海洋生态与环境科学重点实验室知识库

自2009年起，渤海秦皇岛近岸海域每年夏季都会暴发大规模褐潮(Brown tide)，养殖贝类的摄食受到强烈抑制，出现大规模滞长和死亡等现象，造成了巨大的经济损失。褐潮暴发时，微型和微微型浮游植物占主要优势，但目前对其组成和变化规律的认识仍有不足，不利于阐明褐潮发生机制，亟待开展研究。随着DNA测序技术的进步，扩增子高通量测序法在解析微型和微微型浮游植物多样性方面表现出明显优势。本研究对扩增子高通量测序方法进行了优化，并利用该方法，对秦皇岛近岸海域“褐潮区”前后近3年的浮游植物样品进行了分析，较为系统地研究了秦皇岛近海微型和微微型浮游植物的种类组成与动态变化情况，得到如下结果和科学认识： 
1．通过对美国国立生物技术信息中心(National Center for Biotechnology Information，NCBI)数据库中三个高通量测序目标靶区已有序列的生物信息分析和野外浮游植物样品的DNA提取和测序分析，对海洋浮游植物样品的高通量测序方法进行了优化，明确了扩增子高通量测序的靶序列，确定了浮游植物样品中基因组DNA的提取方法。基于NCBI数据库中的序列信息，以及对同一浮游植物样品的核糖体18S rDNA V1V2、V4和V9三个靶区高通量测序结果的对比，分析了三个靶区对不同浮游植物种类的分辨能力。结果表明，V1V2、V4和V9区对不同种类浮游植物的分辨能力存在差别。V1V2区和V4区进化速率适中，对有害藻华原因种分辨能力较强，但V1V2区无法准确分辨褐潮原因种抑食金球藻。V9区与V4区相比过于高变，许多物种的序列信息互相交错，无法准确区分，在种水平上鉴别浮游植物的能力较低。考虑NCBI数据库18S rDNA V4区参考序列数量较多，且对常见有害藻华原因种具有较强的区分能力，因此，本研究选用18S rDNA V4区作为浮游植物样品高通量测序的目标靶区。同时，针对室内构建的模拟浮游植物样品和自胶州湾采集的野外浮游植物样品，从提取基因组DNA的浓度、纯度和完整性三方面，对比分析了CTAB法、SDS法和Sucrose法三种基因组DNA提取方法的优劣，结果表明CTAB法更适用于海洋浮游植物样品基因组DNA的提取。
2．通过对2013~2014年度秦皇岛近海“褐潮区”浮游植物样品的高通量测序分析，揭示了褐潮区浮游植物群落的种类组成与动态变化特征，进一步确认了抑食金球藻是秦皇岛近海褐潮的主要原因种。本研究分别于2013年和2014年褐潮暴发期间，在褐潮区采集微型和微微型浮游植物样品22个，采用高深度的扩增子高通量测序方法对18S rDNA V4区进行了测序分析。平均每个样品获得有效数据超过20万条，发现了超过2,000个微型和微微型浮游植物的OTU(Operational taxonomic unit)，分属硅藻、甲藻、海金藻和绿藻等17个浮游植物类群。2013年和2014年褐潮暴发前后浮游植物优势类群的演替过程相似，褐潮暴发前的优势类群为甲藻、青绿藻、隐藻和硅藻，至褐潮暴发时海金藻成为第一优势类群，褐潮暴发后优势类群趋于多样化，主要包括甲藻、海金藻、金藻、隐藻和青绿藻等。褐潮发生时，抑食金球藻的OTU相对丰度均超过50%，进一步确认了抑食金球藻是秦皇岛近海褐潮的主要原因种。对比两年度的调查结果，发现2014年褐潮暴发时间比2013年提前了约一个月，而且2014年甲藻类在物种数和相对丰度上比2013年都更具优势。通过计算抑食金球藻与其他浮游植物之间的皮尔森相关系数，发现了许多与抑食金球藻具有相关性的OTU，其中一些属于稀有OTU(相对丰度< 0.01%)，为进一步揭示褐潮成因提供了新的视角。
3．自秦皇岛近海成功分离培养了一株抑食金球藻，通过多种手段进行了藻种鉴定确认，基于对中、美两国分离培养的多株抑食金球藻DNA序列分析和野外浮游植物样品的高通量测序结果，发现抑食金球藻存在遗传多态性。使用梯度稀释法，于2015年在秦皇岛近岸海域成功分离得到一株抑食金球藻藻株（MEL50），并通过挑取单细胞方法分离得到了一株单克隆培养株MEL51，基于形态学特征、色素组成和DNA序列等证据进行了藻种的鉴定确认。针对MEL 50、MEL 51以及美国抑食金球藻CCMP 1984和CCMP 1850四个藻株，构建了克隆文库，获得了核糖体18S rDNA V4区、28S rDNA D1D2区和ITS区的序列信息，分析结果表明抑食金球藻核糖体基因存在多态性，V4区更适合作为靶区进行抑食金球藻遗传多态性分析。针对18S rDNA V4区进行的浮游植物样品高通量测序结果表明，秦皇岛近岸海域的抑食金球藻在V4区存在多个基因型，中国株系MEL50和美国株系CCMP1984和CCMP1850都属于基因型I。
综上，本研究采用扩增子高通量测序方法，分析了2013年和2014年秦皇岛褐潮区微型和微微型浮游植物组成及其动态变化情况，进一步确认了抑食金球藻是秦皇岛近岸海域褐潮的主要原因种，挖掘并分析了与抑食金球藻相关的藻种。通过比对中、美两国抑食金球藻藻株的核糖体rDNA部分序列信息，以及对秦皇岛近海浮游植物样品的高通量测序结果，揭示了抑食金球藻的遗传多态性，为深入探索秦皇岛近海褐潮成因提供了基础依据。

