| Analysis of phytogenies and relationships among phytoplankton using sequences of the ribosomal DNA (rDNA) sequences are important complements for the traditionally morphological identification, and can sometimes give researchers valuable results. On the other hand, studies on the quantitatively rapid detection using the divergent sequences of rDNA are one of the most common focuses on phytoplanktonic ecology.In this dissertation, sequences of rDNA from eleven important phytoplanktonic strains collected at Jiaozhou Bay are amplified, cloned, sequenced and compared, and the phylogenetic relationships among them are analyzed using the SSU rDNA sequences. It is found that sequences of rDNA from three Chaetoceros strains show high level of the total sequence similarities (all above 99%), and show close relationships. In contrast, sequences from two strains Gymnodinium are divergent (the sequence similarities of the small subunit (SSU) rDNA, the ITS1, the 5.8S rDNA, the ITS2 and the large subunit (LSU) rDNA are 76.5%, 25.4%, 54.2%, 23.2%, 56.5%, respectively), though their phylogenetic positions are relatively close.On the basis of the researches above, further studies are carried out as following:1. Development of a quantitative method for the rapid detection of Skeletonema costatum --Real-time fluorescent quantitative PCR (RFQ-PCR) .The divergent sequences of rDNA from S. costatum are used to design primers meeting the requirements of the RFQ-PCR. Seven pairs of primers are designed and designated as Primer 1 (F/R) ~ Primer 7 (F/R), respectively, among which primer 1 (F/R), primer 2 (F/R), primer 5 (F/R), primer 6 (F/R) showed high level of specificities to S. costatum. Then, the PCR products primed by primer6 (F/R) are sequenced. Sequences comparisons show that the sequence amplified from S. costatum is very different to that from other microalga. Thus, the sequence from S.costatum is used to design TaqMan 6 for RFQ-PCR. Experiments of nucleic acid hybridization show that TaqMan 6 only hybridizes to the PCR products amplified from S. costatum, but not to products from other microalga. These results indicate that Primer6 (F/R) and TaqMan6 can be used to establish the RFQ-PCR method for the quantitative detection of S. costatum.Thereafter, the RFQ-PCR method for the detection and enumeration of S. costatum cells is established with Primer6 (F/R) and TaqMan6. The regression curve for enumeration is delineated according to the development of the fluorescent densities in the RFQ-PCR with the increasing number of S. costatum cells. The regression equation is y = -3.3427x + 43.443, in which x indicates the log10 of cell number, and y indicates the CT values, with R2 of 0.9788. The standard estimated error of the CT values (Sy.x) is 0.6741, thus 95% of the accurate CT values are included in the range of y 1.3212. The range of the CT values that can be detected accurately is 20.17 1.3212 ~ 33.54 1.3212, corresponding to 106-9623 0.3952 ~ 102.9625 0.3952 of cells. This equation is proved to be credible by the RFQ-PCR using the samples whose cell number is known.2. The primary classification of a Gymnodinium sp. (GYN-15), whose species name was undetermined.The total rDNA sequence from GYN-15 is of 2658 bp length, spanning 1748 bp of the 3' end of the SSU rDNA, the complete sequence of the ITS1-5.8S rDNA-ITS2 region, and 336 bp of the 5' end of the LSU rDNA. The BLASTn results show that GYN-15 is closely related to a symbiont of anemones, S. californium, and the free-living strain, Gymnodinium varians. Sequence comparison show that the similarities among each part of the sequences from these three strains are all above 99%. Phylogenetic reconstruction with Neighbor-joining (NJ) method using sequences of variable regions (V1+V2+V3) of SSU rDNA indicated that GYN-15, S. californium and G. varians form a new clade with 100% bootstrap support. Based on these results, it is believed that GYN-15 may belong to the genus Symbiodinium. In addition, the branch formed by GYN-15 and other two Symbiodinium strains...
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