明尼苏达被毛孢对大豆孢囊线虫寄生适应性进化机制

Parasitism represents a major interspecies interaction and its evolution is not well understood. Local adaptation and density-dependent parasitism are the evolutionary phenotypes in a parasite/host system. Serial passage experiments are a form of experimental evolution that can monitor molecular and phenotypic evolution in real time and provide insights into the causes and consequences of parasite evolution. Soybean cyst nematode (SCN, Heterodera glycines) is the devastating pest of soybean and Hirsutella minnesotensis is a dominant parasite and potential biocontrol agent of SCN. The parasitism evolution of H. minnesotensis (2 isolates) adapted to nematode populations (4 SCN and 1 Caenorhabditis elegans) will be conducted under the controlled condition. This proposal is going 1) to continuously serial infecting different nematode populations with each of two H. minnesotensis isolates for at least 50 infection cycles in 12-well tissue plates; 2) to continuously serial planting soybean at least for 12 generations in the pots with inoculations of different SCN populations and H. minnesotensis isolates; 3) to evaluate the local adaptation and density dependent parasitism of selected adaptive populations on different nematode populations in tissue plate assay and greenhouse pot experiment; 4) to compare the genomes of those selected populations of H. minnesotensis by using SNP, Indel, CNV etc.; 5) to identify key genes involving the parasitism evolution by comparative genomes coupled with the transcriptome data, and gene function determination. The minimum output of this study is to identify key genes from the whole genome for parasitism evolution. The maximum output is to illustrate the process and molecular basis of local adaptation and density dependent parasitism. The achievements will not only provide new insight for parasitism adaptive evolution but also can be applied in the biological control of pests.

寄生是生物防治的核心种间关系之一，寄生适应性进化表现为本地适应性和密度依赖制约关系。本项目以不同地域来源的线虫生防真菌明尼苏达被毛孢(Hm, Hirsutella minnesotensis)和大豆孢囊线虫(SCN, Heterodera glycines)以及秀丽隐杆线虫(Ce, Caenorhabditis elegans)为研究对象，在组培板中进行Hm对SCN继代侵染实验，获得至少50代适应不同地域SCN种群及Ce的菌株；在温室盆栽大豆条件下，建立Hm对SCN侵染实验，获得至少12代大豆条件下适应不同地域SCN种群的Hm；通过对不同线虫种群交叉接种和不同真菌接种浓度下真菌对线虫寄生率测定，明确Hm对SCN的寄生适应性、本地适应性及密度依赖制约关系；通过Hm群体基因组和转录组分析，揭示Hm寄生SCN关键基因，明确Hm对SCN寄生适应性进化分子机制，本研究具有重要的理论和实际意义。

寄生是生物防治的核心种间关系之一，寄生适应性进化表现为本地适应性和密度依赖制约关系。本项目主要研究内容是以不同地域来源的线虫生防真菌明尼苏达被毛孢(Hm, Hirsutella minnesotensis)和大豆孢囊线虫(SCN, Heterodera glycines)以及秀丽隐杆线虫(Ce, Caenorhabditis elegans)为研究对象，通过组培板及温室种植大豆进行Hm对SCN继代侵染实验，明确Hm对SCN的寄生适应性、本地适应性及密度依赖制约关系；通过Hm群体基因组和转录组分析，揭示Hm寄生SCN关键基因，明确Hm对SCN寄生适应性进化分子机制。我们首先完成了明尼苏达被毛孢基因组分析，初步明确了其线虫内寄生特征；群体遗传学分析发现中国种群和美国种群差异较大，中国种群遗传多样性均显著高于美国种群(Tajima's D < 0)，中国种群经历扩张或正选择，美国种群经历瓶颈效应或平衡选择。中美种群的交配型基因不同，中国为mat-1-1而美国为mat-1-2，推测明尼苏达被毛孢为异宗配合，但未发现有性阶段；完成了50个Hm群体基因组重测序，GWAS分析鉴定出与寄生性相关的8个强烈分化位点，并通过基因功能研究验证了一个候选基因与产孢和寄生性有关；同时，通过构建融合GFP菌株，明确了Hm侵染SCN及Ce的侵染历程。在继代侵染方面，继代侵染30代后菌株对不同寄主线虫进行交叉接种，发现没有明显的寄生性提高，经过认真分析发现可能的原因是通过移液枪吸取寄生后的线虫（不能避免带有Hm孢子）转接，从而导致实验失败。因此我们重新构建了新的继代侵染体系，并且修改挑取1条侵染线虫为挑取10条寄生的线虫，并于2017年的年度总结中提出这部分研究内容延期。目前新建立的侵染体系已经在组培坂上完成了30 代的继代侵染和温室4次大豆种植周期，进一步的继代侵染和交叉接种工作正在进行。本研究已经在Genome Biology and Evolution发表论文1篇，1篇在审稿中，在Scientific Reports发表论文1篇。共培养研究生6人，博士后1人。