Coupling 16S-ITS rDNA clone libraries and automated ribosomal intergenic spacer analysis to show marine microbial diversity: development and application to a time series
Coupling 16S-ITS rDNA clone libraries and automated ribosomal intergenic spacer analysis to show marine microbial diversity: development and application to a time series
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DOI：
10.1111/j.1462-2920.2005.00835.x
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发表时间：
2005-09-01
影响因子：
5.1
通讯作者：
Fuhrman, JA
中科院分区：
文献类型：
--
作者：
Brown, MV;Schwalbach, MS;Fuhrman, JA
我们概述了一种方法，同时评估多层次的微生物多样性模式，利用16 S-ITS rDNA克隆库加上自动核糖体基因间间隔区分析（ARISA）。从512个克隆的序列数据允许估计与沿海海洋环境中的细菌相关的ARISA片段长度。我们将92%的ARISA峰（每个包含> 1%的总扩增产物）与来自克隆文库的相应长度匹配。这些具有推定鉴定的峰平均占总扩增群落DNA的83%。在16 S rDNA相似性<98%时，大多数分类群显示ARISA片段长度> 10 bp的差异，很容易检测到，表明ARISA分辨率接近“种”水平。来自ARISA的原绿球藻丰度谱与原绿球藻细胞计数强烈相关（r（2）= 0.86），表明ARISA数据与定义的分类内的实际细胞丰度大致成比例。42个月的ARISA谱分析阐明了微生物存在和丰度的模式，提供了对特定生物的群落变化和生态位的见解，包括原绿球藻属、γ变形菌门和放线菌门内分类群的生态模式耦合。进化枝特异性ARISA协议被开发用于SAR 11和海洋蓝细菌，以解决模糊的鉴定和进行重点研究。16 S-ITS数据可以通过ITS序列分析和微多样性检查高分辨率地鉴定生物体。
We outline an approach to simultaneously assess multilevel microbial diversity patterns utilizing 16S-ITS rDNA clone libraries coupled with automated ribosomal intergenic spacer analysis (ARISA). Sequence data from 512 clones allowed estimation of ARISA fragment lengths associated with bacteria in a coastal marine environment. We matched 92% of ARISA peaks (each comprising > 1% total amplified product) with corresponding lengths from clone libraries. These peaks with putative identification accounted for an average of 83% of total amplified community DNA. At 16S rDNA similarities < 98%, most taxa displayed differences in ARISA fragment lengths > 10 bp, readily detectable and suggesting ARISA resolution is near the 'species' level. Prochlorococcus abundance profiles from ARISA were strongly correlated (r (2) = 0.86) to Prochlorococcus cell counts, indicating ARISA data are roughly proportional to actual cell abundance within a defined taxon. Analysis of ARISA profiles for 42 months elucidated patterns of microbial presence and abundance providing insights into community shifts and ecological niches for specific organisms, including a coupling of ecological patterns for taxa within the Prochlorococcus, the Gamma Proteobacteria and Actinobacteria. Clade-specific ARISA protocols were developed for the SAR11 and marine cyanobacteria to resolve ambiguous identifications and to perform focused studies. 16S-ITS data allowed high-resolution identification of organisms by ITS sequence analysis, and examination of microdiversity.