[논문]Phylogenetic Analysis of Phyllospadix iwatensis Based on Nucleotide Sequences Encoding 18S rRNA and ITS-1

Kim, Jong-Myoung; Choi, Chang-Geun

doi:10.5657/fas.2010.13.4.272

[국내논문] Phylogenetic Analysis of Phyllospadix iwatensis Based on Nucleotide Sequences Encoding 18S rRNA and ITS-1 원문보기

Fisheries and aquatic sciences, v.13 no.4, 2010년, pp.272 - 277

Kim, Jong-Myoung (Dept. of Marine Biomaterials and Aquaculture, Pukyong National University) , Choi, Chang-Geun (Dept. of Ecological Engineering, Pukyong National University)

Abstract ▼ AI-Helper

Seagrasses are marine angiosperms of ecological importance in providing shelter and food to aquatic species as well as maintaining the carbon cycle on earth. Phyllospadix iwatensis is a seagrass of the family Zosteraceae and is distributed along the eastern coast of Korea. The nucleotide sequences of P. iwatensis nuclear genes encoding 18S ribosomal RNA (rRNA) and internal transcribed spacer-1 (ITS-1) were determined for molecular phylogenetic analysis. Genomic DNA was isolated from P. iwatensis and used for PCR amplification of 18S rRNA and ITS-1. Examination of the 18S rRNA sequence of P. iwatensis showed a close (99% similarity) relationship to Zostera noltii, another genus of Zosteraceae, but a distant (84% similarity) evolutionary relationship to other macroalgal Laminariales species. Further discrepancies found in ITS-1 nucleotide sequences between closely related species indicate that the sequence information could be used for species identification.

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제안 방법

, 1994), although its use has sometimes been known to be hindered by discrepancies due to the presence of duplicate copies. To carry out a molecular-marker- based phylogenetic analysis of P. iwatensis, DNA regions encoding 18S rRNA and its neighboring ITS sequences were analyzed for the first time in this study.

대상 데이터

Restriction endonucleases were purchased from Bioneer (Daejeon, Korea). Kits used for plasmid purification, gel extraction, and PCR purification were purchased from NucleoGen (Seoul, Korea).

이론/모형

A phylogenetic tree was constructed using the neighbor-joining method in MEGA v 4.0 (Tamura et al., 2007). An assessment of tree reliability was conducted using a bootstrap method with 1000 replicates.
, 2007). An assessment of tree reliability was conducted using a bootstrap method with 1000 replicates.
, 2007). In this study, genomic DNA was isolated from ground Phyllospadix using the STE and CTAB buffer method (Valera-Alvarez, 2006). The quality of the isolated genomic DNA was confirmed by the presence of high-molecular-weight genomic DNA upon agarose gel electrophoresis (data not shown).

성능/효과

, 1999). Amplification of the regions corresponding to 18S rRNA and ITS-1 results in the synthesis of approximately 1.8 kb and 300 bp, respectively, fragments which are similar to the sizes of the corresponding regions in other macroalgal species including Ecklonia cava and Costaria costata (data not shown). Amplified fragments were purified from agarose gel, cloned into a pGEM-T vector, and then transformed into E.
Nucleotide sequences were obtained from at least two independent clones in which the sequences were confirmed by analysis from both directions. Analysis of P. iwatensis 18s rRNA using BlastN indicated the highest similarity to the18s rRNA of other seagrasses including Zostera noltii and Posidonia oceanica. P.
iwatensis 18s rRNA using BlastN indicated the highest similarity to the18s rRNA of other seagrasses including Zostera noltii and Posidonia oceanica. P. iwatensis 18S rRNA showed a 99% similarity to that of Z. noltii, another genus in Zosteraceae, and 98% similarity to that of P. oceanica, belonging to the family Posidoniaceae in the same order. Comparison of the 18s rRNA sequences of Agarum clathratum Dumortier and Costaria costata belonging to the same family, Laminariaceae, and Ecklonia cava Kjellman of the Alariaceae in the same order, Laminariales, showed almost completely identical 1.
oceanica, belonging to the family Posidoniaceae in the same order. Comparison of the 18s rRNA sequences of Agarum clathratum Dumortier and Costaria costata belonging to the same family, Laminariaceae, and Ecklonia cava Kjellman of the Alariaceae in the same order, Laminariales, showed almost completely identical 1.8 kb sequences, with divergence at only one position (data not shown). In contrast, the 18s rRNA sequence of P.
To further examine the phylogenetic differences among closely related species, DNA sequences encoding P. iwatensis ITS-1 with high variability were determined and compared with those of other members of the family Zosteraceae (Fig. 2). In contrast to the relatively highly conserved 18s rRNA sequences, the ITS-1 fragments showed higher sequence variability in the region, as shown by an 87% similarity to that of P.
2). In contrast to the relatively highly conserved 18s rRNA sequences, the ITS-1 fragments showed higher sequence variability in the region, as shown by an 87% similarity to that of P. torreyi and 70% similarity to that of Z. noltii. This provides useful information, not only for the detailed evolutionary analysis of P.
4) compared with those of other macroalgae and seagrasses. Species belonging to the family Laminales were hardly distinguished by their almost identical 18srRNA sequences, as shown by Costaria costata, Agarum clathratum, members of the family Laminariaceae, and even Ecklonia cava, belonging to the neighboring family, Alariaceae, in the same order, Laminariales (Fig. 3). However, P.
This was followed by Posidonia oceanica in the same order, Alismatales, and Potamogeton pusillus, a closely related but freshwater aquatic. These results were consistent with previous morphology-based classifi-cations and indicated that 18S rRNA can be used to analyze the evolutionary relationships of Phyllospadix. The results also indicated that seagrasses are phylogenetically closer to the species in Chlorophytae (Ulva pertusa) than to the species in Divisions Rhodophyta and Phaeophyta.
These results were consistent with previous morphology-based classifi-cations and indicated that 18S rRNA can be used to analyze the evolutionary relationships of Phyllospadix. The results also indicated that seagrasses are phylogenetically closer to the species in Chlorophytae (Ulva pertusa) than to the species in Divisions Rhodophyta and Phaeophyta.
4). The results showed the closest relationship to another member in the genus, P. torreyi, followed by Z. noltii, another member of the same family. In particular, the evolutionary relationships among members of the order Laminariales (E.
costata), which were indistinguishable using 18S rRNA markers, were clearly differentiated using ITS- 1 comparison. In addition, the evolutionary position of P. iwatensis was further distinguished from P. torreyi, in the same genus, and Zostera noltii, in the same family, showing a clear distinction between P. iwatensis and other seagrasses.

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