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小黄鱼和日本鳗鲡群体遗传结构及本地适应性研究
刘炳舰
学位类型博士
导师刘进贤
2017-05-20
学位授予单位中国科学院大学
学位授予地点北京
学位专业海洋生物学
关键词小黄鱼 日本鳗鲡 群体遗传结构 群体基因组 本地适应性
摘要小黄鱼(Larimichthys polyactis)和日本鳗鲡(Anguilla japonica)是中国乃至东亚地区非常重要的海洋经济鱼类,然而受全球气候变化及过度捕捞等人类活动的影响,中国沿海的渔业资源正面临全面衰退的严峻现状,该两种海洋鱼类也不例外,尤其是日本鳗鲡已经被世界自然保护联盟红色名录收录,状态定为“濒危”。群体遗传学(population genetics)可以用来检测物种的遗传变异水平和群体遗传结构,探讨各种进化因素的影响机制,以及阐明种群适应环境的遗传学机制,为渔业资源的管理和保护提供重要的理论依据。前期关于小黄鱼和日本鳗鲡的群体遗传学研究由于受到遗传标记种类及数目的限制,基于不同类型的遗传标记所获得的研究结果存在一定的分歧甚至相互矛盾。因此,为了更好的保护和利用这两种重要的海洋渔业资源,亟需通过更加精细的分子遗传标记对这两种海洋鱼类群体遗传特征进行解析。在本研究中,我们分别基于较高密度的微卫星位点和基于简化基因组测序技术(RAD-seq)筛选的全基因组层面的SNP位点,对小黄鱼和日本鳗鲡进行了群体遗传结构和本地适应性方面的研究。
主要研究结果如下:
  1. 采用磁珠富集法,成功构建了小黄鱼基因组微卫星文库,设计并筛选出17个具有稳定多态性的微卫星位点。对所筛选的微卫星位点进行多态性评价,结果显示:17个微卫星位点等位基因数目从4到21个不等,观测杂合度范围为0.4583-0.9167,期望杂合度范围为0.7154-0.9566,所有位点经Bonferroni校正后有2个位点显著偏离哈温平衡(Hardy-Weinberg Equilibrium),并且经Micro-Checker软件分析,结果显示这2个微卫星位点可能含有无效等位基因。17个位点之间,均不存在连锁不平衡现象。
  2. 通过15个多态性微卫星位点对小黄鱼四个地点不同采样时间的样品进行群体遗传分析发现,小黄鱼的群体遗传多样性在时间尺度上并没有明显的降低,等位基因丰富度也没有明显变化。但是,基于两两群体间的FST,主成分分析(PCoA),STRUCTURE群体聚类分析和分子方差分析(AMOVA)等对群体结构解析时,首次检测到了小黄鱼群体在时间尺度上遗传组成的显著变化,该遗传分化可能是受到遗传漂变、过度捕捞或者环境因子突变的影响。
  3. 利用15个多态性微卫星位点分析了小黄鱼15个地理群体的遗传多样性与种群遗传结构。15个微卫星位点在15个小黄鱼群体中均检测到较高的多态性,通过中性位点的群体遗传分析结果显示,中国沿海的小黄鱼并不存在明显的遗传分化,表明小黄鱼群体间具有非常高的基因流,且不存在地理隔离模式和近期的瓶颈效应。但是通过离散位点筛选方法筛选到一个适应性位点(Lpol03),该位点可能与谷氨酸受体相关。通过该离散位点的分析,检测到了高基因流下可能的本地适应性信号,并且小黄鱼群体间可能存在隐存的遗传结构。基于离散位点检测到的遗传结构,暗示了中国沿海的小黄鱼可能存在两个比较独立的越冬群体,且具有多条可能的洄游路线。
  4. 基于简化基因组测序技术(RAD-seq)分析了采集自中国和日本沿海的日本鳗鲡11个自然地理群体的264个个体,所有个体经过建库测序共产生4,149,717,848原始数据(Raw reads),数据经过过滤后与已发表的日本鳗鲡基因组草图比对,应用SAMTOOLS软件共检测到45,552,012个SNP位点。经过严格质量过滤,共保留37,700个SNP位点用于群体基因组学分析。基于保留的所有SNP位点分析结果显示11个日本鳗鲡群体间遗传分化水平很低(FST=0.002012),表明日本鳗鲡群体间具有很高的基因流。基于ADMIXTURE的遗传结构分析结果显示,11个日本鳗鲡群体间并不存在显著的遗传结构,但是从DAPC聚类图和NETVIEW遗传关系网络图上能够看出其中几个群体中的部分个体存在一定的分化趋势,可能是受到了遗传漂变、洋流或产卵批次的影响。
  5. 通过两种基于FST值的筛选方法和一种基于等位基因频率与环境因子关联的方法,共筛选到355个离散位点(outliers),功能注释显示这些离散位点所在的基因主要与生长发育、细胞代谢、催化活性等细胞组成和生物过程相关,显示中国近海日本鳗鲡不同群体在单世代可检测到一些与本地适应性相关的信号。KEGG通路分析注释到10条代谢通路,主要包括Wnt信号通路、卵母细胞减数分裂通路、心肌细胞中的肾上腺素通路、钙离子信号通路、MAPK信号通路等。这些代谢通路参与了日本鳗鲡繁殖、发育、学习记忆、渗透压调节等生命过程,可能与其对分布范围内不同环境因子(如水温、盐度等)的适应相关。
相关研究结果系统阐明了小黄鱼和日本鳗鲡的种群遗传特征和本地适应性可能的遗传机制,对于小黄鱼和日本鳗鲡资源的合理保护和管理,以及深入了解海洋生物适应环境的遗传学机制具有重要的指导价值和科学意义。
其他摘要The small yellow croaker(Larimichthys polyactis )and the Japanese eel(Anguilla japonica)are two of the most economically important marine fishery species in China and East Asian countries, however, the abundance of these species had severely decreased due to overfishing as well as other anthropogenic factors. Especially for the Japanese eel, the abundance of Japanese eel had sharply decreased more than 90% since 1970s, and in March 2014, the Japanese eel was included in the IUCN(International Union for the Conservation of Nature)Red List of Threatened Species, with a current status as ‘endangered’(http://www.iucnredlist.org/details/166184/0).Population genetics can be used to detect the genetic structure of marine fish, thereby providing important insights into the sustainable management and utilization of fisheries resources. Limited source of markers has somehow constrained genetic studies of L. polyactis and A. japonica, and previous studies based on various types of genetic markers have obtained discrepant or even conflicting results. Hence, it is urgently needed to assess population genetic structure, genetic diversity, and possible regions of genome under selection for L. polyactis and A. japonica, in order to better manage and conserve these two species.
The main results are as follows:
