【作者】 陈露；

【导师】 李青；

【作者基本信息】 华中农业大学 ， 基因组学， 2020， 博士

【摘要】 玉米是世界上最重要的作物之一,也是遗传学研究的模式物种之一。在过去的几个世纪里,通过结合传统育种方法和现代分子生物学技术,玉米的产量已经有了显著的提升。随着气候的不断变化、耕地的不断减少、人口的不断增长,玉米的遗传改良仍然面临着巨大的挑战。对现有的遗传变异基因库进行扩充,理解玉米细胞生命活动的遗传调控机制,是进行现代玉米遗传改良的基础。本研究利用不同的玉米遗传材料和基因组学测序数据,从不同角度对玉米基因组的变异和遗传调控进行了研究。基于玉蜀黍属所有亚种基因组重测序数据,本论文构建了玉蜀黍属的遗传变异图谱,并以此为基础分析了玉蜀黍属的进化历史,进一步对玉蜀黍属的环境适应性机制进行了解析。另外本研究也基于栽培玉米转录组数据,分析了玉米环状RNA的分子特性和形成机制,对玉米转录水平的调控方式进行了扩展。最后,本研究基于玉米单细胞DNA甲基化测序数据,对玉米单孢子的甲基化模式进行了分析,进一步解析了配子发育细胞异质性产生的原因。主要结果如下:1.玉蜀黍属基因组变异与适应性进化为了扩大现代玉米遗传改良变异库,探索玉蜀黍属不同亚分类群适应环境的分子机制,本研究对玉蜀黍属所有亚分类群的基因组测序数据进行了分析。通过对690份玉米和野生大刍草各亚种基因组的分析,本研究鉴定到了超过65M的单核苷酸多态性(SNPs)、1000个以上的倒位变异位点,揭示了玉蜀黍属进化过程中亚种的分化时间与群体大小。通过对玉蜀黍属不同亚分类群的正选择基因进行研究,发现了玉蜀黍属在适应不同环境时群内选择的证据,包括对生命周期和耐涝性位点的选择。此外基于全基因组选择分析,鉴定了1,387个高原适应性位点和2,057个高纬度适应性位点,发现了激素通路相关基因在大刍草高原适应过程中所起的关键作用。为了探索大刍草高原适应过程中与环境互作的遗传位点,利用8个深度下34种土壤特性和海拔高度共计235个性状进行了全基因组关联分析研究,发现了36个与土壤环境互作的数量性状控制位点和一个与海拔高度直接相关的位点。对海拔高度相关位点候选基因Zm WOX11进行功能验证发现,Zm WOX11可以通过响应土壤中离子,调控根系生长,从而影响大刍草的高原适应性。这部分的工作不仅提供了一个高精度的玉蜀黍属遗传变异图谱,也进一步剖析了玉蜀黍属的进化和适应。通过结合全基因组选择分析和关联分析,鉴定了一批有价值的、与适应有关的变异位点,发现了激素通路在大刍草适应高原环境中的重要性,为通过对激素通路和土壤互作因子进行改良,培育环境适应性玉米新品种这一方向提供了理论基础和实验证据。2.玉米环状RNA的鉴定及功能分析为了加深对玉米功能基因组的认知,解析环状RNA调控表型变异的分子机制,本研究利用环状RNA特异的转录组测序数据、已发表的玉米线性转录组测序数据,对玉米环状RNA进行了大规模的鉴定和系统性分析。通过整合适用于玉米基因组环状RNA鉴定流程,本研究得到了2,804条高质量的环状RNA。进一步通过与线性位点进行比较分析,本研究发现产生环状RNA的位点与线性位点在基因组特性上具有明显差别,并且环化外显子侧翼显著的富集了LINE/L1元件(LLE)和其反向互补对(LLERCPs)。重点对LINE/L1元件(LLE)和其反向互补对(LLERCPs)进行分析发现,环状RNA的累积量随着LLERCPs数目的增加而进一步增加,但线性转录本的表达量却随之下降。此外,研究发现由LLERCP介导形成的环状RNA与表型变异显著相关,其中一个候选环状RNA(circ1690)与玉米穗位高的调控相关。这部分的结果发现玉米环状RNA在基因组中普遍存在,其形成可能与玉米基因组中特定类型的转座子相关。本研究提出了转座子可以通过介导环状RNA的产生,来对基因表达和表型变异进行调控的新方式。3.玉米四分体单细胞甲基化图谱的建立和重编程产生的异质性位点分析为了对玉米减数分裂过程中细胞表观遗传的变化进行解析,本研究基于实验室新开发的单细胞DNA甲基化建库方法,对玉米四分体单细胞小孢子进行了测序。研究首先开发了一种可提高单细胞甲基化测序数据利用率的方法——BRIF-MAP,并基于BRIF-MAP获得了高覆盖度的玉米四分体单细胞小孢子的甲基化图谱。通过对玉米单细胞小孢子甲基化图谱进行系统分析发现,同一个四分体的四个小孢子间甲基化水平类似,不同四分体之间的甲基化水平存在显著的差别,即不同四分体间存在明显的细胞异质性。进一步比较子代四分体小孢子和亲本小孢子的甲基化水平,发现四分体间的异质性与DNA甲基化的重编程相关。相较于亲本,子代四分体中平均有1.33%甲基化位点经历了重编程,其中平均93.8%的位点都为甲基化的获得。这些重编程产生的异质性位点在不同的基因组组分上具有一定的偏好性,在不同遗传背景下的比例类似。本研究通过利用群体基因组数据,鉴定到了大量新的遗传变异,扩大了可用于现代玉米改良的遗传变异基因库,为适应性玉米新品种的改良方向提供了理论基础和实验证据。本研究也利用转录组和表观组数据,通过对玉米环状RNA和单细胞DNA甲基化的分析,进一步增强了对玉米细胞生命活动调控复杂性的理解。更多还原

