IOCAS-IR  > 海洋生态与环境科学重点实验室
中华滨螺群体遗传结构及适应性进化研究
李梦雨
Subtype博士
Thesis Advisor刘进贤
2022-05-19
Degree Grantor中国科学院大学
Place of Conferral中国科学院海洋研究所
Keyword中华滨螺,生态连通性,群体遗传结构,本地适应性,热应激
Abstract

  海洋生物群体连通性受到水文环境、地质地貌和物种自身生物学特性等多种因素的影响。中国沿海,特别是长江三角洲区域自然资源丰富,有复杂的水文环境和多样的潮间带环境。由于受到人类活动的强烈影响,长江三角洲沿岸现既包括泥质浅滩,又包括人工硬基质的岩相环境,潮间带海洋生物的分布范围已经发生了改变,是西南黄海生态系统连通性研究的天然实验室。同时,我国海岸线的纬度跨度大,广布物种分布的不同海域间存在明显的环境梯度。而潮间带是受到人类活动和全球气候变化影响的敏感区域,环境因素,特别是温度条件对广布潮间带生物不同地理群体对本地环境适应性的产生具有重要影响。中华滨螺(Littoraria sinensis, Philippi, 1847)属于广温性岩相潮间带生物,是西北太平洋沿岸最常见的软体动物之一,并在长江三角洲地区人工硬质堤坝上出现新地理群体。其分布范围内存在明显的环境异质性,特别是不同纬度地理群体生存环境的温度条件存在显著差异。因此,中华滨螺既可作为长江三角洲区域海洋生态系统连通性研究的标志生物,也是潮间带生物本地适应性进化机制研究的理想对象。

  本研究采用线粒体ND6(mtDNA ND6)和核基因片段(nuDNA)两种分子标记,探究了中国沿海中华滨螺谱系地理格局,查明了长江三角洲沿岸新形成的中华滨螺群体的来源,探讨了不同季节黄海沿岸流的运输方向和影响范围;使用全基因组重测序方法,解析了中华滨螺自然群体的精细遗传结构和本地适应性进化模式;并联合同质园实验和转录组学分析,解析了中华滨螺南北方群体的高温应激基因表达模式,加深了对潮间带生物的本地适应和适应可塑性的认识。主要研究结果如下:

1、解析了中华滨螺谱系地理格局,探讨了长江口与西南黄海生态系统的连通性模式。

  通过mtDNA ND6和nuDNA两种分子标记的扩增测序,揭示了中华滨螺长江三角洲南北自然群体间的显著遗传分化;单倍型网络显示有一个谱系为南方自然群体特有谱系,另一谱系同时包含南北方自然群体,但主要为北方自然群体。长江三角洲沿岸新形成群体以33.5°N为界,分为显著分化的两组(江苏南部群体和江苏北部群体)。南部群体全部个体均由南方自然群体扩散而来,北部群体至少有27%的个体来源于南方自然群体,并在连云港发现南方自然群体来源个体。结合中华滨螺繁殖特点,本研究结果支持存在全年北向的苏北潮致沿岸流且影响范围可以到达连云港,冬季南向沿岸流局限在33.5°N以北的水域。提示全年时间尺度上,长江口海域都是黄海江苏沿岸海域重要的物质能量来源地,推测沿岸流交汇情况和生境不连续是33.5°N处的生物系统地理学分界线形成的重要影响因素。本研究结果为探究西南黄海与长江口生态系统连通性提供参考,也为黄海生态系统管理和灾害治理提供理论依据。

2、构建了中华滨螺高质量染色体水平参考基因组序列,解析了中华滨螺不同地理群体的精细遗传结构和本地适应性进化机制。

  通过第三代单分子测序技术(ONT)和高通量染色体构象捕获技术(Hi-C)对中华滨螺基因组进行了测序组装和注释,构建了中华滨螺高质量染色体水平基因组。对南北方共6个自然群体进行了全基因组重测序和群体基因组学分析,群体遗传结构分析结果将所有群体分为三个遗传组群,潍坊群体单独成组,青岛和日照群体为构成一个遗传分组,南方自然群体为一个遗传分组,三组间存在显著遗传分化。通过本地适应性进化分析筛选得到了404个离散位点,共注释到241个受选择作用影响的基因,富集分析结果发现这些基因与转录调控、染色体结构稳定,细胞粘附和物质能量代谢等功能和过程相关,提示环境温度是中华滨螺本地适应性的重要影响因子。基因组结构变异分析揭示了两个与群体间分化相关的染色体倒置区,2号染色体倒置区与潍坊群体特有分化有关,6号染色体倒置区与南北方自然群体间分化相关,提示两个倒置区可能受到温度选择作用。相关研究结果揭示了中华滨螺对温度这一环境因子存在本地适应性,受选择基因区域和染色体倒置区同时发挥作用,为潮间带生物对多变环境的温度适应研究提供了参考。

