白豆杉的种群表观基因组学研究：剖析气候和土壤的叠加效应

Pseudotaxus chienii (Cheng) Cheng (Taxaceae) is an endangered gymnosperm species endemic to China. In this project, we propose to investigate the epigenetic variations in natural populations of P. chienii and disentangle the synergistic effects of climate and soil on its population epigenetic differentiation. Techniques such as methylation-sensitive amplified polymorphism (MSAP), reduced representation bisulfite sequencing (RRBS), and methylation filtration (MF) will be used to detect methylated DNA. Meanwhile, a hierarchical Bayesian statistical approach, together with generalized linear regression and linear mixed models will be applied to identify the climate/soil variables that determine the population epigenetic structure. The project will mainly focus on the following objectives: (1) to assess population epigenetic compositions and test the evolutionary correlation between epigenetic, morphological, and genetic variations at population level by using MSAP analysis; (2) to characterize the composition, sequence context, and distribution of DNA methylation across the whole genome based on RRBS data, measure methylation levels, and identify single-methylation variants (SMVs); (3) to examine the roles of climate/soil variables and their interactions on population epigenetic structure, aiming to identify the most important variables and determine the MSAP loci and SMVs by which climate/soil variables exert directional forces to drive epigenetic differentiation; and (4) to sequence methylation filtered clones in order to resolve genes and genomic regions of P. chienii that are subject to methylation pattern changes under different environmental conditions; to detect methylation polymorphisms and single-nucleotide polymorphisms (SNPs) via bisulfite sequencing and conventional sequencing, investigate their clinal variation patterns, and seek the nucleotide site associated with local environment. These studies may provide valuable information for developing an efficient ex-situ conservation strategy for P. chienii in the context of global changes.

白豆杉为我国特有濒危裸子植物。本项目拟采用甲基化敏感扩增多态性MSAP、简化代表性重亚硫酸盐测序RRBS和甲基化过滤文库MF技术，基于贝叶斯统计分析，研究其种群表观遗传变异，解析种群表观遗传分化中气候和土壤的叠加效应。研究内容有：（1）借助MSAP分析种群的表观遗传组成；探究种群表观遗传变异和形态、遗传变异的进化关联；（2）利用RRBS在全基因组范围研究白豆杉DNA甲基化的组成、序列背景及分布；检测种群的甲基化水平，鉴定单甲基化变异SMVs；（3）剖析气候/土壤变量及其相互作用对种群表观遗传结构造成的影响，明确起主导作用的变量，探究气候/土壤因子借对哪些MSAP座位和SMVs的定向作用以驱动表观遗传分化；（4）构建MF文库并测序；检测哪些基因及基因组区域，其甲基化因环境变化而改变；分析甲基化多态性和单核苷酸多态性的梯度变异，识别做出局域性响应的核苷酸位点。项目对白豆杉迁地保育具重要意义。

本项目对白豆杉开展了种群表观基因组学研究，剖析了驱动白豆杉天然种群发生适应性表观遗传分化的关键气候、环境及其他生态因子。已完成以下研究内容并获得一些重要结果：第一，采用简化基因组甲基化测序（Methyl-RAD）技术，对来自不同地域的白豆杉个体的甲基化在全基因组水平进行了检测。第二，利用甲基化敏感扩增多态性（MSAP）技术，揭示了种群的表观遗传变异水平、表观遗传变异在种群内和种群间的分配及种群表观遗传分化程度和地理距离之间的相关性，并将之与相应的遗传变异格局进行了对比分析。第三，对生长于不同气候和土壤条件的种群，自主测定了各种群的20个土壤成分，从WorldClim获得了气候变量数据，另外还获取了其他5种生态因子的数据；据此鉴别出了仅同气候和土壤因子相关以及既同土壤因子又同降雨量相关的表观适应性座位；发现与遗传变异相比，表观遗传变异与干旱季节的降雨量有更强的关联。厘清了环境和空间协变量对遗传和表观遗传变异的作用。另外，还结合白豆杉的生物、生态学特性，明确了影响其种群表观基因组分化式样形成的原因。项目的研究结果发表在BMC Genomics、BMC Plant Biology、Frontiers in Genetics、Ecology and Evolution、Scientific Reports和科学通报等重要SCI国际及国内学术期刊，被国内外其他研究组多次引用。