Large-scale brown tides of Aureococcus anophagefferens have been recorded from 2009 in the coastal waters of Qinhuangdao, Bohai Sea. Feeding activities of cultured scallops were strongly inhibited during the brown tides, and resulted in huge economic losses. During the brown tides, nano- and pico-sized phytoplankton were dominant groups, but the knowledge on the species composition and their dynamics was quite limited. Therefore, it’s necessary to perform studies on phytoplankton communities. Recently, the rapid development of high-throughput sequencing technology showed potential advantages in studies on nano- and pico-sized phytoplankton communities. In this study, the high-throughput sequencing method was optimized and used to analyze the phytoplanton samples collected from the brown tide zone in Qinhuangdao coastal waters in 2013, 2014 and 2016. The composition and dynamic of phytoplankton communities were systematically investigated, and the major results and findings were listed as followed:

1. The method of high-throughput sequencing was optimized for the analysis of phytoplankton samples. The targeted region was selected based on the analysis of corresponding sequences in NCBI, and methods for genome DNA extraction was selected and optimized. Based on the sequence information in NCBI, and the comparison of high-throughput sequencing results of 18S rDNA V1V2, V4 and V9 in the same phytoplankton sample, the resolution of the three targeted regions was compared. The results show that the V1V2 region and the V4 region have relatively moderate evolution rate, and are capable of resolving most causative species of harmful algal blooms. The V1V2 region, however, could not accurately distinguish A. anophagefferens from other pelagophyte species. Compared with the V1V2 and the V4 regions, the V9 region is too hyper-variable. The V9 sequence of many species overlap with each other, and couldn’t be distinguished from each other. Considering that a large pool of 18S rDNA V4 sequences have been deposited in NCBI, and its capability in distinguishing causative species of harmful algal blooms, the V4 region was selected as the target for high-throughput sequencing of field phytoplankton samples in Qinhuangdao coastal waters. Based on the analysis of simulated and natural phytoplankton samples, three methods (CTAB, SDS and Sucrose) for extraction of genome DNA were compared, and the CTAB method was selected in this study based on the yield, purity and integrity of DNA extracted from phytoplankton samples.

2. Based on the high-throughput sequencing analysis of phytoplankton samples from the “brown tide zone” in Qinhuangdao coastal waters from 2013 to 2014, the composition and succession of phytoplankton communities were revealed. The results further confirmed that pelagophyte A. anophagefferens is the major causative species of brown tide in the coastal waters of Qinhuangdao. In this study, 22 samples of nano- and pico-sized phytoplankton were analyzed by high-throughput sequencing (18S rDNA V4) in 2013 and 2014, respectively. On average, more than 200,000 tags were obtained per sample and more than 2,000 OTUs (Operational taxonomic units) were found, belonging to 17 groups of phytoplankton, including diatoms, dinoflagellate, pelagophyte and green algae etc. During the brown tide, there was a similar succession pattern of major phytoplankton groups in 2013 and 2014. The main dominant groups of nano- and pico-sized phytoplankton were dinoflagellates, prasinophytes, cryptophytes and diatoms prior to the occurrence of brown tide, which shifted to a community dominated by pelagophytes, and finally a community dominated by more diversified phytoplankton groups including dinoflagellates, pelagophytes, chrysophytes, cryptophytes and prasinophytes etc. For both years, A. anophagefferens was the most dominant species during the bloom period and its maximum relative abundance exceeded 50 percent. The results further confirmed that A. anophagefferens is the major causative species of brown tide. By comparing the results of the two years, the outbreak time of brown tide in 2014 was about one month earlier than that in 2013, and an increasing dominance of dinoflagellates could be observed in the Qinhuangdao coastal waters. By calculating the Pearson correlation coefficient between A. anophagefferens and other phytoplankton species, the species closely related to the dynamics of A. anophagefferens were found, including some rare OTUs (relative abundance < 0.01%). 

3. A strain of A. anophagefferens isolated from Qinhuangdao coastal waters was identified by morphological observation, pigment analysis and sequencing results. Based on the analysis of partial rDNA sequences of four A. anophagefferens strains from China and the United States, as well as the results of high-throughput sequencing of phytoplankton samples from Qinhuangdao coastal waters, the genetic diversity of A. anophagefferens was revealed. The strain MEL50 was isolated by serial-dilution method in 2015, and strain MEL51 was isolated from MEL 50 by single-cell isolation method. Both strains were identified as A. anophagefferens by morphological observation, pigment analysis and molecular information. The clone libraries of 18S rDNA V4, 28S rDNA D1D2 and the ITS region were constructed for the four strains MEL 50, MEL 51, CCMP 1984 and CCMP 1850. Genetic diversity were revealed in 18S rDNA V4 region through high-throughput sequencing of phytoplankton samples collected from the Qinhuangdao coastal waters. Both the Chinese strain MEL50 and the American strains CCMP1984 and CCMP1850 belong to the genotype I.
In summary, the composition and dynamic of nano- and pico-sized phytoplankton assemblages in Qinhuangdao coastal waters in 2013 and 2014 were analyzed by high-throughput sequencing. Pelagophyte A. anophagefferens was further confirmed as the causative species of brown tides, and many species related to A. anophagefferens were revealed. Potential genetic diversity was found within A. anophagefferens. The results provide strong evidence to better understand the mechanism of brown tide in the coastal waters of China.