Efficient Linkage Mapping Methods for Schistosoma mansoni

曼氏血吸虫的高效连锁作图方法

• 批准号: 8386269
• 负责人: Tim J Anderson
• 金额: $ 26.48万
• 依托单位: TEXAS BIOMEDICAL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-01 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8386269
• 关键词: Alleles; Chromosome Mapping; DNA; Development; Drug effect disorder; Drug resistance; Gene Frequency; Generations; Genes; Genetic; Genetic Determinism; Genome; Genome Mappings; Genomics; Genotype; Goals; Helminths; Human; In Vitro; Individual; Left; Libraries; Life; Location; Logistics; Malaria; Maps; Measurement; Measures; Methods; Molecular; Monitor; Mus; Nature; Parasite resistance; Parasites; Parents; Pharmaceutical Preparations; Phenotype; Plasmodium; Praziquantel; Praziquantel resistance; Quantitative Trait Loci; Reading; Reporting; Research Personnel; Resistance; Rodent; Schistosoma; Schistosoma mansoni; Schistosome Parasite; Schistosomiasis; Specificity; Staging; Testing; Toxoplasma; Trypanosoma; Virulence; Work; Yeasts; base; cost; genetic analysis; genetic linkage analysis; genome sequencing; genome-wide; insight; killings; next generation; pathogen; response; tool; trait

DESCRIPTION (provided by applicant): Linkage mapping has proved to be an extremely powerful approach for identification of functional genes in protozoan parasites (Toxoplasma, Plasmodium, Trypanosomes), allowing identification of genes underlying important biomedical traits such as host specificity, virulence and drug resistance. Using classical linkage mapping we have identified a strong quantitative trait locus (QTL) (LOD = 21) for Oxamniquine resistance on chr 6 providing proof-of-principal that linkage mapping is also feasible for Schistosoma mansoni. However, classical linkage mapping is extremely labor intensive, logistically challenging and expensive, because both phenotypes and genotypes must be measured in individual F2 generation parasites. New X-QTL (or linkage group selection) methods, developed by researchers working on rodent malaria and yeast, promise to further expand the power of linkage mapping, because many 1000s of progeny can be effectively analyzed. In X-QTL analysis large pools of drug selected (or unselected) F2 progeny are quantitatively genotyped to identify selected genome regions. As this method does not require phenotyping and can effectively examine 1000s of pooled progeny, we believe it is well suited to S. mansoni. Our central goal is (a) to develop and validate efficient X-QTL methods for linkage analysis of S. mansoni and (b) to use these methods to identify the genes underlying important biomedical traits in this pathogen. We focus on drug resistance traits as these traits have a genetic basis and are biomedically important. Reduced cure rate following treatment with praziquantel (PZQ), the mainstay of Schistosomiasis control, has been reported from multiple foci and has a genetic basis; furthermore resistance to the second line drug oxamniquine (OXA) also occurs in nature. Initially, we will focus on OXA. We will validate X-QTL methods by comparing the QTLs identified by X-QTL with those identified using classical linkage mapping. In addition, we will use X-QTL to fine map the genome region containing OXA resistance. In aim 2 we will apply X-QTL approaches to map genome regions that underlie resistance to PZQ, using next generation sequencing both to genotype parental parasites and to accurately measure genome wide allele frequencies in large pools of treated or untreated F2 progeny. This work will be conducted using reduced representation libraries generated using sequence capture methods. By focusing on just 5Mb of the 363Mb genome we will be able to sequence pools to high read depth, measuring allele frequencies and QTL location with great accuracy and at low cost. Successful identification of genes that underlie drug response will allow development of molecular tools for monitoring resistance spread, provide insights into the mode of drug action, allow development of modified compounds that can kill resistant parasites. Most importantly, this project will establish efficient X-QTL linkage mapping approaches in the S. mansoni toolkit and set the stage for identification of the genetic determinants of a wide range of biomedically important traits in the major helminth pathogen infecting humans. PUBLIC HEALTH RELEVANCE: Schistosome parasites vary in biomedically important traits including virulence, host specificity, and resistance to drug treatment. We aim to develop efficient, economical methods for genetic mapping in S. mansoni that are applicable to a wide range of biomedically important traits.

描述（由申请人提供）：事实证明，链接映射是一种非常有力的方法，用于鉴定原生动物寄生虫中功能基因（弓形虫，疟原虫，锥虫体），允许鉴定出重要的生物医学特征（例如宿主特异性，病毒性，病毒性和耐药）的基因。使用经典的链接映射，我们已经确定了一个强的定量性状基因座（QTL）（lod = 21），用于chr 6上的oxamniquine耐药性，提供了主要的原理证明，链接映射也适用于Schistosoma Mansoni。但是，经典的连锁映射是劳动力密集的，逻辑上具有挑战性且昂贵的，因为表型和基因型都必须在单个F2生成寄生虫中衡量。由研究啮齿动物疟疾和酵母的研究人员开发的新X-QTL（或链接组选择）有望进一步扩大链接映射的力量，因为可以有效地分析许多1000秒的后代。在X-QTL分析中，对选定（或未选择）F2后代的大量药物库进行了定量基因分型，以鉴定选定的基因组区域。由于这种方法不需要表型，并且可以有效检查1000秒的合并后代，因此我们认为它非常适合Mansoni。我们的核心目标是（a）开发和验证有效的X-QTL方法，用于对曼氏链球菌的链接分析和（b）使用这些方法来识别该病原体中重要的生物医学特征的基因。我们专注于耐药性状，因为这些特征具有遗传基础，并且在生物医学上很重要。已从多个病灶据报道，用Praziquantel（PZQ）（PZQ）治疗后的治愈率降低，并具有遗传基础。此外，对第二线药物类黄酮（OXA）的耐药性也出现在自然界中。最初，我们将专注于Oxa。我们将通过将X-QTL识别的QTL与使用经典链接映射识别的QTL进行比较来验证X-QTL方法。此外，我们将使用 X-QTL以细微绘制含有OXA抗性的基因组区域。在AIM 2中，我们将采用X-QTL方法来绘制对PZQ抗药性的基因组区域，使用下一代测序既是基因型亲本寄生虫，并准确地测量大量处理过的或未经处理过的F2后代的基因组宽等位基因的频繁。这项工作将使用使用序列捕获方法生成的减少表示库进行。通过仅关注363MB基因组的5MB，我们将能够将池对高读取深度进行测序，以高成本和低成本测量等位基因频率和QTL位置。成功识别药物反应的基因将允许开发用于监测耐药性扩散的分子工具，提供对药物作用方式的见解，允许开发可杀死抗性寄生虫的改良化合物。最重要的是，该项目将在S. Mansoni工具包中建立有效的X-QTL链接映射方法，并为鉴定主要蠕虫感染人类的​​主要生物医学重要特征的遗传决定因素奠定了基础。 公共卫生相关性：生物医学上重要的特征在内的棘孢子寄生虫各种，包括毒力，宿主特异性和对药物治疗的抵抗力。我们旨在开发有效的，经济的方法来用于曼氏链球菌中的遗传图，这些方法适用于广泛的生物医学特征。