中国科学院海洋研究所机构知识库

近年来人类活动引起的环境变化对生物多样性产生严重危害，生物多样性的丢失会影响区域乃至全球的生态系统及其服务功能。环境改变通常会引起适应能力差的群体发生衰退、消失，甚至导致整个物种灭绝，而物种内遗传变异作为生物多样性的重要组成部分可以减缓这些有害效应，直接决定着生态系统的稳定性。另一方面，生物通过迁移、表型可塑性和适应性进化等方式应对有害环境变化，群体内及群体间遗传变异水平是适应性进化的关键和基础。因此，人类活动极大地重塑了物种内中性和适应性遗传变异的存在模式，在此背景下评估种内遗传多样性水平和分布模式有利于保护自然群体野生资源，特别是对渔业经济物种的合理开发与管理有重要指导意义，而探究生物如何快速适应环境变化的遗传机制更能为进化生物学提供新的见解。大银鱼（Protosalanx hyalocranius）隶属于胡瓜鱼目、银鱼科、大银鱼属，曾是我国许多沿海河口地区和内陆湖泊的重要渔业物种，开展大银鱼群体遗传学及本地适应性研究有两方面重要意义：（1）水利工程修建、水体污染及过度捕捞等人类活动导致大银鱼野生资源严重衰退，特别在鸭绿江口、黄河口和长江口等河口地区已经丧失经济利用价值，而且大银鱼被移植到我国众多水库湖泊，作为入侵物种严重危害本地生物的多样性水平，同时也造成大银鱼群体间基因流增加，近交程度升高，不利于遗传变异的维持，开展群体遗传学研究有利于其野生资源的合理开发和有效保护。（2）大银鱼作为一种适应性极强的物种，是研究复杂性状适应性进化遗传机制的理想材料，首先大银鱼存在河口洄游和淡水陆封两种生态类型，可以适应高盐和低盐两种生境；其次移植过程扩展了大银鱼分布范围，适应了一些与原生地差异较大的极端环境，例如在高纬度极寒的黑龙江河流建立了稳定群体。因此，从以上两个方面开展适应性进化的遗传机制研究，可以评估大银鱼进化潜力，对进化生物学有重要启示作用。而目前大银鱼的群体遗传学研究中，只是利用单个分子标记或针对的仅为几个群体甚至少数个体，亟需从更高层面对大银鱼整个分布范围内群体展开遗传学研究，本研究通过线粒体DNA、微卫星标记和群体基因组学三种手段全面衡量大银鱼种内遗传变异水平、群体间遗传结构，并探究适应性进化的分子机制，主要研究结果如下：

1、设计引物扩增了大银鱼线粒体DNA一个控制区片段（668 bp），基于一个样本的简化基因组测序数据开发得到9个多态性微卫星位点，采用以上两种标记评估大银鱼6个地理群体遗传变异水平，平均单倍型多样性和核苷酸多样性分别为0.737和0.0029，巢湖的单倍型多样性（0.634）和核苷酸多样性（0.0019）在所有群体中最低，杂合度分析显示巢湖、辽河口和鸭绿江口群体的期望和观测杂合度较低，黑龙江入侵群体有较高遗传多样性水平。遗传结构分析显示，太湖同其他群体间D_est（大于0.0982）明显高于其余群体间D_est值，洪泽湖同两个河口群体间存在显著遗传差异，而巢湖同河口群体间不存在遗传分化，黑龙江群体同洪泽湖之间遗传关系最近，STRUCTURE结果中除了辽河口和鸭绿江有相似遗传构成，遗传变异的分布模式基本按照群体来源区分，主成分分析结果同D_est和STRUCTURE相一致。Bottleneck分析显示，辽河口和鸭绿江口群体可能经历了近期瓶颈效应。DIYABC模拟显示，黑龙江入侵群体来源于太湖和洪泽湖两个群体，且洪泽湖贡献更高比例遗传变异。以上结果表明，人类活动造成河口地区大银鱼更为严重的种群衰退，河口洄游型群体比淡水陆封群体遗传多样性更低，淡水群体中，巢湖遗传多样性水平最低，更易受到人类活动干扰，应在保护中重点关注；淡水群体同河口群体的遗传关系远近反应了各自从祖先群体分离的时间早晚；相比于原生地群体，黑龙江入侵群体的遗传变异并没有显著减少，表明多来源移植过程可以在入侵群体保持较高遗传多样性水平。

