for the deaths of hundreds of thousands of individuals every year, is a challenge. The parasite's life cycle is complex, alternates between the mosquito vector and human host and includes obligate sexual reproduction. In order to generate and phenotype recombinant parasites, the complete life cycle from asexual blood stage-to-sexual blood stage-to-recombinant formation in the mosquito-to-liver stage and back to asexual blood stage must occur in the laboratory. Previously, the liver stage-to blood stage transition was only possible in splenectomized chimps and due to the ethical and financial roadblocks to chimp research only three genetic crosses were ever performed before the NIH banned chimp research. We have developed a human-liver chimeric mouse model that allows for P. falciparum liver stage development and transition to blood stage infection and have used this model successfully to recover progeny from P. falciparum genetic crosses. The ease of use of this mouse model will now allow us to create numerous well-informed crosses. This Research Project will initially define variables and factors that will allow us to maximize the recovery of unique recombinant parasites. We predict that with >60 unique recombinant progeny, we will be able to fine map traits of interest to genome regions containing just a few candidate genes. Furthermore, we will use recombinant progeny for backcrossing experiments to determine genes involved in the parasite's ability to avoid selfing and promote hybridization as well as cytoplasmic genome incompatibility observed in crossing experiments. Accelerated hybridization could drive recombination between parasite strains and thus could speed the spread of drug resistant genes through a parasite population. Conversely, cytoplasmic genome incompatibility could prevent parasites strains from recombining. The mating mechanisms analyzed within this project are of great importance in the context of the overall P01 grant, which aims to more fully understand artemisinin resistance and resistance to partner drugs, including piperaquine, which currently are a significant concern in malaria elimination efforts. Finally, we will establish bulk segregant analyses coupled with genome sequencing and quantitative trait loci mapping to enable more efficient mapping of genes linked to phenotypes and this will be especially important for analysis of emerging piperaquine resistant parasites that have now been documented in Southeast Asia. The successful completion of this Research Project will greatly enhance P. falciparum genetics research and will further our understanding of parasite drug resistance, with regard to its emergence, evolution and spread.