本研究以淡水生态系统中常见的萼花臂尾轮虫（Brachionus calyciflorus）为模式生物，探讨了母体效应在其不同繁殖类型母体中的作用模式及其对子代形态性状的传代影响，为以后在分子水平上深入探究母体效应的作用及其机理奠定基础。本研究还初步探讨了母体效应对子代全基因组甲基化类型及水平的影响，并基于 SLAF 简化基因组测序技术进行了萼花臂尾轮虫 SNPs 位点开发。研究工作的主要成果概述如下：

     1、萼花臂尾轮虫不同繁殖方式中捕食者诱导的母体效应响应模式的差异

     母体效应在生物对异质环境的适应性响应中发挥着重要作用，但不同母体类型如何影响后代的适应性响应模式, 还知之甚少。本研究探究了捕食者信号诱导的母体效应在萼花臂尾轮虫两种繁殖方式的母体（非混交雌体和混交雌体）中的作用模式（性状、方向和强度）是否存在差异。结果发现：首先，捕食者信号诱导的母体效应通过非混交雌体和混交雌体均可以对子代表型产生影响。营孤雌繁殖的非混交雌体母体对子代跨代表型响应的敏感性影响显著，但这一现象在有性繁殖的混交雌体母体中未发现。其次，非混交雌体中母体效应对子代性成熟时间和初始生命期望的影响显著，但混交雌体的母体效应对子代性成熟时间、 种群的内禀增长率和世代净增值率的作用更为显著。 此外， 当子代环境中不存在捕食胁迫时，两种繁殖方式中捕食者信号诱导的母体效应对子代性成熟时间的作用方向完全相反。 本研究验证了萼花臂尾轮虫中捕食者信号诱导的母体效应存在繁殖母体类型特异性和子代响应性状特异性。

       2、捕食者诱导的母体效应对萼花臂尾轮虫干母传代方式的影响

       尽管近期有研究模型预测在广泛的异质性环境条件下，传代效应所带来的多代表观遗传稳定性可能影响子代的适合度，然而仍鲜有实证研究。单巢类轮虫干母代内表型可塑性的抑制可能通过更持久的传代效应使子代产生持久的表型响2应，予以弥补克隆种群在异质性环境中的适合度。本研究以卜氏晶囊轮虫-萼花臂尾轮虫为捕食者-猎物模型，以该模型中萼花臂尾轮虫防御形态的变化作为母体效应的响应指标，探究了捕食者信号能否诱导干母产生传代效应及其作用模式。实验结果表明： 1 捕食者信号诱导的母体效应不仅可以通过干母影响 F1 代侧后棘刺长度的发育，对子代体型大小的传代模式也存在显著影响； 2 F1 代侧后棘刺长度受干母环境母体效应的影响显著，但 F1 代的体型大小则不然； 3 捕食者信号诱导的母体效应对干母子代侧后棘刺长度的影响仅维持了一代，不存在传代应，但子代体型大小却随传代数的变化呈现明显的传代效应，至少可以持续 7代以上。本研究结果揭示了干母经历捕食者信号胁迫会对子代体型产生持久的传代效应，并且这种传代效应存在性状异质性，代内可塑性响应强的性状可能不存在显著的传代效应。

       3、萼花臂尾轮虫母体和子代经历的捕食者信号对子代全基因组甲基化类型及水平的影响

       目前，人们对母体效应的表观遗传机制尚知之甚少。本研究在前期研究的基础上，利用简化基因组甲基化技术进一步探究了混交雌体所产休眠卵孵化出的干母环境中捕食者信号胁迫对子 1 代全基因组甲基化水平的影响。结果发现，萼花臂尾轮虫中存在 5mC 胞嘧啶甲基化，并且与多数无脊椎动物相似，其全基因组甲基化水平远低于脊椎动物， CHH（H 代表碱基 A、 C 或 T 的任意一种）作为主要的甲基化类型，占比约 80%。当子代环境中存在捕食者信号时，其全基因组甲基化水平低于子代环境中不存在捕食者信号的处理。相关性分析的结果显示，母体环境中的捕食者信号胁迫可能会改变子代 DNA 甲基化的调控模式。本研究首次开展轮虫适应性表型（代内或代间）可塑性的表观遗传调控的研究，为未来在分子水平上进一步研究适应性跨代可塑性与表观遗传调控的适应性进化模式开辟了窗口。

