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松江鲈遗传标记开发与保护遗传学研究
李玉龙1,2
学位类型博士
导师刘进贤
2017-05
学位授予单位中国科学院大学
学位授予地点北京
学位专业海洋生态学
关键词保护遗传学 群体基因组学 Rad-seq 松江鲈 遗传结构 本地适应性
其他摘要
    鉴定保护单元对于濒危物种的监测和管理至关重要。群体的遗传特征,包括群体遗传结构、遗传多样性水平、基因交流、本地适应性等,是保护单元制定的重要理论依据。遗传多样性是物种维持进化潜力的重要因素。本文的研究对象松江鲈(Trachidermus fasciatus)隶属于鲉形目、杜父鱼科、松江鲈属,是一种小型底栖降海产卵洄游性鱼类。受人类活动的影响,如修建水坝、过度捕捞、环境污染、产卵场破坏等,目前松江鲈群体数量已大大减少,其栖息地已呈片段化分布。鉴于此原因并考虑到其文化、生态及进化学上的价值,在1988年中国政府将其列为国家二级保护动物。阐明群体遗传特征对于松江鲈保护措施的制定和实施具有重要意义,但是目前关于松江鲈保护遗传学和群体遗传学方面的研究仍然存在分歧,特别是关于中国松江鲈群体遗传结构的研究结果并不清晰,更是缺乏基因组方面的研究,极大地限制了其管理和保护工作。本文采用了多态性的微卫星分子标记和更高分辨率的群体基因组学手段对松江鲈群体系统地开展了保护遗传学研究,以期为其保护单元的划分提供理论依据。主要研究结果如下:
    1、采用磁珠富集法开发了25个松江鲈高多态性微卫星位点。选取富阳群体24个个体进行基因分型,结果显示不同位点等位基因数目范围为6至28,期望杂合度范围为0.54至0.97,观测杂合度范围为0.42至1.00,多态信息含量范围为0.50至0.95,表明这些微卫星位点具有高度的多态性。
    2、利用简化基因组学RAD-seq技术对丹东和富阳两个松江鲈群体共27个个体开发了基因组范围的SNP标记,并开发了一种优化后的RAD序列组装方法,提高了组装的准确率和效率。针对27个个体成功组装了RAD参考序列176,619条,其平均序列长度为572 bp,N50为603 bp,GC含量为41.6%。其中96.7% 的RAD位点分别组装为单个contig,98.0% 的参与组装的reads可正确比对到参考序列上,表明了序列的可靠性。最终保留了174,017条高质量contigs用于SNP开发,共开发了1,612,103个SNP标记,经质量过滤后保留44,066个SNP位点用于群体遗传学分析。基于基因组SNP标记的群体遗传学分析结果显示两个群体间存在显著的遗传分化(FST = 0.0705,P < 0.01),从侧面反映了组装序列的有效性。另外本研究也移植了STACKS软件的bounded error SNP提取模型用于双末端测序数据中,弥补了STACKS在双端数据分析中的不足。
    3、采用16个高多态性微卫星位点对10个松江鲈群体共232个个体进行了群体遗传学研究。结果表明10个群体均具有较高的遗传多样性水平,其观测杂合度范围为0.734至0.872,期望杂合度范围为0.835至0.947,多态信息含量范围为0.793至0.924。但青岛群体显示了相对较低的遗传多样性水平和较小的有效群体大小。遗传结构分析结果表明群体间存在显著的遗传分化,10个群体可划分为7个遗传组群,其中丹东、大连和秦皇岛群体可划分为一个组群,东营和潍坊群体为一个组群,荣成、文登、青岛、富阳、有明海群体分别为单独的组群;AMOVA显示不同组群间存在显著遗传分化(FCT = 0.042,P < 0.01)。除东营和潍坊群体间遗传分化不显著外,其他群体间皆存在显著遗传分化(P < 0.01),整体FST为0.048。近期群体间的迁移率分析结果表明松江鲈群体间基因交流水平较低,且群体间的遗传分化不符合距离隔离模式。研究结果揭示了栖息地片段化和小种群的遗传漂变作用可能是松江鲈不同群体间产生遗传分化的主要原因。
    4、采用简化基因组学RAD-seq技术对中国松江鲈8个地理群体共180个个体开展了群体基因组学研究。共挑选32个个体采用优化的组装方式组装了RAD参考序列共262,533条,其平均长度为517 bp,N50为566 bp,GC含量为41.6%。最终保留237,947条含有酶切位点的contigs,共检测到3,436,092个SNP位点,经质量过滤后保留10,153个高质量SNP位点。群体遗传结构分析结果显示8个群体可划分为6个遗传组群,且AMOVA结果表明不同遗传分组间的分化水平相较于微卫星的结果更为明显(FCT = 0.081,P < 0.01)。其中丹东和大连群体为一个遗传组群,东营和潍坊群体为一个遗传组群,荣成、文登、青岛、富阳群体分别为单独的遗传组群。除东营和潍坊群体间遗传分化不显著外,其他群体间皆存在显著遗传分化(P < 0.01),并且表现出相较于微卫星更高的遗传分化水平(整体FST为0.103)。青岛群体同样显示相对较低的遗传多样和较高的遗传分化水平,暗示了其保护的优先级。本地适应性分析共筛选到离散位点414个,其中18个既是高分化的离散位点又与环境因子(温度)相关联。414个离散位点GO功能注释显示其与多个生物过程、细胞组分、分子功能相关,主要参与代谢、离子结合、应激、催化活性等。对18个离散位点采用进一步的定位和功能注释结果发现多个与能量代谢,特别是与脂肪酸的合成与代谢相关的功能基因和代谢通路。这表明松江鲈本地适应性可能与不同地理群体所处的环境温度相关,与能量代谢相关的基因可能在松江鲈适应环境温度中发挥重要作用。适应性遗传结构分析的结果也表明处于相似水温的地理群体聚为一组,其中丹东、大连、东营、潍坊群体为一组,荣成、文登群体为一组,青岛群体为一组,富阳群体为一组。基于中性位点的遗传结构与基于全部位点的遗传结构一致,表明松江鲈群体间的遗传结构主要是由于中性进化力量作用,暗示遗传漂变作用是群体间遗传分化产生的主要原因之一。
    综上,本文对松江鲈开发了微卫星和基因组SNP两种分子标记,基于两种分子标记的结果均显示松江鲈群体间已产生显著遗传分化,并且可能存在与环境温度关联的本地适应性现象。研究结果表明中国松江鲈8个群体(丹东、大连、东营、潍坊、荣成、文登、青岛、富阳)可以划分为7个管理单元(除东营和潍坊群体可作为一个管理单元外,其他群体为单独的管理单元)和4个适应性单元(丹东、大连、东营、潍坊群体为一个单元,荣成、文登群体为一个单元,青岛、富阳群体分别为单独的单元)。结果暗示了人类活动影响导致的松江鲈栖息地片段化和遗传漂变作用是遗传分化产生的主要原因。建议不同群体进行原地保护以维持其进化潜力,避免种质混乱。相关的研究结果丰富了松江鲈遗传资源库,阐明了群体间的遗传结构,揭示了其本地适应性机理,填补了松江鲈基因组学研究的空白,为我国松江鲈保护单元的科学划分提供了理论依据。