1.      Seventeen polymorphic microsatellite loci were developed and characterized for L. polyactis using magnetic beads enrichment procedure. The number of alleles per locus ranged from 4 to 21, and the observed and expected heterozygosities varied from 0.4583-0.9167 and 0.7154-0.9566, respectively. Two loci showed significant deviations from Hardy–Weinberg equilibrium, most likely because of null alleles. These highly polymorphic nuclear markers would be useful for studies of genetic diversity and population genetic structure of the highly exploited L. polyactis
2.      Using fifteen microsatellites markers, we noted a non-significant decrease in genetic diversity between different time periods, and no obvious change in allelic richness was observed. Measures of population structure indicated substantial temporal genetic changes according to population differentiation tests(FST), the PCoA, STRUCTURE and the AMOVA analysis. To our knowledge, this is the first study that showed the temporal genetic variation of the L. polyactis. The temporal genetic variation of L. polyactis populations might be attributed to genetic drift, fishing-induced or ecological changes for this species.
3.      Fifteen microsatellite loci were used to estimate genetic diversity and population structure of fifteen samples along China coast. High genetic diversity and low genetic differentiation among populations were detected with all microsatellites. No evidence of isolation by distance and recent genetic bottleneck events was detected. However, outlier analyses indicated that the locus Lpol03 might be under directional selection, which showed a strong homology with Grid2 gene encoding the glutamate receptor δ2 protein(GluRδ2). Based on Lpol03, two distinct clusters were identified by both STRUCTURE and PCoA analyses, suggesting that there were two overwintering aggregations of L. polyactis, and these results provided new perspectives on the population genetic structure and migratory routine of L. polyactis.
4.      Based on restriction site-assosciated DNA(RAD)sequencing technology, we analyzed 264 Japanese eel(Angullia japonica)individuals from eleven geographic locations. A total of 4,149,717,848 Raw Reads were generated after sequencing, and 45,552,012 SNPs were called using SAMTOOLS. After the quality filtering, a total of 37,700 SNPs were retained, the identified 37,700 SNPs were widely distributed across the draft genome of the Japanese eel. The overall low genetic differentiation found(FST=0.002012, P > 0.05)indicated that most of the genome is homogenized by gene flow. Results of ADMIXTURE revealed that all individuals were grouped into a single cluster when using all the SNPs. While the results of DAPC and NETVIEW suggested that there might some substructure for the 11 populations, which might be attributed to genetic drift for this species.
5.      We tested for local selection by searching for increased population differentiation using FST-based outlier tests and by testing for significant associations between allele frequencies and environmental variables, and a total of 355 outlier SNPs were detected. Functional categorization of the annotated sequences involved in signaling, response to stimulus, metabolic process, biological regulation, catalytic activity and development process, etc.. The KEGG pathway analysis showed some of putative targets of local selection, including genes in several important pathways such as Wnt signaling pathway, Oocyte meiosis, calcium signaling pathway and MAPK signaling pathway.
These results will be useful to inform conservation and management decisions for these two species.
学科领域海洋生物学
文献类型学位论文
条目标识符http://ir.qdio.ac.cn/handle/337002/136639
专题海洋生态与环境科学重点实验室
作者单位中国科学院海洋研究所
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刘炳舰. 小黄鱼和日本鳗鲡群体遗传结构及本地适应性研究[D]. 北京. 中国科学院大学,2017.
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