高通量测序技术研究辽河真核浮游藻类的群落结构特征

真核浮游藻类在河流生态系统中发挥着重要生态功能,本研究对辽河水环境样本的18S rRNA基因的V4可变区进行454焦磷酸测序,对获得的OTU代表性序列进行物种注释.比较注释后的生物种类信息与光学显微镜观察结果的异同,并将OTUs的代表性序列与NCBI中真核浮游藻类的18S rRNA基因序列进行系统发育分析,此外研究了真核浮游藻类群落结构特征及其环境影响指标.研究共获得约167 901条18S rRNA基因V4可变区的高质量序列,注释为424个OTUs,涵盖了134种真核浮游藻类OTUs.与光学显微镜鉴别结果类似,高通量测序结果以硅藻门和绿藻门为优势类群,此外检测到轮藻门、隐藻门、定鞭藻门和金藻纲等显微镜观察结果中未有的类群.系统发育分析可在门这一分类阶元对隐藻门和甲藻门聚类,并将隐藻门在属水平进行聚类和区分,甲藻门可以在部分科或属水平聚类和区分.RDA结果显示,氨氮、活性磷和硝态氮是影响真核浮游藻类群落结构特征最重要的环境因子.本研究为认识辽河水环境中真核浮游藻类的多样性、群落结构及其环境影响因子提供了新的视角,同时也预示着高通量测序技术在辽河流域浮游植物监测和水环境质量指示中的应用潜力.

Community Structure Characteristics of Eukaryotic Planktonic Algae in Liaohe River Through High-throughput Sequencing

Eukaryotic phytoplankton plays an important ecological function in river ecosystem. The 18S rRNA gene V4 variable region of the environmental samples in Liaohe river was sequenced by using 454 pyrosequencing technology. The reprehensive sequences of OTUs were annotated, and then the OTUs list was compared to the species list obtained by microscope observation. The phylogenetic tree was constructed based on the reprehensive sequences of OTUs and 18S rRNA genes of eukaryotic phytoplankton algae obtained from GenBank. The community structure characteristics of eukaryotic phytoplankton and its environmental impact indicators were further studied. In this study, we got 167901 high-quality sequences of 18S rRNA gene V4 region. These sequences were annotated to 424 OTUs, including 134 eukaryotic algae. The results of the high-throughput sequencing showed that bacillariophyta and chlorophyta were the dominant groups of the total phyla. The high-throughput sequencing also revealed the presence of Charophyta, Cryptophyta, Haptophyta and Chrysophyceae which were not observed by optical microscope. The phylogeny analysis clustered the Cryptophyta and Pyrroptata at phylum level. The different genera of Cryptophyta and part of families or genera of Pyrroptata were clustered and distinguished by phylogeny analysis. The results of RDA showed that NH₄⁺-N, PO₄³-P and NO₃^- were the most important environmental factors that affected the community structure. This study provided a new perspective to understand the diversity, community structure and influence factors of eukaryotic phytoplankton in Liaohe river. The study also indicated that high-throughput sequencing has great application potential in investigation of phytoplankton and the water environment quality assessment in Liaohe river.