2、获取洪泽湖大银鱼雌雄各20尾的重测序数据，比对到已发表的大银鱼参考基因组，通过比较性别间测序深度差异，筛选到scaffold195上一个雌性特有的基因组区域，长度为598 bp，BLASTp比对后发现scaffold4上存在与雌性特异区域同源的序列，两者之间存在总长230 bp的片段缺失，针对单拷贝雌性特异区和双拷贝同源序列的保守区域设计PCR引物，扩增结果显示雌性中包含两个条带，雄性中只有一个条带，可以简便高效地鉴定个体性别。本研究开发的基于PCR快速鉴定大银鱼遗传性别的技术，不仅可以判定性成熟前群体性别比例，以排除不均一性比对遗传结构解析的干扰，还可用于大银鱼性别决定机制的研究。

3、已发表的两个大银鱼基因组完整性一般，均为scaffold水平，在基因组结构变异检测时有较大局限性。本研究结合三代PacBio长读长测序和HiC测序技术，组装了大银鱼染色体水平高质量参考基因组序列，基因组大小445 Mb，包含28条染色体，scaffold N50达17 Mb，该参考基因组序列是开展大银鱼及近缘物种群体基因组学及比较基因组学研究的有利工具。

4、将6个群体146个样本的重测序数据比对到本研究组装的参考基因组，过滤后获得1,100,500个高质量双等位基因SNP标记，基于全基因组SNP进行遗传特征分析，太湖群体的期望杂合度（0.323）和核苷酸多样性（0.332）最高，黑龙江入侵群体的遗传变异水平较高，巢湖和两个河口群体有效群体较小，分别为1281.1（辽河口）、1271.5（鸭绿江口）和868.4（巢湖）。纯合片段（ROH）统计显示黑龙江群体基因组上的ROH数目和长度均显著低于原生地群体。Admixture遗传结构分析显示，除了两个河口群体间有相似的遗传组成，各地理群体间遗传组分构成差异明显。河口与淡水群体间遗传差异均显著（P＜0.01），其F_ST范围为0.00920（辽河口和巢湖间）至0.18231（辽河口和太湖间），两个河口洄游群体间无显著分化。太湖同其余五个群体间遗传分化指数高于剩余群体间一个数量级。主成分分析将太湖、洪泽湖和黑龙江群体相互分开，两个河口洄游群体的个体聚集在一起且同太湖、洪泽湖和黑龙江三个淡水群体分隔较远，而来自巢湖的个体和两个河口群体重合在一起。基于F_ST构建的群体间NJ树呈现出与Admixture以及PCA相一致的结果。群体历史动态模拟显示，太湖、洪泽湖和巢湖分别起源于三次独立的淡水入侵事件。以上结果表明，淡水群体同河口群体间遗传距离远近反应了各自从祖先群体分离时间，太湖群体形成时间最早，更长的隔离时间导致太湖同河口群体间较大遗传差异，随后从共同祖先分离的洪泽湖和巢湖则分别对应中等程度和较小的遗传差异，三次独立的淡水定居事件是平行演化研究的理想材料；黑龙江群体有较高遗传多样性水平，近交程度并没有比源头群体显著升高，表明黑龙江群体建立过程中不存在奠基者效应，适合于开展大银鱼对黑龙江环境的适应性进化研究。

5、基于群体基因组学策略和生物信息学技术，揭示了大银鱼淡水定居的平行演化模式，筛选到64,909个淡水适应离散位点，这些SNP等位基因频率在河口和淡水群体间存在巨大频率差异，两个河口群体中平均频率为0.09，而在三个淡水群体中平均频率远高于河口群体，最大值为0.57（洪泽湖），最小值为0.30（巢湖）。基因功能注释显示离子转运、钙离子信号通路以及生长发育等相关基因在淡水适应过程发挥重要作用。阐明了大银鱼适应黑龙江极端环境的遗传机制，共有50,513个入侵适应离散位点，注释结果显示温度感受、免疫反应和低氧响应等相关基因协助大银鱼应对低温和低氧的黑龙江生境。LDna分析检测到6个染色体倒置，其中LG18上的倒置长为8.26 Mb，共有99.95％淡水适应的离散位点位于这个倒置区，LG21和LG26上长度分别为5.22 Mb和1.80 Mb的倒置包含了大部分入侵适应性位点。由于三个淡水陆封群体从祖先群体的分离是较近时间尺度的过程，而黑龙江入侵群体的形成时间也不超过三十年，表明LG18、LG21和LG26上的三个染色体倒置可以作为固有遗传变异基因库，协助大银鱼快速适应环境改变。

