Efficient Linkage Mapping Methods for Schistosoma mansoni

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）中功能基因的一种非常有效的方法，可以鉴定重要生物医学性状（如宿主特异性、毒力和耐药性）的基因。使用经典的连锁作图，我们已经确定了一个强大的数量性状位点（QTL）（LOD = 21）的抗氯喹的chr 6提供证据的原则，连锁作图也是可行的曼氏血吸虫。然而，经典的连锁作图是极其劳动密集型的，逻辑上具有挑战性和昂贵的，因为表型和基因型必须在单个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的新X-QTL（或连锁群选择）方法有望进一步扩大连锁作图的能力，因为可以有效地分析数千个后代。在X-QTL分析中，对药物选择的（或非药物选择的）F2子代的大库进行定量基因分型以鉴定选择的基因组区域。由于这种方法不需要进行表型分析，并且可以有效地检测1000个混合后代，我们认为它非常适合于S。mansoni我们的主要目标是：（a）建立和验证有效的X QTL连锁分析方法; mansoni和（B）使用这些方法鉴定该病原体中重要生物医学性状的基因。我们专注于耐药性性状，因为这些性状具有遗传基础，并且在生物医学上很重要。吡喹酮（PZQ）是血吸虫病控制的主要药物，在多个疫源地报告了治疗后治愈率降低，并具有遗传基础;此外，自然界中也存在对二线药物奥曲喹（OXA）的耐药性。首先，我们将专注于OXA。我们将通过比较X-QTL与经典连锁作图法所鉴定的QTL来验证X-QTL方法。此外，我们将使用 X-QTL精细定位含有OXA抗性的基因组区域。在目标2中，我们将应用X-QTL方法来定位基因组区域，这些区域是PZQ抗性的基础，使用下一代测序对亲本寄生虫进行基因分型，并准确测量处理或未处理的F2后代的大池中的全基因组等位基因频率。这项工作将使用使用序列捕获方法生成的简化代表性文库进行。通过专注于363 Mb基因组中的仅5 Mb，我们将能够以高读取深度对池进行测序，以高精度和低成本测量等位基因频率和QTL位置。成功鉴定药物反应基因将允许开发用于监测耐药性传播的分子工具，提供对药物作用模式的见解，允许开发可以杀死耐药寄生虫的修饰化合物。最重要的是，本项目将建立有效的X-QTL连锁作图方法。mansoni工具包，并为鉴定感染人类的主要蠕虫病原体中广泛的生物医学重要性状的遗传决定因素奠定了基础。

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.