       4、基于 SLAF 简化基因组技术的萼花臂尾轮虫 SNPs 位点开发及遗传分化分析

       萼花臂尾轮虫作为母体效应研究的优良模式生物，尚缺乏基因组水平的高效分子标记用于进一步深入探讨母体效应在种群进化、生态适应方面的作用及其机理。本研究在实验室前期基于萼花臂尾轮虫线粒体 CoI 序列分子标记分型的基础3之上，利用特异性位点扩增片段测序（Specific Locus Amplified FragmentSequencing, SLAF-seq）技术对五个实验室用于个体适应和种群进化研究的萼花臂尾轮虫品系进行 Illumina 高通量测序。从 H4、 H6、 H1、 D1 和 LY 五个品系中共测得 125.83Mreads 数据，总计获得 1318,772 个 SLAF 标签。五个样品的平均测序深度为 63.49?。共获得 66,392 个群体 SNPs。系统发育树结果显示，同域分布的 H1、 H4 和 H6 品系具有更近亲缘关系，而 D1 和 LY 的亲缘关系更近。五个品系之间存在较高的遗传分化，可能来自于 5 个不同的原始祖先群体。季节性宽生态位的 H6 品系遗传多态性最高，其次为 LY。季节性窄生态位品系 H4 和H1 的遗传多态性最低。 LY 与 D1 间可能存在一定程度的基因渗入。本研究利用SLAF-seq 技术开发了萼花臂尾轮虫 SNPs 分子标记， 检测到大量 DNA 多态性。并再次于同域分布的萼花臂尾轮虫中验证了“扩散-基因流悖论”。 本项结果为未来以萼花臂尾轮虫为模式生物深入开展种群进化与相关生态研究提供了有价值的分子标记和基因组数据。

       The present thesis studied the differences of maternal effects between different type of mathers in the parthenogenetic cycle and the sexual cycle and the role of transgenerational effects on stem female?s defensive morphology using the monogonant rotifer Brachionus calyciflorus, a common species in freshwater ecosystems, as the model animal. This study also preliminarily discussed variable patterns of the whole-genome DNA methylation of progeny under the influence of maternal effects induced by predator-cue. Furthermore, we have identified rich DNA polymorphism by the SLAF-seq method in B. calyciflorus. Our principal results are as the following:

      1、In this work, we aimed to examine the different response patterns of predator-cue induced maternal effects between amictic mother in the parthenogenetic reproduction cycle and mictic morther in the sexual reproduction cycle in the rotifer B. calyciflorus. Firstly, we found that predator-cue induced maternal effects affected the offspring?s phenotypes both in the parthenogenetic reproduction cycle and the sexual reproduction cycle. However, when amictic mother F0 experienced a predator-cue environment, her offspring F1 produced longer posterolateral spine F2 at a low concentration of predator-cue than those F1without predator-cue environment of F0. Secondly, for parthenogenetic mothers, maternal effects mainly affected the offspring?s age at maturity and life expectancy at hatchings, while maternal effects of the mictic mother significantly influenced the offspring?s age at maturity, their population?s intrinsic growth rate and net reproductive rate. Furthermore, when there was no predator-cue in the offspring?s environment, the directions of maternal effects induced by predator-cue on the offspring?s age at maturity were different between two maternal types. Our result found that predator-cue induced maternal effects differed much between different maternal types in two reproduction cycles and that the offspring?s response phenotypes were specificity.

