海洋生态与环境科学重点实验室知识库

人类活动引起的选择压力会迅速改变物种的进化轨迹，加快物种的进化速率，但其潜在的遗传机理仍不清楚。捕捞和驯化都是人类活动驱动物种进化的经典案例。遗传学认为捕捞作为有效的选择力量诱导了鱼类群体的进化，但缺乏野生群体中捕捞诱导进化的直接证据。与家养动物相比，大多数水产养殖物种的选育历史较短，保留了较高的遗传多样性。因此，鱼类养殖群体为探讨养殖活动下人工选择对物种驯化早期的遗传机制提供了机会。

大黄鱼（Larimichthys crocea）主要分布在中国沿海地区，是受人类活动影响最为严重的海洋物种之一。自20世纪50年代末，大黄鱼开始遭受持续大规模的捕捞，目前大黄鱼野生资源已严重衰竭。为了保护大黄鱼野生资源同时满足人们对大黄鱼的需求，大黄鱼养殖技术于20世纪80年代研发成功，随后开展了长期、大规模的大黄鱼增殖放流活动。基于长期大规模捕捞压力以及较短的选育、养殖历史，大黄鱼是研究捕捞诱导鱼类进化及人工选择作用下物种快速适应性进化遗传机制的理想材料。

本研究采集了中国东海大黄鱼历史样本（20世纪60年代，80年代）和现代野生样本，基于微卫星标记（SSR）、全基因组单核苷酸多态性位点（SNP）和线粒体基因组三种分子标记，通过比较历史样本与现代野生样本的遗传变异，查明大黄鱼野生群体的遗传多样性与遗传结构的时空变化、近亲繁殖和遗传负荷水平；在大黄鱼野生群体中首次证实了捕捞诱导进化的存在并提供了遗传学资料；基于基因组SNP和线粒体基因组分子标记对大黄鱼历史、现代野生和养殖群体进行了深入分析，探明了大黄鱼养殖群体的遗传多样性和遗传负荷水平，并为揭示人工选择影响下大黄鱼养殖群体快速进化的遗传学机制提供了遗传学资料。主要研究结果如下：

1. 查明了东海大黄鱼野生群体在过度捕捞前后遗传多样性及遗传结构的时空变化。

采用7个微卫星位点、660,359个全基因组高质量SNP位点和线粒体基因组序列探究大黄鱼野生群体遗传多样性及遗传结构的时空变化。三种分子标记的遗传多样性分析结果显示，与历史群体相比，大黄鱼现代野生群体的等位基因丰富度、平均期望杂合度、核苷酸多态性、近交系数和有效群体大小等遗传多样性参数均没有出现明显的降低。不同群体间杂合子占比没有显著差异。基于三种分子标记的遗传结构分析显示不同群体间没有显著的遗传分化。线粒体基因组网络图结果显示所有单倍型成星型分布，且不存在不同年代或地理群体特有的谱系分支。综上，大黄鱼野生群体虽然经历了持续的过度捕捞，但其遗传多样性及遗传结构并没有发生明显变化。野生大黄鱼群体仍然具有恢复到过度捕捞前群体规模的潜力，为了避免增殖放流对大黄鱼野生群体的负面效应，我们认为限制捕捞和生境恢复是帮助大黄鱼群体恢复最直接、最有效的管理策略。

2. 查明了大黄鱼野生群体的近交繁殖、遗传负荷水平，并在大黄鱼野生群体中检测到捕捞诱导进化的信号。

本部分选取大黄鱼历史和现代野生群体，通过全基因SNP对其遗传负荷水平和选择性清除信号进行分析。与大黄鱼历史群体相比，大黄鱼现代野生群体具有更多连续纯合片段，说明现代群体存在一定的近交衰退。现代野生群体的遗传负荷在一定程度上高于历史群体，反应出现代野生群体的适合度下降。选择性清除分析共筛选到58个窗口，并注释到84个基因。这些基因包括生长激素受体基因、血红蛋白合成基因及参与神经、智力发育的基因。生长激素受体基因可能与大黄鱼生长加快相关；血红蛋白合成基因与氧运输有关，更高的氧运输效率可以增加大黄鱼耐力，从而提高躲避捕捞的机会；神经、智力发育的基因则可能与大黄鱼的行为相关，在躲避捕捞过程中发挥一定作用。TopGO结果富集到多个与氧运输、热适应、能量、代谢过程相关的基因集。这些筛选到的部分基因和基因集可能与捕捞选择有关，暗示了捕捞可能诱导了大黄鱼野生群体的进化。