3、基于同质园实验和转录组测序技术,揭示了中华滨螺青岛和厦门群体对高温刺激有不同的基因表达响应机制。

  对中华滨螺青岛和厦门群体开展同质园实验,并进行高温刺激和转录组测序分析,结果显示热休克蛋白家族在中华滨螺的热应激反应中发挥重要作用,35℃对北方青岛群体造成的胁迫程度更高。在高温刺激下,青岛群体表现出更高的可塑性和更多的热休克蛋白表达,并有免疫反应,蛋白质泛素化,细胞凋亡和信号转导相关基因上调和细胞粘附相关基因下调。在南方厦门群体中,一些在实验组中未出现差异表达的基因在对照组已有较高表达水平,即其对高温刺激的“预适应”模式。厦门群体实验组主要通过清除变性蛋白,上调蛋白质异构相关基因以响应高温胁迫。通过比较本地适应性分析中离散位点所在基因和染色体区域与差异表达基因集,发现部分离散位点所在基因在未热激状态下已存在差异表达,且高温刺激后部分离散位点所在基因表现出适应可塑性。本研究结果提示了中华滨螺对高温刺激存在多种适应模式,不同地理群体应对温度升高的适应潜力存在差异,为后续探究潮间带生物的高温耐受机制提供可参考资料。

  综上,本文综合采用线粒体、核基因片段、基因组重测序和转录组测序多种分析方法,查明了中华滨螺的谱系地理格局和群体遗传结构,探讨了不同季节长江口区域与西南黄海海域的生态连通性模式和影响因素;筛选了中华滨螺基因组中受选择作用的基因和染色体倒置区,探讨了其本地适应性遗传机制;揭示了高温胁迫下中华滨螺的可塑性,比较了南北方群体间适应可塑性差异。研究结果表明中华滨螺存在本地适应性分化,并对高温条件存在转录可塑性适应。本研究结果可以为生态系统的科学管理与保护提供理论支持,并有助于了解潮间带广温性生物对全球气候变化的适应策略。

Other Abstract

  The population connectivity of marine organisms is affected by a variety of factors, such as the hydrological environment, geological landforms, and the biological characteristics of the species themselves. Coastal areas of China, especially the Yangtze River Delta (YRD), are rich in natural resources and have complex hydrological environment. Due to the strong influence of human activities, the YRD coastline is now composed of both muddy shoal and artificial hard substratum. The distribution range of intertidal organisms has changed, and the artificial substratum is a natural laboratory for the studies of ecosystem connectivity in the southwestern Yellow Sea. Meanwhile, the latitude span of Chinese coastline is large, and there are obvious environmental gradients between different sea areas where widespread species distributed. Intertidal zone is a sensitive area affected by human activities and global climate change, environmental factors, especially temperature, play an important role in the adaptation of widespread marine organisms to the local environment. Littoraria sinensis (Philippi, 1847) is one of the most common mollusks in the northwestern Pacific, and new geographical populations of this species appeared on the artificial hard substratum in the YRD. Environmental heterogeneity is obvious in the distribution range of L.sinensis, especially the temperature environment in different latitude populations. Therefore, L. sinensis can be used as a biological marker for the research of the ecological connectivity around the YRD, and it is also an ideal subject for the study of the local adaptive evolution mechanism in intertidal organisms.

  In this study, two molecular markers, mitochondrial ND6 (mt ND6) and anonymous nuclear gene fragment (nuDNA), were used to explore the phylogenetic pattern of L.sinensis along the coast of China. The origin of the new populations, the transportation direction and influence range of the Yellow Sea Coastal Current in different seasons were discussed; whole-genome resequencing was used to analyze the fine-scale genetic structure combined and local adaptation in natural population of L.sinensis; combining with common garden experiment and transcriptomic analysis, the gene expression patterns under heat stress in the northern and southern populations of L.sinensis were analyzed, which deepened the understanding of local adaptation and adaptive plasticity of intertidal organisms. The main findings are as follows:

1. The phylogeographic pattern of L.sinensis, and the connectivity between the Yangtze River Estuary (YRE) and the southwestern Yellow Sea were investigated.