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    Identification of conservation units is crucial for the monitoring and management of endangered species. The genetic characteristics of populations, including population structure, genetic diversity, gene flow and local adaptation etc., are the fundamentals of fashioning conservation units. The extent of genetic variability of a population determines the ability of that population to adapt to the environment through natural selection. The roughskin sculpin, Trachidermus fasciatus Heckel (Scorpaeniformes: Cottidae), is a small, benthic, carnivorous, and catadromous fish species. It was widely distributed along the east coast of China, west and south coast of the Korean Peninsula, and Kyushu coast of Japan. However, due to anthropogenic activities such as overfishing, environmental pollution, construction of dams, and deterioration of spawning grounds, T. fasciatus has declined in abundance and even gone extinct across most of its historical range in China and its current distribution has become highly fragmented in the past decades. Considering its biological, ecological, and endangered status in China, T. fasciatus has been included in the List of the Wildlife under Special State Protection as a second class state protected aquatic animal by the Chinese government in 1988. A better understanding of genetic diversity and population genetic status of T. fasciatus are crucial for designing suitable management guidelines. However recent studies on population structure of T. fasciatus along the coast of China have produced conflicting results. The genome research is still lacking, which will further restrict the management and protection for T. fasciatus. This study focused on the conservation genetics of T. fasciatus by using microsatellites and population genomics techniques, which would be crucial for designing management guidelines. Main results are presented as follows:
    1. We developed 25 highly polymorphic microsatellites by using magnetic beads enrichment procedure. The genotyping results based on 24 individuals from Fuyang suggested high levels of genetic diversity: the number of alleles per locus ranged from 6 to 28, polymorphic information content ranged from 0.50 to 0.95, and the expected and observed heterozygosities ranged from 0.54 to 0.97 and 0.42 to 1.00 respectively.
    2. We developed genome-wide SNPs for 27 individuals in two populations of T. fasciatus based on RAD-seq. An optimized high-efficient assembly method for multi-individuals was developed. And the results suggested its high accuracy and efficiency, a total of 176,619 contigs were generated with mean length of 572 bp and N50 of 603 bp, and the GC content was 41.6%. Most of the loci (96.7%) were each assembled into one contig, 98.0% of the input reads were properly mapped to the generated contigs. 174,017 contigs were retained for downstream SNP discovery and population analysis. A total of 1,612,103 SNPs were detected based on the above RAD contigs, of these 44,066 SNPs were retained after quality filtering. The genetic divergence between the two populations was high and significant (FST = 0.0705, P < 0.01), suggesting the efficiency of RAD contigs. Moreover, in order to overcome the limitation of utilization of paired reads in STACKS, the bounded error SNP calling model in STACKS was also modified to call SNPs from paired reads.