综上，本研究基于线粒体DNA、微卫星标记和群体基因组学三种手段评估了人类活动影响下大银鱼不同群体的遗传多样性水平，解析了群体间遗传结构及造成遗传差异大小的因素，揭示了大银鱼定居湖泊生境的平行演化模式及适应淡水环境的遗传机制，阐明了黑龙江入侵群体的来源及大银鱼对黑龙江极端环境快速适应性进化的遗传机制，并探清了染色体倒置在快速适应性进化中作为固有遗传变异基因库发挥的重要作用。相关研究结果不仅有助于大银鱼野生资源的合理开发保护、入侵事件的有效防治管理，同时为生物快速适应环境变化的遗传机制提供新的见解。

Recently, biodiversity of the Earth system has been heavily impacted by human-induced environmental changes. Loss of biodiversity impacts ecosystems and the services they provide not only on local region, but also on global scales. Those populations that are poorly suited to new conditions would often been harmed by environmental changes, leading to population declines, extirpation, and even extinction. However, as one of the key parts of biodiversity, intraspecific diversity can counteract those negative effects, defining the stability of ecosystem directly. Also, natural populations could response to harmful environmental changes through migration, plasticity and adaptive evolution. And genetic variation within or among populations plays key roles in the process of adaptive evolution. Therefore, while human activities reshaped the pattern of neutral and adaptive variations within species, evaluating the level and structure of intraspecific genetic variation is crucial for protecting natural population resources, especially for those fishery species. The clearhead icefish, Protosalanx hyalocranius, is an important commercial fishery species in many coastal regions and inland lakes of China. There were two reasons why we conducted the studies on population genetic and local adaptation for P. hyalocranius: (1) Wild populations of P. hyalocranius have markedly declined in recent years due to human activities such as overexploitation, hydro projects and water pollution. Wild resources of P. hyalocranius have lost commercial viability in many estuaries such as Yalu River Estuary, Yellow River Estuary, Yangtze River Estuary and so on. Besides, the clearhead icefish has been widely introduced into lakes or reservoirs on a large scale in China. Introduced P. hyalocranius may become invasive species and reduce the biodiversity of native species. Gene flow and inbreeding were also increased among P. hyalocranius populations, which was harmful for intraspecific genetic diversity. Hence, population genetic study of P. hyalocranius is beneficial to the exploitation and protection of its wild resources. (2) P. hyalocranius can adapt to various conditions, including two aspects. Firstly, P. hyalocranius exists in two distinct life history forms, could inhabits in both salt water and freshwater. Invasive population of P. hyalocranius adapted to some environments that were distinct from its native ranges. For instance, P. hyalocranius formed stable invasive population in Heilong River, which is an extremely cold-water condition. Exploring the genetic basis of above two adaptive processes is essential to assessing its evolutionary potential and would provide new insights into evolutionary biology. In the present study, mitochondrial DNA, microsatellite markers and population genomic strategies were used to evaluating the genetic variation, population structure and genetic basis of adaptive evolution. Main results are as follows:

1. A 668 bp sequence of the mitochondrial partial control region was obtained for analyses. Nine highly polymorphic microsatellites were developed using RAD-seq data. Based on mt CR sequence, the average haplotype diversity and nucleotide diversity among six populations were 0.737 and 0.0029 respectively. Haplotype diversity (0.634) and nucleotide diversity (0.0019) of Chaohu were lowest. The observed and expected heterozygosity of Chaohu Lake, Liao River Estuary and Yalu River Estuary were relatively low. Genetic diversity was high in the invasive Heilong River population. The pairwise D_est values between Taihu Lake and other five populations were relatively large among all comparisons (> 0.0982) and statistically significant. There was no genetic differentiation between Chaohu Lake and anadromous populations. STRUCTURE analysis indicated that genetic variation was partitioned by geography, except for a similar genetic component between two anadromous populations. Results of PCA analysis were consistent with D_est and STRUCTURE. Bottleneck analysis showed that two anadromous populations may have experienced a recent genetic bottleneck. The DIYABC analysis indicated the invasive Heilong River population formed with contributions of both Taihu and Hongze Lake. In conclusion, human activities have caused native anadromous populations declined more severely than freshwater populations. Chaohu Lake suffered more impacts from human activities and should be paid more attention. The genetic relationships between freshwater and anadromous populations reflected the divergent time between them. The invasive Heilong River population showed relatively high genetic diversity level when compared with native freshwater populations, indicating that multiple introductions could retain more genetic diversity.