  Two molecular markers, mtDNA ND6 and nuDNA were amplified and sequenced. Significant genetic differentiation was detected between natural populations in the north and south of the YRD. The network of haplotypes showed that one lineage was southern specific clade and the other one lineage contained northern and southern natural populations, but northern natural populations were dominant. The newly colonized populations along the YRD were bounded by 33.5° N and were divided into two groups with significant differentiation. All individuals in the south group along the YRD and at least 27% of the north group were from southern natural population to the south of YRE, and southern origin individuals were found in Lianyungang, Jiangsu Province. Combined with the breeding habits of L.sinensis, the results of this study support the existence of the northward Subei tide-induced coastal current in the whole year, and the north effect boundary could reach Lianyungang. The southward coastal current in winter is limited in the area to the north of 33.5°N. It is suggested that on the time scale of the whole year, the YRE serves as an important source of material and energy for the coastal waters of the southwestern Yellow Sea. The intersection of coastal currents and habitat discontinuity are key factors for the formation of the phylogeographic boundary at 33.5°N. The results of this study provide insights into the ecological connectivity between the southwestern Yellow Sea and the YRD, which can be crucial for the ecosystem and disaster management in Yellow Sea.

2. A high-quality chromosome-level reference genome of L. sinensis was constructed, the fine-scale genetic structure and local adaptive evolution were explored.

  By using the third generation single molecule sequencing technology and high-throughput chromosome conformation capture technology, the genome of L.sinensis was sequenced, assembled and annotated, a high-quality chromosome-level reference genome of L. sinensis was constructed. Whole-genome resequencing and population genetic analysis were performed in 6 natural populations to the north and south of YRE. Population genetic structure analyses showed that six populations could be divided into three genetic clusters. One cluster contained Weifang population, one cluster was formed by Qingdao and Rizhao populations, and the southern natural populations formed the third cluster. Genetic differentiation among the three clusters were significant. 404 outlier SNP loci were identified and 241 candidate genes that potentially under local selection were annotated. After Gene Ontology (GO) enrichment analyses, we found that these genes were related to regulation of transcription, chromosome structure stability, cell adhesion, and material and energy metabolic process, which demonstrated local adaptation to temperature in different populations. Chromosomal structural variation analysis revealed two chromosomal inversion regions associated with population differentiation. The Chr2 inversion region was related to the specific divergence of Weifang population and Chr6 inversion was related to divergence between northern and southern natural populations, which suggested these two inversions were affected by temperature natural selection. This study showed that there were local adaptation to temperature in different populations of L.sinensis, genes under selection and chromosomal inversion might play a role in this process at the same time. Our results could provide a reference for the study in genetic mechanism of temperature adaptation to changing environments in intertidal organisms.

3. Based on the common garden experiment and transcriptome sequencing technology, the different gene expression response to high temperature stress in Qingdao and Xiamen populations were investigated.

  After common garden experiment in Qingdao and Xiamen populations, we increased the temperature and performed RNA-seq of L.sinensis. The results showed that heat shock proteins played an important role in the heat stress response of L.sinensis. 35℃ caused higher degree of stress to the northern Qingdao population than southern Xiamen population. Qingdao population showed higher plasticity under high temperature. They upregulated more heat shock proteins and upregulation genes were associated with immune response, protein ubiquitination, apoptosis and signal transduction related functions. They downregulated cell adhesion related genes. Several genes, which were not differentially expressed in southern populations after heat stress, showed higher expression under control conditions. This suggested the "pre-adaptation" mode serves as the defense against high temperature stress in the southern Xiamen population. Heat-stressed samples in Xiamen population responded mainly by removing denatured proteins and upregulating genes related to protein isomerism. By comparing the candidate genes under selection and chromosomal inversion regions in the local adaptive analysis with the differentially expressed gene sets, we found that some of the candidate selected genes were differentially expressed in the control group, and some of the candidate selected genes showed adaptive plasticity after heat stress. It is suggested that there were various adaptation modes of L.sinensis to heat stress, and different geographical populations have different adaptation potential for high temperature. The results of this study can provide reference materials for the following study of the high temperature tolerance mechanism of intertidal organisms.