3. 探明大黄鱼养殖群体的遗传多样性、近交繁殖和遗传负荷水平，并为揭示人工选择作用下大黄鱼养殖群体快速进化的遗传机制提供了遗传学资料。

基于基因组SNP和线粒体基因组对大黄鱼历史、现代野生和养殖群体进行群体基因组学分析。基因组SNP和线粒体基因组数据分析揭示出大黄鱼养殖与野生群体在遗传多样性及遗传结构方面存在显著差异；养殖群体的连续纯合片段及遗传负荷水平显著高于野生群体，说明养殖群体具有较低的适合度和进化潜力；选择性清除分析发现了多个可能与人工选择相关的基因，例如多药抗药性相关蛋白基因，光感受器锚定重复蛋白基因、热休克蛋白基因等。这些结果可能是人工选择诱导大黄鱼养殖群体快速进化的遗传证据。

综上，本研究采用三种分子标记技术，查明了大黄鱼野生群体遗传多样性和遗传结构的时空变化趋势，探明其近交繁殖和遗传负荷水平，并探讨了大黄鱼应对持续捕捞的适应性机制；揭示了大黄鱼养殖群体的遗传多样性和遗传负荷水平，以及为人工选择压力下大黄鱼养殖群体快速进化的遗传机制提供了遗传学资料。相关研究结果丰富了我们对大黄鱼遗传多样性现状以及渔业活动影响下大黄鱼快速进化遗传机制的认知。本研究为保护和恢复野生大黄鱼资源提供了合理评价和理论支持。研究结果填补了西北太平洋地区鱼类时空遗传多样性和进化机制研究的空白，为该区域商业鱼类资源的可持续开发与保护研究提供了重要参考。同时对深入了解捕捞诱导进化以及人工选择对物种驯化早期的遗传影响具有重要科学意义。

Anthropogenic selection pressures might rapidly transform the evolutionary trajectory and dramatically accelerating evolutionary changes of many species, but the underlying genetic mechanism remains unclear. Representative examples of anthropogenic pressures driven evolution come from fishery and domestication. Even though genetic theory provided strong evidence that fishing could be a potent driver of evolutionary changes, a clear empirical validation of fishing-induced evolution in wild stocks would be required. As compared with livestock, most aquaculture species had a short or even a recent history of domestication, and some of which were even just newly introduced into aquaculture and often had high genetic variation. Thus, aquaculture species is a representative case to reveal genetic mechanism of artificial selection on the early stages of species domestication.

Large yellow croaker (Larimichthys crocea) which major spread over the coast of China is one of the most heavily threatened marine species by anthropogenic activities. It was so severely depleted due to continuous overfishing since late-1950s. In order to protect and satisfy people’s demand for large yellow croaker, large scale and long-term restocking programs of the species had been applied in the East China Sea since hatchery was successful in the 1980s. Based on the continuous overexploitation and short history of selective breeding, large yellow croaker provides a unique opportunity to test for genetic mechanism of fishing-induced evolution and rapid evolution under artificial selection.

In the present study, historical (1960s and 1980s) and contemporary wild samples were collected from the East China Sea. Molecular markers include SSR, whole genomic SNPs and mitochondrial genome were used to compare the genetic variation between historical and contemporary wild samples, in order to assess the spatio-temporal genetic structure, inbreeding, genetic load of wild samples. We have proved the existence of fishing-induced evolution and provided genetic data for revealing evolutionary mechanism for the wild stocks of large yellow croaker. In addition, based on molecular markers of whole genomic SNPs and mitochondrial genome, we deeply analyzed the genomic dataset of historical, contemporary wild and cultivated samples, exploring the genetic diversity and genetic load of cultivated population of large yellow croaker. The results provided genetic data for revealing the genetic mechanism of rapid evolution of large yellow croaker under the influence of artificial selection. The main results were listed as follows:

1. We investigated the temporal and spatial genetic diversity and genetic structure changes before and after overfishing for large yellow croaker wild stocks in the East China Sea.

