| 引用本文: | 王一奇,丁翔翔,宋会银,陈楠生.青岛近海球形棕囊藻的宏条形码分析.海洋与湖沼,2023,54(5):1351-1362. |
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| 青岛近海球形棕囊藻的宏条形码分析 |
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王一奇1,2,3,4, 丁翔翔1,2,3,4, 宋会银1,2,3,5, 陈楠生1,2,3
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1.中国科学院海洋研究所海洋生态与环境科学重点实验室 山东青岛 266071;2.崂山实验室 海洋生态与环境科学功能实验室 山东青岛 266237;3.中国科学院海洋大科学研究中心 山东青岛 266071;4.中国科学院大学 北京 100049;5.江汉大学 湖北武汉 430056
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| 摘要: |
| 自1997年球形棕囊藻(Phaeocystis globosa)赤潮首次在我国南海海域暴发以来, 该赤潮在我国南海和渤海的多个海域频繁暴发, 表明球形棕囊藻在我国海域的分布比较广泛。尽管如此, 在此之前开展的分类和生态调查研究均未发现我国海域球形棕囊藻的存在。近年来基于分子标记扩增和高通量测序的宏条形码分析检测到了球形棕囊藻, 显示出宏条形码分析对球形棕囊藻鉴定的优势。然而, 由于包括球形棕囊藻在内的定鞭藻的18S rDNA序列具有较高的G(Guanine, 鸟嘌呤)C(Cytosine, 胞嘧啶)含量等特点, 导致针对球形棕囊藻的宏条形码分析结果在很大程度上依赖分子标记的选择或PCR扩增引物的选择。以青岛栈桥海域样本为研究对象, 评估了扩增通用分子标记18S rDNA V4的两套常用PCR引物(即Stoeck引物和Song引物)对球形棕囊藻的宏条形码分析结果的影响, 并利用Song引物对青岛近海2021年冬季突发性球形棕囊藻赤潮样本开展了宏条形码分析。分析发现不同引物对定鞭藻门物种的分析结果具有显著影响。利用Stoeck引物的宏条形码分析没有获得任何对应的定鞭藻的ASV(amplicon sequence variant, 扩增子序列变异); 与此相比, 利用Song引物的宏条形码分析鉴定了76个定鞭藻ASVs, 说明Song引物能够有效扩增定鞭藻的18S rDNA V4序列。利用Song引物对青岛栈桥海域2021年9月逐日样本展开的宏条形码分析发现了两个注释为球形棕囊藻的ASVs (ASV_2和ASV_6), 表明青岛栈桥海域不仅存在球形棕囊藻, 而且具有一定的遗传多样性。利用Song引物对青岛近海2021年冬季突发性球形棕囊藻赤潮样本的宏条形码分析也发现了ASV_2和ASV_6, 并且发现这两种ASVs的相对丰度显示出不同的时间变化过程, 表明它们代表了不同的球形棕囊藻株系。其中ASV_2代表的球形棕囊藻株系随着西海岸赤潮暴发呈现出先增加后减少的趋势, 相对丰度较高, 而ASV_6代表的球形棕囊藻株系变化幅度较小, 相对丰度较低, 表明ASV_2所代表的球形棕囊藻株系是青岛近海2021年冬季球形棕囊藻赤潮暴发过程中的主要贡献者。研究表明, 引物的选择对基于常用分子标记18S rDNA V4展开的针对球形棕囊藻的宏条形码分析非常重要; 选择合适的引物不仅可以有效跟踪球形棕囊藻和其他浮游植物,还能够区分和跟踪不同球形棕囊藻株系的动态变化过程。 |
| 关键词: 球形棕囊藻 赤潮 分子标记 PCR引物 宏条形码分析 |
| DOI：10.11693/hyhz20230100014 |
| 分类号:Q949.2 |
| 基金项目:国家自然科学基金项目,42176162号;中国科学院战略性先导科技专项(B类),XDB42000000号;中国科学院率先行动“百人计划”;泰山学者特聘专家计划。 |
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| METABARCODING ANALYSIS OF PHAEOCYSTIS GLOBOSA IN QINGDAO COASTAL REGIONS |
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WANG Yi-Qi1,2,3,4, DING Xiang-Xiang1,2,3,4, SONG Hui-Yin1,2,3,5, CHEN Nan-Sheng1,2,3
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1.CAS Key Laboratory of Marine Ecology and Environmental Science, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China;2.Functional Laboratory for Marine Ecology and Environmental science, Laoshan Laboratory, Qingdao 266237, China;3.Center for Ocean Mega-Science, Chinese Academy of Sciences, Qingdao 266071, China;4.University of Chinese Academy of Sciences, Beijing 100049, China;5.Jianghan University, Wuhan 430056, China
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| Abstract: |
| Since the first occurrence of Phaeocystis globosa bloom in the South China Sea in 1997, P. globosa blooms have been occurring frequently in various ocean regions in China including the South China Sea and the Bohai Sea, suggesting that P. globosa is a widely distributed harmful algal bloom species in the coastal regions of China. However, neither previous morphology-based taxonomical studies nor ecology-based investigations has detected the presence of P. globosa in Chinese ocean regions. In recent years, metabarcoding analysis based on molecular marker amplification and high-throughput sequencing has detected P. globosa, demonstrating the competitive advantages of metabarcoding analysis for identification of P. globosa. However, due to the high G (Guanine) C (Cytosine) content of the 18S rDNA sequences of Haptophyta species including P. globosa, metabarcoding analysis for P. globosa highly depends on the selection of molecular marker or PCR primers. Using sea water samples collected from Zhanqiao Pier, Qingdao, the influence of two sets of PCR primers was evaluated for amplifying the universal molecular marker 18S rDNA V4 (i.e., the Stoeck primers and the Song primers) for the metabarcoding analysis of P. globosa. Then Song primers were utilized for metabarcoding analysis conducted on samples collected from the sudden P. globosa bloom in the Qingdao coastal regions in winter of 2021. Comparative analysis showed that different primers affected greatly on the analysis results for Haptophyta species. When using the Stoeck primers for metabarcoding analysis, no ASV corresponding to Haptophyta were found. In contrast, 76 Haptophyta ASVs were identified in metabarcoding analysis using the Song primers, which explaining that the Song primers could effectively amplify the sequences of 18S rDNA V4 of Haptophyta. Two ASVs (ASV_2 and ASV_6) annotated as P. globosa were found in daily samples from Zhanqiao Pier waters in September 2021 by using the Song primers, indicating not only the presence of P. globosa in Qingdao ocean region, but the presence of P. globosa genetic diversity. Metabarcoding analysis using the Song primers on samples collected from the sudden P. globosa bloom in the Qingdao coastal regions in winter of 2021 also detected ASV_2 and ASV_6, and found that the relative abundance of these two ASVs showed different time courses, suggesting that they represent different P. globosa strains. The P. globosa strain represented by ASV_2 showed a trend of first increase followed by a decrease, and high relative abundance, while the P. globosa strain represented by ASV_6 showed minor changes and low relative abundance. These results suggest that the P. globosa strain represented by ASV_2 was responsible for the outbreak of P. globosa bloom in the Qingdao coastal regions in 2021 winter. This study showed that primer selection is important for conducting metabarcoding analysis targeting P. globosa when using 18S rDNA V4 that is a universal molecular marker. The selection of appropriate primers can not only effectively track P. globosa, but also distinguish and track the dynamic change process of different P. globosa strains. |
| Key words: Phaeocystis globosa bloom molecular marker PCR primers metabarcoding analysis |
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