  In conclusion, this study which combined mitochondrial marker, nuclear gene fragment, whole-genome resequencing and transcriptome sequencing investigated the phylogenetic pattern and population genetic structure of L.sinensis, and the ecological connectivity patterns and influencing factors between the YRE and the southwestern Yellow Sea in different seasons were discussed. We detected 241 candidate genes that under natural seleciton and two chromosomal inversion regions in the genome of L.sinensis, and discussed the genetic mechanisms of local adaptation. The plasticity of L.sinensis under heat stress and the differences in adaptive plasticity between northern and southern populations were discussed. We indicated that there is local adaptive differentiation and adaptation to temperature through transcriptional plasticity in L.sinensis. The results of this study can provide theoretical support for the scientific management and protection of marine ecosystems, and provide insights into the adaptation strategies of eurythermal intertidal organisms.

Language中文
Table of Contents

目 录

第一章 绪论……1

1.1 海洋生态连通性简述……1

1.1.1 海洋生物群体连通性……1

1.1.2 西北太平洋生态系统连通性概况……3

1.1.3 长江口附近连通性模式及形成原因……4

1.2 生物对环境适应性……7

1.2.1 基因组适应性进化的研究方法与现状……8

1.2.2 转录水平适应性分化的研究方法与现状……12

1.3 中华滨螺的研究概述……14

1.3.1 中华滨螺生物学特征……14

1.3.2 中华滨螺群体遗传学研究现状……15

1.4 本研究的目的与意义……16

1.4.1 研究的目的与意义……16

1.4.2 研究思路、预期成果与技术路线……17

第二章 中华滨螺谱系地理学及群体间连通性模式研究……19

2.1 引言……19

2.2 材料与方法……20

2.2.1 样品采集与DNA提取……20

2.2.2 mtDNA ND6片段扩增与测序……22

2.2.3 nuDNA片段位点开发与扩增……22

2.2.4 群体遗传多样性分析……23

2.2.5 群体遗传结构分析……23

2.3 研究结果……24

2.3.1 mtDNA ND6及nuDNA片段扩增……24

2.3.2 遗传多样性与遗传结构分析……25

2.4 讨论……31

2.4.1 中华滨螺群体间遗传分化和幼虫扩散……31

2.4.2 长江口与黄海间生态连通性……32

2.4.3 生物扩散的影响因素……33

2.4.4 潜在的适应性分化位点……34

2.5 本章小结……34

第三章 中华滨螺群体基因组学及本地适应性研究……37

3.1 引言……37

3.2 材料与方法……38

3.2.1 参考基因组构建……38

3.2.2 样品采集、DNA提取及全基因组重测序……40

3.2.3 SNP提取与位点质量过滤……41

3.2.4 群体遗传学分析……41

3.2.5 离散位点筛选与环境因子关联分析……42

3.2.6 基因组结构变异检测……42

3.3 研究结果……43

3.3.1 基因组组装与注释……43

3.3.2 基因组重测序、SNP提取及筛选……43

3.3.3 群体遗传结构分析……47

3.3.4 离散位点筛选与环境因子关联分析……49

3.3.5 基因组结构变异……51

3.4 讨论……57

3.4.1 中华滨螺参考基因组……57

3.4.2 中华滨螺群体遗传结构……57

3.4.2 适应性离散位点……58

3.4.3 基因组结构变异……60

3.5 本章小结……61

第四章 高温应激转录组差异表达研究……63

4.1 引言……63

4.2 材料与方法……64

4.2.1 样品采集与同质园实验……64

4.2.2 高温胁迫与转录组测序……65

4.2.3 转录组数据处理与基因表达量分析……65

4.2.4 差异表达基因筛选与功能分析……66

4.2.5 受选择位点与差异表达……67

4.3 研究结果……67

4.3.1 转录组测序及质控……67

4.3.2 转录组PCA分析……68

4.3.3 差异表达基因的筛选……71

4.3.4 差异表达基因的功能分析……76

4.3.5 选择位点基因的表达分化……81

4.4 讨论……81

4.4.1 中华滨螺热应激可塑性……82

4.4.2 中华滨螺热应激适应可塑性……82

4.4.3 低纬度群体高温预适应……84

4.4.4 适应性进化与表达可塑性……85

4.5 本章小结……87

第五章 研究总结与展望……89

5.1 研究总结……89

5.2 主要创新点……90

5.3 研究不足之处……90

5.4 展望……90

参考文献……91

附 录……109

致谢……123

作者简历及攻读学位期间发表的的学术论文与研究成果……125

Document Type学位论文
Identifierhttp://ir.qdio.ac.cn/handle/337002/178287
Collection海洋生态与环境科学重点实验室
Recommended Citation
GB/T 7714
李梦雨. 中华滨螺群体遗传结构及适应性进化研究[D]. 中国科学院海洋研究所. 中国科学院大学,2022.
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