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引用本文:黄平平,赵峰,徐奎栋.近海水体环境DNA沉降对沉积物中纤毛虫分子多样性评估的影响.海洋与湖沼,2020,51(3):602-612.
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近海水体环境DNA沉降对沉积物中纤毛虫分子多样性评估的影响
黄平平1,2, 赵峰1, 徐奎栋1,2
1.中国科学院海洋研究所 海洋生物分类与系统演化实验室 青岛 266071;2.中国科学院大学 北京 100049
摘要:
在陆架海区沉积物中,DNA高通量测序技术可检获大量浮游寡毛类和舞毛类等非底栖纤毛虫,这些源于水体的环境DNA(eDNA)如何影响对沉积物中纤毛虫分子多样性的评估,以及影响程度如何尚不明确。本研究选取了黄海冷水团中的两个站位,通过提取水体和沉积物中DNA和RNA,并采用直接提取法和洗脱法提取沉积物DNA,结合DNA和cDNA测序技术,探讨了水体和沉积物中纤毛虫分子多样性的关系。研究表明,基于洗脱DNA法、直接提取DNA法和cDNA法获得的沉积物中纤毛虫OTUs数分别为451、312和324个,其中211个OTUs同时由三种方法检获;而164个OTUs仅通过洗脱DNA法检获,其中89%为相对丰度低于0.1%的稀有类群。直接提取DNA法所获的寡毛类和舞毛类序列数占比达46%,而在洗脱DNA法和cDNA法中占比仅为12%和10%。沉积物中检获的43%-71%的舞毛类和寡毛类OTUs与浅层水(<40m)共有,仅19%-29%与深层水(>40m)共有,且这些共有的OTUs可同时在浅层水中检获。本研究发现,对沉积物中纤毛虫分子多样性评价造成影响的浮游类群主要来自浅层水,洗脱DNA与cDNA测序均可显著降低浮游类群eDNA的影响。鉴于洗脱DNA法较之cDNA法操作更简便,且避免了反转录过程导致的偏差,因此推荐用于对近海沉积物中纤毛虫等真核微生物的分子多样性研究。
关键词:  纤毛虫  海洋水体  海洋沉积物  分子多样性  DNA高通量测序  cDNA高通量测序
DOI:10.11693/hyhz20191100235
分类号:Q958
基金项目:国家自然科学基金项目,41876171号,41476144号。
EFFECTS OF SEDIMENTATION OF DNA FROM OVERLYING WATERS ON THE EVALUATION OF CILIATE MOLECULAR DIVERSITY IN OFFSHORE SEDIMENTS
HUANG Ping-Ping1,2, ZHAO Feng1, XU Kui-Dong1,2
1.Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China;2.University of Chinese Academy of Sciences, Beijing 100049, China
Abstract:
A high proportion of Choreotrichia and Oligotrichia, which are typically planktonic ciliates, has been frequently detected from marine sediments by DNA high-throughput sequencing. However, few of them could be observed with the morphological method. The influence of these environmental DNA (eDNA) in waters on assessing the ciliate diversity in sediments and the extent to which ciliates linking the sediments and overlying waters are far from being known. Based on the sediment and overlying water samples collected from two stations in the Yellow Sea Cold Water Mass, the ciliate molecular diversity in sediments in relation to that in the upper water layers was evaluated by using DNA and cDNA (complementary DNA) sequencing. The DNA in sediments was extracted by directly extraction and elution methods. Results show that the highest ciliate diversity in sediments was obtained by the treatment of DNA elution, which yielded 451 OTUs. By contrast, the direction extraction of DNA and the cDNA method detected only 312 and 324 OTUs, respectively. Among them, 211 OTUs were simultaneously detected by the three methods. The DNA elution method was more effective in the detection of rare taxa. Among the 164 OTUs exclusively detected by the DNA elution method, about 89% of them were rare taxa in relative abundance lower than 0.1%. DNA sequencing with the direct extraction of DNA from the sediments yielded a high proportion of Choreotrichia and Oligotrichia, which contributed about 46% of the total sequences, while DNA sequencing with the eluted DNA and cDNA sequencing yielded only about 12% and 10% of the total sequences, respectively. About 43%-71% of the total OTUs of Choreotrichia and Oligotrichia detected from the sediments were shared with those in the upper water layers (<40m), while only 19%-29% of these OTUs were shared with those in the lower water layers (>40m). Generally, these OTUs shared in the lower water layers and sediments could be obtained in the upper water layers. The planktonic eDNA that influenced the evaluation of the molecular diversity of ciliates in sediments was mainly originated from the upper water layers. For the assessment of the sediment ciliate diversity, both cDNA sequencing and DNA sequencing with the DNA elution could highly reduce the influence of the planktonic eDNA. Therefore, DNA sequencing with the DNA elution is more appropriate for the study of molecular diversity of ciliates and other microeukaryotes in sediments. Compared with cDNA sequencing, the DNA elution method is much easier to operate and can avoid some biases resulted from reverse transcription.
Key words:  ciliate  sea water  marine sediment  molecular diversity  DNA high-throughput sequencing  cDNA high-throughput sequencing
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