The dinoflagellate Alexandrium minutum is often associated with harmful algal blooms (HABs). This species consists of many strains that differ in their ability to produce toxins but have similar morphology, making ide...The dinoflagellate Alexandrium minutum is often associated with harmful algal blooms (HABs). This species consists of many strains that differ in their ability to produce toxins but have similar morphology, making identification difficult. In this study, species-specific rRNA probes were designed for whole-cell fluorescence in situ hybridization (FISH) to distinguish A. minutum from two phylogenetic clades. We acquired the complete SSU to LSU rDNA sequences (GenBank accession numbers JF906989-JF906999) of 11 Alexandrium strains and used these to design rRNA targeted oligonucleotide probes. Three ribotype-specific probes, M-GC-1, M-PC-2, and M-PC-3, were designed. The former is specific for the GC clade ("Global clade") of A. minutum, the majority of which have been found non-toxic, and the latter two are specific for the PSP (paralytic shellfish poisoning)-producing PC clade ("Pacific clade"). The specificity of these three probes was confirmed by FISH. All cells in observed fields of view were fluorescently labeled when probes and target species were incubated under optimized FISH conditions. However, the accessibility of rRNA molecules in ribosomes varied among the probe binding positions. Thus, there was variation in the distribution of positive signals in labeled cells within nucleolus and cytosol (M-GC-1, M-PC-3), or just nucleolus (M-PC-2). Our results provide a methodological basis for studying the biogeography and population dynamics of A. minutum, and providing an early warning of toxic HABs.展开更多
Prorocentrum donghaiense is a dinoflagellate that is widely distributed in the East China Sea and has become increasingly involved in Harmful Algal Blooms (HABs). Therefore, it is necessary to study this dinoflagellat...Prorocentrum donghaiense is a dinoflagellate that is widely distributed in the East China Sea and has become increasingly involved in Harmful Algal Blooms (HABs). Therefore, it is necessary to study this dinoflagellate to monitor HABs. In this study, 13 pairs of primers specific to P. donghaiense (within its internal transcribed spacer (ITS) regions) were designed for SYBR Green I real-time PCR. As the SYBR Green I real-time PCR could not identify P. donghaiense in a specific manner, a Taqman real-time PCR method was developed by designing a set of specific primers and a Taqman probe. A 10-fold serial dilution of recombinant plasmid containing ITS regions of P. donghaiense was prepared as standard samples and the standard curve was established. Additionally, we quantified the genomic DNA in P. donghaiense cells and utilized this DNA to prepare another 10-fold serial dilution of standard sample and accordingly set up the standard curve. The mathematic correlation between the cell number and its corresponding plasmid copy number was also established. In order to test the efficiency of the real-time PCR method, laboratory samples and P. donghaiense HAB field samples were employed for identification and quantitative analysis. As to laboratory samples, as few as 102 cells of P. donghaiense could be quantified precisely utilizing both centrifugation and filtration techniques. The quantification results from field samples by real-time PCR were highly similar to those by light microscopy. In conclusion, the real-time PCR could be applied to identify and quantify P. donghaiense in HABs.展开更多
基金Supported by the National Natural Science Foundation of China(No.41006082)the Postdoctoral Science Foundation(No.20090461272)the National Natural Science Foundation of China for Creative Research Groups(No.40821004)
文摘The dinoflagellate Alexandrium minutum is often associated with harmful algal blooms (HABs). This species consists of many strains that differ in their ability to produce toxins but have similar morphology, making identification difficult. In this study, species-specific rRNA probes were designed for whole-cell fluorescence in situ hybridization (FISH) to distinguish A. minutum from two phylogenetic clades. We acquired the complete SSU to LSU rDNA sequences (GenBank accession numbers JF906989-JF906999) of 11 Alexandrium strains and used these to design rRNA targeted oligonucleotide probes. Three ribotype-specific probes, M-GC-1, M-PC-2, and M-PC-3, were designed. The former is specific for the GC clade ("Global clade") of A. minutum, the majority of which have been found non-toxic, and the latter two are specific for the PSP (paralytic shellfish poisoning)-producing PC clade ("Pacific clade"). The specificity of these three probes was confirmed by FISH. All cells in observed fields of view were fluorescently labeled when probes and target species were incubated under optimized FISH conditions. However, the accessibility of rRNA molecules in ribosomes varied among the probe binding positions. Thus, there was variation in the distribution of positive signals in labeled cells within nucleolus and cytosol (M-GC-1, M-PC-3), or just nucleolus (M-PC-2). Our results provide a methodological basis for studying the biogeography and population dynamics of A. minutum, and providing an early warning of toxic HABs.
基金supported by the National Basic Research Program of China (973 Program) (No.2011CB403602)the National High Technology Research and Development Program of China (863 Program) (No.2007AA09200111)the National Marine Public Welfare Research Project (201205031-02)
文摘Prorocentrum donghaiense is a dinoflagellate that is widely distributed in the East China Sea and has become increasingly involved in Harmful Algal Blooms (HABs). Therefore, it is necessary to study this dinoflagellate to monitor HABs. In this study, 13 pairs of primers specific to P. donghaiense (within its internal transcribed spacer (ITS) regions) were designed for SYBR Green I real-time PCR. As the SYBR Green I real-time PCR could not identify P. donghaiense in a specific manner, a Taqman real-time PCR method was developed by designing a set of specific primers and a Taqman probe. A 10-fold serial dilution of recombinant plasmid containing ITS regions of P. donghaiense was prepared as standard samples and the standard curve was established. Additionally, we quantified the genomic DNA in P. donghaiense cells and utilized this DNA to prepare another 10-fold serial dilution of standard sample and accordingly set up the standard curve. The mathematic correlation between the cell number and its corresponding plasmid copy number was also established. In order to test the efficiency of the real-time PCR method, laboratory samples and P. donghaiense HAB field samples were employed for identification and quantitative analysis. As to laboratory samples, as few as 102 cells of P. donghaiense could be quantified precisely utilizing both centrifugation and filtration techniques. The quantification results from field samples by real-time PCR were highly similar to those by light microscopy. In conclusion, the real-time PCR could be applied to identify and quantify P. donghaiense in HABs.
