核糖体RNA基因是原生动物分子鉴定、进化与生态研究中一个极为重要的分子标记。申请人近期的研究首次发现纤毛虫原生动物基因组中rRNA基因的拷贝数与多态性总体上高于其他真核生物类群、多态性与拷贝数呈正相关，并指出该现象可能影响原生动物分子生态、种类鉴定及系统演化推断的准确度。本项目拟在此工作基础上，应用单细胞定量PCR与焦磷酸测序测序，分析不同类群纤毛虫基因组中rDNA拷贝数与多态性在个体、种群及种间水平的变异，弄清rDNA拷贝数及序列变异的主要来源；明确不同营养及环境（温度、盐度、溶氧）条件下rRNA基因表达的比例及基因型；评估rDNA多态性对原生动物物种鉴定、系统进化推断及分子多样性研究的影响。研究成果将为原生动物分子分类、生理与生态适应机制、rRNA基因的表观遗传学、分子生态研究提供第一手数据，也为rDNA非协同进化类群的系统进化提供重要参考。

The ribosomal RNA operon is one of the most important biomarker in molecular taxonomy, evolutionary and molecular ecological studies. Recently, for the first time, the applicant has found that: 1) the copy numbers and sequence polymorphisms of rRNA gene in ciliated protozoa (unicellular eukaryotes) are generally higher than these in all other eukaryotes including plants, animals and fungi; 2) the sequence polymorphism is significantly correlated with the copy number, and 3) thus provide evidence that the relative abundance of ciliates in environmental samples could be overestimated due to their high rDNA copy number per cell, and that the estimation of species richness and phylogeny of ciliates in environmental samples could be problematic because of their high sequence polymorphism in a single cell. Despite these advances, little is known about the situation at the RNA level, and more data from diverse ciliate taxa are needed to strengthen these new findings..Following the line of the previous study, this project proposes to determine the variations of the number and polymorphisms of rDNA copies among individuals, populations, species and higher taxa, by using single-cell based quantitative PCR and deep sequencing technologies. By doing so, the source of main variation can be identified. As the ciliates unlikely need so many rDNA copies (mostly over 100,000) for faster transcription, some rRNA gene must not be actively expressed. Therefore, how many and which rDNA copies are expressed under certain conditions are interesting questions, which are also hot topics on epigenetics of rRNA genes. In this project, we are going to generate such important data by comparing the numbers of rRNA transcripts and rDNA in newly formed duaghter cells and mature individuals, using reverse-transcription PCR and pyrosequencing. With these data, the hypothesis of high copy number – high sequence polymorphism can be further tested, and the effect of high polymophosims of rDNA on rDNA-based phylogenetic inference can be investigated.