We employed 7 microsatellite loci, 660,359 SNPs and mitochondrial genomic sequences to reveal the spatio-temporal changes of genetic diversity and genetic structure for large yellow croaker wild stocks. The results of the three molecular markers showed non-significant decline of allelic richness, average expected heterozygosity, π, inbreeding coefficient and effective population size for contemporary wild populations comparing with historical populations. Various populations had similar proportion of heterozygous sites. The results of genetic structure analysis revealed non-significant difference among various samples. The mitochondrial network showed starlike distribution for all of the haplotypes, and no specific lineages were observed in different samples. In conclusion, the long-term overfishing had not given rise to either significant loss in genetic diversity or changes in spatio-temporal genetic structure for large yellow croaker stocks in the East China Sea. We suggested that fishing restrictions and habitat restorations should be the most direct and effective management strategy for recovering the large yellow croake.

2. We revealed the inbreeding and genetic load of wild stocks of large yellow croaker. We also detected the existing of fishing-induced evolution in large yellow croaker wild stocks and provided genetic data for revealing its evolutionary mechanism.

Based on whole genomic SNPs, genetic load and selective sweep were analyzed for historical and contemporary wild samples of large yellow croaker. The results showed the contemporary wild populations has higher inbreeding compared with historical populations. Besides, we also detected the genetic load of contemporary wild samples was significant higher than historical samples, which suggested the fitness of contemporary wild stocks have decreased. Selective sweep analysis was performed to screen 58 windows and annotate 84 genes. These genes included growth hormone receptor gene, hemoglobin synthesis genes and neurological and intellectual development genes. The growth hormone receptor gene might be related to accelerated growth of large yellow croaker. Hemoglobin synthesis genes were associated with oxygen transport. Individuals with higher oxygen utilization were more likely to have hypoxia endurance ability, in order to improve the chances of avoiding fishing. Genes for neurological and intellectual development might be related to the behavior of large yellow croaker and played an important role in avoiding fishing. Meanwhile, GO annotation analysis also revealed multiple GO terms that involved in oxygen transport, heat adaptation, energy and metabolic processes. These genes and GO terms might related to fishing selection, which demonstrated the existence of fishing-induced evolution in large yellow croaker wild stocks.

3. We revealed the genetic diversity, inbreeding and genetic load of large yellow croaker cultivated stocks and provided genetic data for exploring the genetic mechanism of rapid evolution under the influence of artificial selection.

Based on the whole genomic SNPs and mitochondrial genome, we performed genetic analysis for historical, contemporary wild and cultivated samples. Genetic diversity and genetic structure for both whole genomic SNPs and mitochondrial genome dataset revealed significant difference between cultivated and wild samples. Total ROH and genetic load of cultivated samples was significantly higher than wild samples, indicating the inferior fitness and evolutionary potential of cultivated samples. Selective sweep analysis revealed some interesting genes that might be associated with artificial selection, such as multidrug resistance-associated protein gene, photoreceptor ankyrin repeat protein gene and heat shock protein gene. These results might be direct evidence for rapid evolution induced by artificial selection in large yellow croaker cultivated stocks.

In conclusion, three molecular markers were used in the present study to identify the temporal and spatial changes of genetic diversity and genetic structure of wild stocks of large yellow croaker and explore its inbreeding and genetic load. Besides, we discussed the adaptive mechanism of wild stocks under the continuous fishing and revealed the genetic diversity, genetic load and provided genetic data for exploring the genetic mechanism of rapid evolution of cultivated stocks under the pressure of artificial selection. The results enrich our cognition to genetic diversity and genetic mechanism of rapid evolution for large yellow croaker under the influence of fishery activities, which should be significative for protecting and restoring wild resources of large yellow croaker. The results fill in the blank in the genetic effects of overexploitation for commercial species in the Northwestern Pacific Ocean, which could have implications for management of fishery resources in this area. Meanwhile, it is of great significance to further understand fishing-induced evolution and the genetic mechanism of artificial selection on the early stage of domestication.

