Efficient Linkage Mapping Methods for Schistosoma mansoni

描述(由申请人提供)：连锁作图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有效的方法，使鉴定寄主专一性、毒力和抗药性等重要生物医学特征的基因成为可能。利用经典连锁定位技术，我们在中国血吸虫CHR-6上定位了一个与奥沙尼喹抗性相关的强QTL(LOD=21)，为连锁定位在曼氏血吸虫中也是可行的提供了理论依据。然而，经典的连锁作图是劳动密集型、后勤挑战和昂贵的，因为表型和基因型都必须在单个F2代寄生虫中进行测量。由研究啮齿动物疟疾和酵母的研究人员开发的新的X-QTL(或连锁群选择)方法有望进一步扩大连锁图谱的力量，因为可以有效地分析数千个后代。在X-QTL分析中，对大量药物选择(或未选择)的F2后代进行定量基因分型，以确定选择的基因组区域。由于这种方法不需要表型，而且可以有效地检测数千个汇集后代，因此我们认为它非常适合曼氏血吸虫。我们的中心目标是(A)开发和验证用于曼氏血吸虫连锁分析的有效的X-QTL方法，以及(B)使用这些方法来鉴定这种病原体的重要生物医学性状的基因。我们把重点放在耐药特征上，因为这些特征有遗传基础，在生物医学上很重要。血吸虫病控制的主要药物吡喹酮(PZQ)治疗后治愈率下降，已有多个病源地的报道，并有遗传基础；此外，自然界中也存在对二线药物奥沙宁(OXA)的耐药性。最初，我们将重点关注OXA。我们将通过比较由X-QTL识别的QTL和使用经典连锁图谱识别的QTL来验证X-QTL方法。此外，我们还将使用 X-QTL用于精细定位包含OXA抗性的基因组区域。在目标2中，我们将应用X-QTL方法来定位对PZQ的抗性背后的基因组区域，使用下一代测序既可以对双亲寄生虫进行基因分型，也可以准确地测量大量处理或未处理的F2后代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表示库来进行。通过只关注363Mb基因组中的5Mb，我们将能够对池进行高阅读深度的测序，以极高的精度和低成本测量等位基因频率和QTL定位。成功识别药物反应的基础基因将有助于开发监测耐药性传播的分子工具，提供对药物作用模式的洞察，允许开发能够杀死抗药性寄生虫的修饰化合物。最重要的是，该项目将在曼氏血吸虫工具箱中建立有效的X-QTL连锁作图方法，并为鉴定感染人类的主要蠕虫病原体的一系列重要生物医学特征的遗传决定因素奠定基础。

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.