2. To discover female-specific genomic regions, we compared whole genome re-sequencing data of both males and females that collected from Hongze Lake. A female specific genomic region (598 bp) was identified on scaffold195 and contained a putative FOXI gene, which was homologous to another FOXI gene on scaffold4. There was significant length difference between these two sequences with total length of 230 bp. According to the consensus flanking sequence of the haploid female specific region and its paralogous diploid sequences, a set of PCR primers was designed. The genetic sex of individual could be identified through PCR and traditional agarose gel electrophoresis with one band for males and two bands for females. This method could be used to assess sex ratio in P. hyalocranius population before sexual maturation, and have applications in further studies of the mechanism of sex determination in this species.

3. Two published whole genome of P. hyalocranius were both scaffold level. Their relatively low integrity makes them unsuitable for structural variation detection. Combing third generation sequencing and HiC technology, a high-quality chromosome-level genome was assembled for P. hyalocranius. We constructed a 445 Mb P. hyalocranius genome with 28 chromosomes. The contig N50 size was 2.5 Mb and scaffold N50 was 17 Mb. This genome should be an important tool for population genomic studies of P. hyalocranius and its related species.

4. By re-sequencing the genomes of 146 clearhead icefish individuals from six populations, 1,100,500 high-quality bi-allelic SNPs were obtained across 28 chromosomes of the reference genome. Genetic diversity analysis showed that expected heterozygosity (H_E) and nucleotide diversity (Pi) in Taihu Lake were a little higher than the other five populations, with 0.323 for H_E and 0.332 for Pi. Genetic diversity of the invasive Heilong River population was not significantly lower than others. The effective population size of Chaohu Lake and two anadromous populations were low. ROH analysis indicated that length and number of ROH within invasive Heilong River were significantly less than its source populations. Admixture results indicated that genetic variation was partitioned by geography, except for a similar genetic component between Liao River Estuary and Yalu River Estuary. The fixation indexes (F_ST) of all anadromous-freshwater population pairs were statistically significant (P < 0.01), with a range from 0.00920 (Liao River Estuary versus Chaohu Lake) to 0.18231 (Liao River Estuary versus Taihu Lake). Two anadromous population were genetically close to each other (F_ST = -0.00003; P = 0.012). F_ST between Taihu Lake and the other five populations were a magnitude larger than the rest of population pairs. Principal component analysis (PCA) separated Taihu Lake, Hongze Lake, and Heilong River from the others. Individuals from two anadromous populations were grouped together. However, individuals from Chaohu Lake overlapped with those from two anadromous populations, which was consistent with NJ tree based on F_ST. The DIYABC analysis suggested three native freshwater populations were derived from their common ancestor independently. In conclusion, the genetic distance between freshwater and anadromous populations reflected the divergent time of freshwater population from their ancestor. Closer to the estuary and older of Taihu Lake made the clearhead icefish arrived and settled here easier and more ancient than other lakes, leading to large genetic difference with other populations. Hongze Lake and Chaohu Lake derived from ancestor successively, corresponding to moderate and low genetic differentiation respectively. Three independent freshwater-resident events were ideal system for studying parallel adaptation. In addition, the founder effect might be absent in Heilong River. Heilong River population was suitable for illustrating genetic basis of rapid adaptation to extreme environment in Heilong River.

5. Based on population genomic strategies and bioinformatics technology, parallel adaptive pattern of freshwater-resident was uncovered in P. hyalocranius. A total of 64,909 candidate outliers were detected in parallel freshwater adaptation. Large allele frequency shifts were observed for freshwater adaptive outliers. The allele frequency of outliers in the anadromous group was generally low with an average of 0.09. In contrast, the average allele frequency of freshwater outliers in all freshwater populations was much higher than that in anadromous group, with a maximum in Hongze Lake (0.57) and minimum in Chaohu Lake (0.30). Gene annotations of those outliers identified many genes that contributed to repeated freshwater adaptation, including ion transport, Ca²⁺-related signaling and growth factor genes. A total of 50,513 outliers were detected in invasive adaptation. Gene annotations indicated that some temperature stimulus, immune and hypoxia related genes were associated with response to cold temperature and hypoxia stress. LDna analysis detected six putative chromosome inversions. Among them, the chromosome inversion on LG18 covered about 8.26 Mb, on which 64,876 freshwater adaptation outliers (99.95%) were located. The chromosome inversions on LG21 and LG26, which were associated with invasive adaptation in Heilong River, covered about 5.22 Mb and 1.80 Mb respectively. Total 49469 of the 50513 (98%) invasive adaptation outliers were located in the two inversions. The three native freshwater populations were isolated within a short timeframe. And invasive Heilong River population established stable population for a shorter period (less than 30 years). These results suggested that the inversions on LG18, LG21 and LG26 server as genetic pools of standing variation, facilitating P. hyalocranius adapt to environment changes rapidly.

