Institutional Repository of Key Laboratory of Marine Ecology & Environmental Sciences, CAS
伪菱形藻属物种基因组内 18S rDNA V4 区序列多样性解析 | |
王绘 | |
学位类型 | 硕士 |
导师 | 陈楠生 |
2024-05-06 | |
学位授予单位 | 中国科学院大学 |
学位授予地点 | 中国科学院海洋研究所 |
学位名称 | 理学硕士 |
关键词 | 伪菱形藻 宏条形码分析 基因组内变异 核糖体基因 时空动态分布 |
摘要 | 中文摘要: 伪菱形藻属(Pseudo-nitzschia)中近一半物种是能够合成软骨藻酸(domoic acid,DA)毒素的有害藻华(harmful algal bloom,HAB)物种,可以对海洋生态系统、海洋生物甚至人类的健康产生危害。随着高通量测序技术的发展,宏条形码分析成为监测海洋环境中产毒伪菱形藻物种的新技术,跟踪监测海洋生态系统中的伪菱形藻物种组成及其时空动态变化。但是,有证据表明很多藻类物种的基因组内可能存在较高的分子标记序列多样性(intragenomic variations,IGVs),这种现象可以导致在宏条形码分析过程中对伪菱形藻物种多样性的高估。本研究将评估IGVs的存在如何影响宏条形码分析结果的解析。 本研究的主要结果包括: (1)为全面了解我国近海伪菱形藻多样性和时空动态分布,通过靶向扩增、高通量测序和生物信息学分析,使用18S rDNA V4分子标记,利用基于扩增子序列变异子(amplicon sequence variants,ASVs)的宏条形码分析技术对山东近海四个季度航次样本开展分析,聚焦探讨山东近海海域的伪菱形藻物种多样性和时空动态变化。共检测到了40个ASVs,鉴定出8个伪菱形藻物种,包括四个该海域的未鉴定物种(P. multistriata、P. micropora、P. galaxiae、P. cuspidata),显示了宏条形码方法在航次调查研究中具有一定的优势。不过,以往鉴定的伪菱形藻物种中有两个物种(P. multiseries和P. fraudulenta)在本次宏条形码分析中未得到鉴定,可能表明这四个航次并没有覆盖所有的海域,或没有覆盖所有的季节。另外,分子标记的分辨率的局限也是产生该差异的原因之一。本研究发现不同伪菱形藻物种具有不同的时间和空间分布差异。不仅如此,不同伪菱形藻物种(比如P. pungens和P. galaxiae)的不同品系具有不同的时空分布差异。 (2)利用宏条形码技术解析了伪菱形藻物种的基因组内变异(IGVs)。开展了10个伪菱形藻物种的单个株系的基于通用分子标记18S rDNA V4的宏条形码分析,结果表明每个伪菱形藻株系都存在较高的IGVs。针对有害藻华物种多纹伪菱形藻(P. multistriata),每个株系都有超过100个的ASVs,其中一个是相对丰度较高的ASV(即优势ASV),还有大量丰度较低的ASVs(即非优势ASVs)。所有多纹伪菱形藻株系的优势ASV均100%相同,每一个株系中存在大量非优势ASVs可能表明核糖体基因均质化不完全。多纹伪菱形藻不同株系间的非优势ASVs集合不完全相同,但是具有很高的重叠。 其他9个伪菱形藻物种的IGVs与多纹伪菱形藻株系的分析结果类似,每一个株系的ASVs中通常包括一个优势ASV以及大量的非优势ASVs。除P. pungens之外,每一个物种的所有株系的优势ASV都完全相同,同一物种不同株系的非优势ASVs相似度很高。P. pungens的株系可根据其优势ASV的不同区分为两组,分别以不同的ASV为优势(即ASV_7和ASV_8),表明P. pungens的遗传多样性较高,并且18S rDNA V4可以将它们区分开。此外大多数伪菱形藻属物种的大多数株系还包含大量与其他伪菱形藻物种优势18S rDNA V4序列相同的非优势18S rDNA V4序列,这些结果表明,在伪菱形藻物种中发现的大多数18S rDNA V4拷贝共存在于所有伪菱形藻物种的共同祖先中,并且不同物种基因组中某些拷贝的相对丰度可能通过同质化逐渐占优势,而其他拷贝在进化中逐渐转为非优势。 综上,广泛应用于宏条形码分析的通用分子标记18S rDNA V4在伪菱形藻物种中存在较高的IGVs,其中每个株系中相对丰度最高的ASV(即优势ASV)代表物种多样性(种间多样性)或遗传多样性(种内多样性)。除此之外,每一个株系中还存在数目很高、丰度较低的非优势ASVs。研究结果表明,尽管宏条形码分析在揭示生态系统中物种组成即其时空动态研究中具有重要价值,增加对IGVs的认识是准确解释宏条形码分析结果的保证,从而增加伪菱形藻物种的生态监测和有害藻华预防的准确性。 |
其他摘要 | 英文摘要: Species of the diatom Pseudo-nitzschia genus are mostly cosmopolitan phytoplankton. Nearly half of the species are harmful algal bloom (HAB) species capable of synthesizing domoic acid toxin, harmful to the marine ecosystems, marine organisms and even human health. With the development of high-throughput sequencing technology, metabarcoding analysis may be a new technique for monitoring Pseudo-nitzschia species in marine environment, and tracking the composition and spatiotemporal dynamic changes of Pseudo-nitzschia species in marine ecosystems. However, the evidence has shown that there may be a high level of intragenomic molecular marker sequences variations (intragenomic variations, IGVs) in many species, which may lead to overestimation of the diversity of Pseudo-nitzschia species in metabarcoding analysis. This study will verify this scientific hypothesis and evaluate how the presence of IGVs affects metabarcoding analysis. In this study, the main research findings are as follows: (1) In order to comprehensively understand the diversity and spatiotemporal dynamic distribution of Pseudo-nitzschia in coastal waters of China, using targeted amplification, high-throughput sequencing, and bioinformatics analysis, this study carries out metabarcoding analysis based on the amplicon sequence variants (ASVs) of the Shandong coastal waters with four quarterly voyage samples based on 18S rDNA V4, focusing on the species diversity and spatiotemporal dynamic changes of Pseudo-nitzschia species in Shandong offshore waters. 