【Abstract】 Maize is one of the world’s most valuable crops and genetic model organisms.Through the combination of traditional breeding methods and modern molecular biology technique,the yield of maize has achieved remarkable improvments in the past centuries.Under the rapid change of climate,shrinking arable land and growthing population,genetic improvement of maize still facing huge challenges.Expanding the available genetic variation gene pool and understanding the genetic regulation mechanism of maize cell life activities are the basis of modern maize genetic improvement.This study explored the complexity of maize genetic variation and genetic regulation from different perspectives by using different maize genetic materials and genomics sequencing data.Based on the genome resequencing data of all subspecies of Zea,this study constructed a genetic variation map of Zea and analyzed the evolutionary history of Zea,further explored the environmental adaptation mechanism of Zea.In addition,based on the transcriptome data of cultivated maize,this study also analyzed the molecular characteristics and formation mechanism of maize circular RNAs,and extended the transcriptional regulation ways.Finally,based on the maize single cell DNA methylation sequencing data,this study analyzed the methylation pattern of maize monospore,and further analyzed the reasons for the heterogeneity in gametes developmental cells.The main findings are: 1.Genomic variation and adaptive evolution of Zea genusIn order to expand the modern maize genetic improvement variation pool and explore the molecular mechanisms of different taxa of Zea genus while adapting to different environments,genome sequencing data of all wild taxa of Zea genus(teosinte)and cultivated maize were studied.By comparing the differences between 690 genomes of teosintes and maize,we identified over 65 M single nucleotide polymorphisms(SNPs)as well as dozens of novel inversions.Our analysis of maize and teosinte genomes revealed the divergence time and effective population size change under the evolutionary of Zea genus.It reveals evidence of selection within taxa displaying novel adaptations including perenniality and a high tolerance to waterlogging by studying the positive selection genes of different taxa of Zea genus.In addition,based on genome-wide selection analysis,1,387 candidate sites related to highland adaptation and 2,057 sites related to high latitude adaptation were identified,and indicated the key role of hormone pathway in highland adaptation.Finally,by conducting environment genome-wide association analysis on 34 soil properties under 8 depth and the living altitude,we complement the candidate regions in plant-soil interaction.36 quantitative trait control sites that interact with soil and one site directly related to altitude were found.In the altitude related site,Zm WOX11 was regarded as the candidate gene.Functional analysis of Zm WOX11 indicate it can regulate root growth by responding to the plasma levels in the soil,and further affecting the high-altitude adaptability of teosinte.This work not only provides a comprehensive genetic variation map of Zea genus,but also gains new understanding of Zea evolution and adaptation.The study also identified numbers of valuable adaptation-related variation sites by combing genome-wide selection and association analysis,revealing the importance of hormone pathways in the teosinte highland adaptation and provides a theoretical basis and experimental evidence for the cultivation of new maize varieties with environmental adaptability through the improvement of hormone pathway related genes and soil interaction factors.2.Identification of maize circular RNAs and their function analysisIn order to deepen the understanding of maize functional genome and analyze the molecular mechanism of the maize circular RNA on phenotype variation,this study used the circular RNA specific and ～900 public access maize transcriptomic sequencing data to carry out large-scale identification and systematic analysis of maize circular RNA.By developing the bioinformatics analysis pipeline for genome-wide identification of circular RNAs,2,804 high-confidence maize circular RNAs were uncovered which show distinct genomic features.Further analyses demonstrated that sequences related to LINE/L1 elements(LLE)and their Reverse Complementary Pairs(LLERCPs)are significantly enriched in the flanking regions of circular RNAs.Interestingly,as the number of LLERCPs increase,the accumulation of circ RNAs varies,whereas that of linear transcripts decrease.Furthermore,genes with LLERCP-mediated circular RNAs are enriched among loci that associated with phenotypic variation.Finally,we showed that circ1690 was related to the variation of ear height.Our study indicates circular RNAs are widespread in maize genome and the biogenesis of maize circ RNAs are related to specific types of transposon.The study also uncovers a potential new way for transposons to modulate transcriptomic and phenotypic variations by mediating the production of circ RNAs.3.Establishment of the methylation map of single cell tetrad of maize and analysis of the heterogeneous sites generated by reprogrammingIn order to analyze the epigenetic changes during maize meiosis,this study sequenced the single cell microspore of maize tetrad based on a newly single-cell DNA metylation sequencing method in our laboratory.BRIF-MAP,a method to improve the utilization rate of single cell methylation sequenceing data was first developed.Based on BRIF-MAP,we uncovered the genome-wide DNA methylation landscape of maize tetrad microspore.Through the systematic analysis of the maize single-cell methylation map,we found the methylation rates among the four microspores in one maize tetrad were similar but significantly vairies among tetrads,indicating a obvious cell heterogeneity.By comparing the DNA methylation level between each single tetrad microspore and it’s particular parent,we found the heterogenetity between tetrads were related to reprograming process during meiosis.Compared with the parents,averagely 1.33% methylation sites in the tetrad underwent reprogramming,among which 93.8% sites gained DNA methylation.The heterogeneous loci generated by reprogramming have certain preference on genomic components but remain similar under different genetic backgrounds.Through the utilization of population genetic data,this study not only identified large numbers of new genetic variations,but also expanded the available gene pool for modern maize improvement and provided a theoretical basis and experimental evidence for the improvement direction of adaptive maize new varieties.This study also used transcriptome and epigenome data to study the maize circular RNAs and single cell DNA methylation,further enhance our understanding of the regulatory complexity in maize cell activities.更多还原