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）：连锁图谱已被证明是鉴定原生动物寄生虫（弓形虫、疟原虫、锥虫）功能基因的一种极其强大的方法，允许鉴定重要生物医学特征（如宿主特异性、毒力和耐药性）的基因。利用经典的连锁定位方法，我们已经鉴定出了一个强数量性状位点（QTL） （LOD = 21），为曼氏血吸虫对奥氨喹的抗性提供了基本证据，证明连锁定位在曼氏血吸虫中也是可行的。然而，经典的连锁定位是非常劳动密集型的，物流上具有挑战性和昂贵的，因为表型和基因型都必须在个体F2代寄生虫中测量。研究啮齿动物疟疾和酵母的研究人员开发了新的X-QTL（或连锁群选择）方法，有望进一步扩大连锁图谱的能力，因为可以有效地分析数千个后代。在X-QTL分析中，大量药物选择（或未选择）的F2后代被定量基因分型，以确定选定的基因组区域。由于该方法不需要表型分型，可以有效地检查1000个汇集的后代，我们认为它非常适合曼索尼血吸虫。我们的中心目标是(a)开发和验证用于曼氏沙门氏菌连锁分析的高效X-QTL方法，以及(b)使用这些方法鉴定该病原体重要生物医学性状的潜在基因。我们重点研究耐药性状，因为这些性状具有遗传基础，在生物医学上具有重要意义。用吡喹酮（控制血吸虫病的主要手段）治疗后治愈率降低，已从多个疫源地报告，并具有遗传基础；此外，对二线药物奥胺喹（OXA）的耐药性也存在于自然界。最初，我们将专注于OXA。我们将通过比较X-QTL鉴定的qtl与使用经典连锁映射鉴定的qtl来验证X-QTL方法。此外，我们将使用