       2 ．Although recent research theoretical models have predicted that under heterogeneous environmental conditions, the stability of multi-generational epigenetic inheritance related to transgenerational effects would have an impact on the fitness of the offspring, it is rarely proved by experimental studies. In rotifer genus Brachionus, inhibition of within-generational phenotypic plasticity in the stem female may be made up of transgenerational effects to adapt for the heterogeneous habits. In our study, Asplanchna brightwellii and B. calyciflorus constituted a predator-prey model. The variations of defensive morphology of B. calyciflorus were taken as parameters of maternal effects to explore whether the predator signal could induce transgenerational effects in the stem female and its action mode. The results show that: 1 the predator signal experienced by stem female could not only affect the length of posterolateral spines of the F1 generation, but also showed significant transgenerational effects of the body size of the progeny; 2 For the F1, predator-cue in stem female?s environment increased the length of posterolateral spines, but decreased body size of F1 by mismatching of the parental-offspring environment; 3 The predator-cue induced transgenerational effects were only detected in body size and lasted for 7th generations at least, while the effect on length of posterolateral spines just maintained for one generation. These results reveal that the stem female experienced predator signal stress could affect the body size of multigenerational offspring, and this transgenerational effects could be specificity on traits. Those traits with strong intra-generation plasticity response may not have significant transgenerational effects.

       3 ．Little is known about the epigenetic mechanism of maternal effects in B.calyciflorus. Our study used reduced representation bisulfite sequencing to achieve the genome-wide analysis of DNA methylation of offspring, to explore the influence of predator-cue experienced by mictic female and stem female on methylation patterns in their offspring B.calyciflorus. The results found that there was 5mC cytosine methylation in B. calyciflorus, which is similar to most invertebrates, and its genome-wide methylation level is much lower than that of vertebrates. CHH (H stands for base A, C or T) as the main type of methylation, was accounting for about 80% in all four treatments. When there was predator-cue in the offspring?s environment, the genome-wide methylation level was lower than that without predator-cue. The results of Pearson correlation analysis showed that maternal6 environment with predator-cue could change the regulation pattern of DNA methylation of the progeny. It is the first study trying to explore epigenetic regulation of adaptive phenotype plasticity (within or between generations) in the rotifer genus, Brachionus, which would open a window for further research on the adaptive evolution of maternal effects and epigenetic regulation.

       4 ．As an excellent model organism for the study of maternal effects, B. calyciflorus lacked efficient genome-wide molecular markers to explore the role and mechanisms of maternal effects in ecological adaptation and evolution. In this study, five strains of B. calyciflorus labeled by the DNA bar code mitochondrial (CoI) DNA sequences were used to develop SNP marks of the rotifer. These strains were sampled initially in different seasons from Xihai Lake, Beijing, and cultured in our laboratory. They were also identified DNA polymorphisms by Specific Locus Amplified Fragment Sequencing technology (SLAF-seq). Using Illumina high-throughput sequencing platform, a total of 125.83M reads data and 1318,772 SLAF tags were developed from the five strains of rotifer (H4, H6, H1, D1, and LY). The average sequencing depth for the five samples is 63.49. We identified a total of 66,392 SNPs. The phylogenetic relationships of five strains were classified into two branches, one consisting of H1, H4, and H6 strains, one consisting of D1 and LY. The genetic diversity of the H6 strain, which owned a long-term seasonal niche was the highest, followed by LY stains. H4 and H1 showed the lowest genetic polymorphism in the short-term seasonal niche. There may be gene infiltration between LY and D1. This work got a big amount of SNP marksusing the SLAF-seq technology, identified rich DNA polymorphisms in rotifer, B. calyciflorus. The dispersal–gene flow paradox in zoo plankton was verified again with the genome-wide DNA of rotifer B. calyciflorus distributed in the homogenous filed. Our result will provide valuable molecular markers and genomic data for future researches.