In summary, mitochondrial DNA, microsatellite markers and population genomic strategies were used in different populations of the clearhead icefish for assessing the genetic diversity, elucidating the population genetic structure and its influencing factors, uncovering the parallel adaptive pattern and genetic basis of freshwater adaptation, detecting the sources of Heilong River population and molecular basis of rapidly invasive adaptation, and finally demonstrating the key role of chromosome inversions in rapid adaptive evolution. These results were crucial to inform conservation and management decisions for P. hyalocranius, could also provide new insights into how populations response to environment changes rapidly.

第1章 绪论  1

1.1 群体遗传学概述    1

1.1.1 群体     1

1.1.2 群体遗传学及其主要研究内容  1

1.1.3 鱼类的群体遗传学研究     2

1.2 分子标记概述和群体基因组学    4

1.2.1 分子标记技术的发展  4

1.2.2 群体基因组学概述     8

1.3 快速适应性进化研究概述    10

1.3.1 群体适应性进化的速度     10

1.3.2 适应性进化的研究策略     10

1.3.3 适应性变异的来源     13

1.3.4 基因组结构变异在适应性进化中的作用  13

1.4 大银鱼研究概述    16

1.4.1 大银鱼的分类地位     16

1.4.2 大银鱼的形态和生物学特征     17

1.4.3 大银鱼的分布和资源现状  19

1.4.4 大银鱼群体遗传学研究现状     21

1.5 本研究的目的及意义、研究思路和拟解决的科学问题    22

1.5.1 研究的目的及意义     22

1.5.2 研究思路     23

1.5.3 拟解决的科学问题     24

第2章 基于线粒体和微卫星的大银鱼群体遗传学研究  25

2.1 引言 25

2.2 材料和方法    27

2.2.1 样品采集     27

2.2.2 基因组DNA提取       28

2.2.3 线粒体DNA和微卫星标记的开发    29

2.2.4 线粒体数据分析  30

2.2.5 微卫星数据分析  31

2.3 实验结果 34

2.3.1 遗传多样性  34

2.3.2 群体遗传结构     42

2.3.3 群体历史动态分析     44

2.4 讨论 49

2.4.1 大银鱼原生群体遗传多样性     49

2.4.2 大银鱼原生群体遗传结构  50

2.4.3 黑龙江入侵群体遗传特征  51

2.5 本章小结 53

第3章 基于全基因组重测序的大银鱼群体遗传结构研究     55

3.1 引言 55

3.2 材料和方法    56

3.2.1 性别鉴定标记开发     56

3.2.2 染色体水平参考基因组组装     58

3.2.3 群体遗传结构分析     59

3.3 实验结果 64

3.3.1 性别鉴定标记开发     64

3.3.2 染色体水平参考基因组组装     66

3.3.3 群体遗传结构研究     67

3.4 讨论 74

3.4.1 基于PCR技术快速鉴定遗传性别    74

3.4.2 大银鱼染色体水平基因组的组装     75

3.4.3 群体遗传结构     76

3.5 本章小结 78

第4章 基于全基因组重测序的大银鱼适应性进化研究  79

4.1 引言 79

4.2 材料和方法    80

4.2.1 适应性位点筛选  80

4.2.2 染色体倒置检测  81

4.2.3 基因功能注释和富集分析  82

4.3 实验结果 82

4.3.1 适应性离散位点筛选  83

4.3.2 染色体倒置检测  88

4.3.3 基因功能注释和GO富集  94

4.4 讨论 104

4.4.1 淡水定居和入侵适应的遗传基础     104

4.4.2 染色体倒置在适应性进化中的作用  105

4.4.3 适应性进化的速度     106

4.5 本章小结 106

第5章 总结与展望     109

5.1 研究总结 109

5.2 主要创新点    110

5.3 研究不足 110

5.4 展望 111

参考文献       113

致  谢    129

作者简历及攻读学位期间发表的学术论文与研究成果    131