第1章  绪论... 1

1.1  过度捕捞对鱼类群体遗传与进化的影响... 1

1.1.1  过度捕捞对鱼类群体遗传多样性、近交及遗传负荷的影响... 1

1.1.2  捕捞诱导进化的研究进展... 3

1.2  人工选择诱导养殖鱼类快速进化的遗传机制... 7

1.2.1  选育对养殖鱼类遗传多样性，近交及遗传负荷的影响... 7

1.2.2  选育对养殖鱼类适应性进化的影响... 8

1.3  历史DNA在鱼类群体遗传与进化研究中的应用... 10

1.3.1  历史样本的获取及历史DNA的提取... 10

1.3.2  历史DNA基因分型技术... 13

1.3.3  历史DNA的应用热点... 14

1.4  大黄鱼研究概述... 16

1.4.1  大黄鱼的生物学特征及分布... 16

1.4.2  大黄鱼历史群体资源及渔业发展... 17

1.4.3  大黄鱼养殖产业的发展与现状... 19

1.4.4  大黄鱼增殖放流... 21

1.4.5  大黄鱼群体遗传学研究概述... 22

1.5  本研究拟解决的科学问题，研究意义以及技术路线... 23

1.5.1  科学问题... 23

1.5.2  研究目标与意义... 23

1.5.3  技术路线... 24

第2章  大黄鱼时空遗传多样性及遗传结构研究... 25

2.1  引言... 25

2.2  材料与方法... 26

2.2.1  样本采集与DNA提取... 26

2.2.2  SSR标记开发与PCR扩增... 27

2.2.3  现代样品重测序文库构建与测序... 29

2.2.4  历史样品重测序文库构建与测序... 31

2.2.6  线粒体基因组提取... 42

2.2.7  大黄鱼遗传多样性分析... 42

2.2.8  大黄鱼群体遗传结构分析... 45

2.3  实验结果... 46

2.3.1  遗传多样性... 46

2.3.2  群体遗传结构... 50

2.4  讨论... 55

2.4.1  大黄鱼遗传多样性时空变化... 56

2.4.2  大黄鱼时空遗传结构... 57

2.4.3  管理与保护... 57

2.5  本章小结... 58

第3章 大黄鱼野生群体适应性进化研究... 59

3.1  引言... 59

3.2  材料与方法... 60

3.2.1  样本采集与DNA提取... 60

3.2.2  重测序文库构建与测序... 61

3.2.3  重测序数据处理与分析... 61

3.2.4  ROH分析... 61

3.2.5  遗传负荷分析... 61

3.2.6  选择性清除及GO富集分析... 62

3.3  实验结果... 63

3.3.1  ROH与近交繁殖... 63

3.3.2  遗传负荷... 63

3.3.3  选择性清除及GO富集... 65

3.4  讨论... 70

3.4.1  大黄鱼野生群体近交衰退与遗传负荷状况... 70

3.4.2  捕捞诱导进化... 72

3.5  本章小结... 74

第4章  大黄鱼养殖群体快速进化的遗传机制研究... 75

4.1  引言... 75

4.2 材料与方法... 76

4.2.1 样本采集与DNA提取... 76

4.2.2  重测序文库构建与测序... 77

4.2.3  重测序数据处理与分析... 77

4.2.4  基于基因组SNP的遗传多样性分析... 77

4.2.5  基于基因组SNP的遗传结构分析... 77

4.2.6  ROH分析... 77

4.2.7  遗传负荷分析... 78

4.2.8  LD衰减分析... 78

4.2.9  选择性清除及GO富集分析... 78

4.2.10  线粒体基因组提取... 78

4.2.11  基于线粒体基因组的遗传多样性分析... 78

4.2.12  基于线粒体基因组的遗传结构分析... 78

4.3  实验结果... 78

4.3.1  基于基因组SNP的遗传多样性... 78

4.3.2  基于基因组SNP的遗传结构... 80

4.3.3  ROH与近交繁殖... 82

4.3.4  遗传负荷... 83

4.3.5  LD衰减... 85

4.3.6  选择性清除及GO富集... 85

4.3.7  基于线粒体基因组的遗传多样性... 90

4.3.8  基于线粒体基因组的遗传结构... 91

4.4  讨论... 94

4.4.1 遗传多样性与群体遗传结构... 94

4.4.2选择性清除及GO富集... 96

4.4.3 增殖放流对野生群体的潜在影响... 98

4.4.4 管理与建议... 99

4.5 本章小结... 99

第5章 研究总结与展望... 101

5.1 研究总结... 101

5.2 主要创新点... 102

5.3 研究不足... 102

5.4 展望... 102

参考文献... 105

致  谢... 125

作者简历及攻读学位期间发表的学术论文与研究成果... 127