    3. Genetic variation for 232 individuals in 10 population of T. fasciatus were assessed by 16 polymorphic microsatellites. Genetic diversity were high within populations, polymorphic information content ranged from 0.793 to 0.924, and the observed and expected heterozygosities ranged from 0.734 to 0.872 and 0.835 to 0.847 respectively. The Qingdao population showed both the lowest levels of genetic diversity and effective population size, suggesting its priority of protection. Results of population structure analysis indicated that there were seven genetic clusters among the sampled populations, with “Dandong”, “Dalian” and “Qinhuangdao” forming into one group, “Dongying” and “Weifang” forming into one group, and the rest five populations (“Wendeng”, “Rongcheng”, “Qingdao”, “Fuyang”, “Ariake Sea”) grouping into distinct clusters respectively. AMOVA suggested significant divergence among the seven groups, and all the pairwise FST between populations were significant (P < 0.01) except for the pair between Dongying and Weifang, the global FST was 0.048. Recent migration rates suggested low levels of gene flow among nine populations of China, and there was no pattern of isolation by distance among populations. These results suggested habitat fragmentation and genetic drift were the main causes for the significant genetic divergence among populations.
    4. Genetic structure and local adaptation for 180 individuals in eight population of T. fasciatus were assessed by population genomic techniques. 32 individuals were selected for RAD contigs assembly based on our optimized assembly method, which yielded a total of 262,533 contigs with mean length of 517 bp and N50 of 566 bp, GC content of 41.6%. A total of 237,947 contigs containing the enzyme cutting sites were retained for downstream analysis, which corresponded to 237,828 RAD loci. A total of 3,436,092 SNPs were detected based on 180 individuals in eight populations of T. fasciatus, of these 10,153 SNPs were retained after quality filtering. Results of population structure analysis indicated that there were six genetic clusters among eight populations, with “Dandong” and “Dalian” forming into one cluster, “Dongying” and “Weifang” forming into one cluster, and the rest four populations (“Wendeng”, “Rongcheng”, “Qingdao”, “Fuyang”) forming into distinct clusters respectively. AMOVA suggested the significant genetic divergence among the six genetic clusters (FCT = 0.081,P < 0.01). Pairwise FST were similar to the results based on microsatellites but with high values, overall FST was 0.103. The Qingdao population owned the lowest genetic diversity and highest genetic differentiation, suggesting the priority of protection. A total of 414 outliers related either to FST or to environmental factors were detected, of these 18 outliers were both FST-outliers and environmental related. GO characterization of 414 Outliers were classified into biological process, cellular component and molecular function, such as metabolism, response, regulation and catalytic activity etc.. Furthermore, we found several genes and pathways related to metabolism or transport, especially the synthesis and metabolism of fatty acids. The results suggested the relevance of ocean temperature with local adaptation of T. fasciatus. Genetic structure analysis based on adaptive loci suggested populations with same ocean temperature grouped together, with “Dandong”, “Dalian”, “Dongying”, “Weifang” forming into one group, “Wendeng” and “Rongcheng” forming into one group, “Qingdao” forming into one group, “Fuyang” forming into one group. The genetic structure based on neutral loci was similar to that based on all loci, suggesting the main causes of genetic structure of T. fasciatus were neutral forces, which implied the strong effects of genetic drift.
    Overall, we have developed two kinds of genetic markers, microsatellites and genome-wide SNPs, both the results based on the above makers indicated the significant genetic structure among populations and local adaptations within populations of T. fasciatus. Our results suggested that the eight populations (“Dandong”, “Dalian”, “Dongying”, “Weifang”, “Rongcheng”, “Wendeng”, “Qingdao”, “Fuyang”) of T. fasciatus in China should be defined into seven MUs (“Dongying” and “Weifang” could be defined as one MUs, and the rest six populations as distinct MUs) and four adaptive groups (“Dandong”, “Dalian”, “Dongying” and “Weifang” should be defined as one adaptive group, “Rongcheng” and “Wendeng” should be defined as one adaptive group, “Qingdao” and “Fuyang” are distinct adaptive groups respectively). Habitat fragmentation and genetic drift due to anthropogenic activities were responsible for the significant genetic differentiation among populations. We highlight the need for in situ conservation efforts for T. fasciatus across its entire distribution range to preserve overall evolutionary potential and to avoid germplasm confusion. The generated genetic markers are valuable genetic resources, and our results have filled the gap of genome research and clarified the genetic structure and the mechanism of local adaptation for T. fasciatus, which are crucial for designing guidelines of conservation units.
 
学科领域海洋生物学 ; 种群生态学
语种中文
文献类型学位论文
条目标识符http://ir.qdio.ac.cn/handle/337002/136645
专题海洋生态与环境科学重点实验室
作者单位1.中国科学院海洋研究所
2.中国科学院大学
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李玉龙. 松江鲈遗传标记开发与保护遗传学研究[D]. 北京. 中国科学院大学,2017.
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