40 ASVs were detected in Shandong coastal waters, and a total of eight Pseudo-nitzschia species were identified. Among them, four species (P. multistriata, P. micropora, P. galaxiae and P. cuspidata) were not identified in previous voyage surveys in this area, indicating that the metabarcoding method has certain advantages in voyage survey research. However, two of the previously identified Pseudo-nitzschia species (P. multiseries and P. fraudulenta) were not identified in this study, and these differences may indicate that the four voyages did not cover all seas or all seasons. In addition, the difference also attributes to the limited resolution of molecular markers. In this study, it was found that different Pseudo-nitzschia species have different temporal and spatial preferences. Moreover, different strains of different Pseudo-nitzschia species, such as P. pungens and P. galaxiae, have different spatiotemporal dynamic preferences. (2) The intra-genomic variations (IGVs) were analyzed by using metabarcoding analysis. This study carried out metabarcoding analysis of ten different Pseudo-nitzschia species based on 18S rDNA V4, a universal molecular marker. The results showed that each Pseudo-nitzschia strain had high IGVs. Among them, of the HAB species P. multistriata, each strain has more than 100 ASVs, in which including an ASV with the higher relative abundance (i.e., dominant ASV) and high number ASVs with lower abundance (i.e., non-dominant ASVs). The dominant ASV of all strains were identical, the large number of non-dominant ASVs in each P. multistriata strain may indicate incomplete ribosomal gene homogenization. The set of non-dominant ASVs among different strains of P. multistriata was not identical, but with a high overlap. Similar to the results of the P. multistriata, each strain of other nine Pseudo-nitzschia species included one dominant ASV and a large number of non-dominant ASVs. The dominant ASV were identical in all strains of each species, and the non-dominant ASVs were highly similar in different strains of the same species, except for P. pungens. The strains of P. pungens can be divided into two groups according to their respectively different dominant ASVs (ASV_7 and ASV_8), indicating that P. pungens has high genetic diversity, and 18S rDNA V4 can distinguish them. Most strains of most Pseudo-nitzschia species also contain large numbers of non-dominant 18S rDNA V4 domains that are identical to dominant 18S rDNA V4 domains of other Pseudo-nitzschia species. These results demonstrated that most 18S rDNA V4 domains identified in Pseudo-nitzschia species co-existed in the common ancestor of all Pseudo-nitzschia species, and the relative abundance of some variants in a particular genome might have gained dominance via homogenization, while other became non-dominant in evolution. In summary, based on 18S rDNA V4, which is widely used in metabarcoding analysis, has a high IGVs in Pseudo-nitzschia species, in which the ASV (i.e., dominant ASV) with the highest relative abundance in each strain represents species diversity (interspecific diversity) or genetic diversity (intraspecific diversity). In addition, there were also high number and low abundance of non-dominant ASVs in each strain. The results suggest that although metabarcoding are of great value in revealing the composition of species in ecosystems, i.e., their temporal and spatial dynamics, still increased understanding of IGVs is a guarantee of accurate interpretation of metabarcoding results, thereby increasing the accuracy of ecological monitoring of Pseudo-nitzschia species and the prevention of harmful algal blooms. |
学科门类 | 理学 ; 理学::海洋科学 |
语种 | 中文 |
目录 | 目 录 表目录
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文献类型 | 学位论文 |
条目标识符 | http://ir.qdio.ac.cn/handle/337002/185280 |
专题 | 海洋生态与环境科学重点实验室 中国科学院海洋研究所 |
推荐引用方式 GB/T 7714 | 王绘. 伪菱形藻属物种基因组内 18S rDNA V4 区序列多样性解析[D]. 中国科学院海洋研究所. 中国科学院大学,2024. |
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