Mapping of picoeucaryotes in marine ecosystems with quantitative PCR of the 18S rRNA gene

被引:490
作者
Zhu, F
Massana, R
Not, F
Marie, D
Vaulot, D
机构
[1] CNRS, Biol Stn, UMR 7127, INSU, F-29680 Roscoff, France
[2] Univ Paris 06, F-29680 Roscoff, France
[3] CSIC, Inst Ciencias Mar, Dept Biol Marina & Oceanog, E-08003 Barcelona, Spain
关键词
coastal ecosystems ecology; fluorescent in situ hybridization; picoplankton; prasinophytes; quantitative PCR;
D O I
10.1016/j.femsec.2004.10.006
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
A quantitative PCR (QPCR) assay based on the use of SYBR Green I was developed to assess the abundance of specific groups of picoeukaryotes in marine waters. Six primer sets were designed targeting four different taxonomic levels: domain (Eukaryota), division (Chlorophyta), order (Mamiellales) and genus (Bathycoccus, Micromonas, and Ostreococcus). Reaction conditions were optimized for each primer set which was validated in silico, on agarose gels, and by QPCR against a variety of target and non-target cultures. The approach was tested by estimating gene copy numbers for Micromonas, Bathycoccus, and Ostreococcus in seawater samples to which cultured cells were added in various concentrations. QPCR was then used to determine that rRNA gene (rDNA) copy number varied from one to more than 12,000 in 18 strains of phytoplankton. Finally, QPCR was applied to environmental samples from a Mediterranean Sea coastal site and the results were compared to those obtained by Fluorescent in situ hybridization (FISH). The data obtained demonstrate that Chlorophyta and more specifically Mamiellales were important in these waters, especially during the winter picoplankton bloom. The timing of major abundance peaks of the targeted species was similar by QPCR and FISH. When used in conjunction with other techniques such as FISH or gene clone libraries, QPCR appears as very promising to quickly obtain data on the ecological distribution of important phytoplankton groups. Data interpretation must take into account primer specificity and the varying rRNA gene copy number among eukaryotes. (c) 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
引用
收藏
页码:79 / 92
页数:14
相关论文
共 53 条
[1]   Complete genome sequence of the apicomplexan, Cryptosporidium parvum [J].
Abrahamsen, MS ;
Templeton, TJ ;
Enomoto, S ;
Abrahante, JE ;
Zhu, G ;
Lancto, CA ;
Deng, MQ ;
Liu, C ;
Widmer, G ;
Tzipori, S ;
Buck, GA ;
Xu, P ;
Bankier, AT ;
Dear, PH ;
Konfortov, BA ;
Spriggs, HF ;
Iyer, L ;
Anantharaman, V ;
Aravind, L ;
Kapur, V .
SCIENCE, 2004, 304 (5669) :441-445
[2]   Divergence and redundancy of 16S rRNA sequences in genomes with multiple rrn operons [J].
Acinas, SG ;
Marcelino, LA ;
Klepac-Ceraj, V ;
Polz, MF .
JOURNAL OF BACTERIOLOGY, 2004, 186 (09) :2629-2635
[3]   Detection of the toxic dinoflagellate Alexandrium fundyense (Dinophyceae) with oligonucleotide and antibody probes:: Variability in labeling intensity with physiological condition [J].
Anderson, DM ;
Kulis, DM ;
Keafer, BA ;
Berdalet, E .
JOURNAL OF PHYCOLOGY, 1999, 35 (04) :870-883
[4]   Quantitative tracing, by Taq nuclease assays, of a Synechococcus ecotype in a highly diversified natural population [J].
Becker, S ;
Fahrbach, M ;
Böger, P ;
Ernst, A .
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 2002, 68 (09) :4486-4494
[5]   Quantitative assessment of picoeukaryotes in the natural environment by using taxon-specific oligonucleotide probes in association with tyramide signal amplification-fluorescence in situ hybridization and flow cytometry [J].
Biegala, IC ;
Not, F ;
Vaulot, D ;
Simon, N .
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 2003, 69 (09) :5519-5529
[6]  
Binder BJ, 1998, APPL ENVIRON MICROB, V64, P3346
[7]   FLOW CYTOMETRIC DETERMINATION OF PHYTOPLANKTON DNA IN CULTURES AND OCEANIC POPULATIONS [J].
BOUCHER, N ;
VAULOT, D ;
PARTENSKY, F .
MARINE ECOLOGY PROGRESS SERIES, 1991, 71 (01) :75-84
[8]   Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays [J].
Bustin, SA .
JOURNAL OF MOLECULAR ENDOCRINOLOGY, 2000, 25 (02) :169-193
[10]   Real-time PCR analysis of Vibrio vulnificus from oysters [J].
Campbell, MS ;
Wright, AC .
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 2003, 69 (12) :7137-7144