Molecular genetic tools are widely used to learn more about the identical characterization of obscure microalgal strains. At the Korea Marine Microalgae Culture Center (KMMCC), the authors deduced the genetic relationship of 41 strains of the genus
Tetraselmis
by analysing a small subunit ribosomal DNA (18S rDNA) sequences. Forty-one strains were seperated into five groups, which showed over a 98-99% similarity to
Tetraselmis striata
or
Tetraselmis
sp. Tsbre. Also, 13 strains among them had an identical genotype to
Tetraselmis striata
while 5 strains had with
Tetraselmis
sp. Tsbre, respectively. The mean size of each strain generally showed the tendency of different variation according to the groups.
INTRODUCTION
Species of
Tetraselmis
in Prasinophyceae are well known as the basic food organisms in aquaculture (Kim and Hur 1998; Park and Hur 2000; Cabrera
et al
. 2005), as an important source for antioxidative substances in pharmacological studies (Laguna
et al
. 1993; Kim
et al
. 2002), and for their inportance in marine ecotoxicological testing (Park
et al
. 2005).
Since the first description of
Tetraselmis
by F. Stein in 1878 (Norris
et al
. 1980), many taxonomical studies on this green motile unicellular algae have been reported. The genus of
Tetraselmis
, first formalized according to the Christensen Criterion (Christensen 1962) has been identified by not only characteristics such as scale, flagellar hair, and basal bodies (Moestrup and Throndsen 1988; Marin and Melkonian 1994; Throndsen 1997), but also by types of pigments (Egeland
et al
. 1997; Latasa
et al
. 2004).
Although, by using light and electron microscope, morphological classification is possible at the species level, taxonomic decisions within the
Tetraselmis
are difficult to make because of the complex process of cellular characterization.
Genetic data based upon the amplification and sequencing of genes are also accumulated to use as a powerful tools for analysis of diverse microalgae. Thus, it is necessary to collect molecular data on
Tetraselmis
to determine whether they are identical strains or not. However, it is still difficult to find molecular studies on
Tetraselmis
.
In this study, 18S rDNA sequence of 41 strains of
Tetraselmis
was analysed to discriminate the genomic variations. The strains were constructed as a phylogenetic tree by comparing them with other known sequences from the National Center for Biotechnology Information (NCBI) database. These variations were also compared with the differences in the mean size of each strain of
Tetraselmis
.
MATERIALS AND METHODS
- Microalgae culture condition
Forty-one strains of
Tetraselmis
were received from the Korea Marine Microalgae Culture Center (KMMCC) (Hur 2008). Information about the strains is given in
Table 1
. The strains were grown in 100 mL of f/2 culture medium (Guillard and Ryther 1962) at 22℃ with continuous light with 60 μmol photons m
-2
s
-1
for 10 days.
The Mean length of 30 specimens of the
Tetraselmis
strain was measured using a light microscope (×400). Significant differences in mean size of the strains were analysed using ANOVA and Ducan tests (SPSS software version 10.1; SPSS Inc., Chicago, IL, USA) at the level of 5%.
Culture history of forty-one strains ofTetraselmisand their GenBank accession numbers for the 18S rDNA sequences
Culture history of forty-one strains of Tetraselmis and their GenBank accession numbers for the 18S rDNA sequences
- Genomic DNA extraction and polymerase chain reactions
The LiCl method was used to extract the genomic DNA of the
Tetraselmis
(Hong
et al
. 1995). The quality and quantity of the extracted genomic DNA were measured by electrophoresis (Mupid
TM
; Advance, Tokyo, Japan) and spectrometry (NanoDrop
®
ND-1000; NanoDrop Technologies, Wilmington, DE, USA), respectively.
Polymerase chain reactions (PCR) (Mullis and Faloona 1987) performed using 10-100 ng of genomic DNA as a template and 0.5 μM of degenerated primers
(Table 2)
derived from a conserved region of 10 species of other known microalgae, listed in
Table 3
. Amplification conditions consisted of one cycle of denaturation at 95°C for 5 min, 30-35 cycles of denaturation at 95°C for 30 s, annealing at 50-55°C for 30 s and extension at 72°C for 1 min 45 s, followed by the final extension at 72°C for 7 min, and then stored at 4°C. About a 1.7 Kb size of PCR product was confirmed at 1% gel electrophoresis and extracted using the AccuPrep
®
Gel Purification Kit (Bioneer, Daejeon, Korea). Purified PCR products were ligated with the pGEM
®
-T Easy Vector Systems (Promega, San Luis Obisco, CA, USA) and then were transformed into the
Eschrichia colil
XL-1 blue. The recombinant plasmids were purified using the AccuPrep
®
Plasmid Extraction Kit (Bioneer, Korea). Selected clones were confirmed by colony PCR and EcoR restriction enzyme digestion (Fermentas, Burlington, Ontario, Canada) followed by DNA sequencing (Genotech, Daejeon, Korea).
List of sequences of oligonucleotides used for the polymerase chain reactions
List of sequences of oligonucleotides used for the polymerase chain reactions
The strains used for the construction of a phylogenetic tree and their GenBank accession numbers of 18S rDNA sequences
The strains used for the construction of a phylogenetic tree and their GenBank accession numbers of 18S rDNA sequences
- Sequence alignments and phylogenetic analysis
The identities of the acquired partial 18S rDNA sequence from
Tetraselmis
strains were confirmed by using the Blast N program (
http://blast.ncbi.nlm.nih. gov
) and were aligned with the other known microalgae 18S rDNA sequences using ClustalW2 (Thompson
et al
. 1994). The accession numbers of the microalgae sequence found in the NCBI GenBank are given in
Table 3
.
To confirm the phylogenetic relationships, a total of 1615 positions in the final dataset were subjected to neighbor-joining (NJ) and maximum-parsimony (MP) analysis using the MEGA v.4.0 (MEGA, Tempe, AZ, USA) (Tamura
et al
. 2007). The evolutionary distance method to construct the NJ tree was calculated with the Kimura 2-parameter (Kimura 1980). The rate variation among sites was modeled with a gamma distribution (0.25-parameter) and all positions containing gaps and missing data were eliminated from the dataset. The MP tree was obtained using the close-neighbor-interchange algorithm (Nei and Kumar 2000) in which the initial trees were obtained with the random additional 100 replications of sequences. All positions containing gaps and missing data were eliminated from the dataset. The reliability of both trees was tested by 2000 replication bootstraps. The 18S rDNA sequence of
Chlorella vulgaris
was used as the outgroup sequence.
RESULTS AND DISCUSSION
To test their generic similarity, the almost complete 18S rDNA sequences were determined for the 36 strains collected from Korean coastal water and 5 strains from foreign areas. Their accession numbers registered at GenBank are given in
Table 1
and the lengths of sequence were varied on the sense and antisense primer used in gene amplification
(Table 2)
. Forty-one strains showed high levels of identity (98-99%) to the correspoding regions of known 18S rDNA sequences such as
Tetraselmis
striata PLY 443 or
Tetraselmis
sp. Tsbre using the Blast N program. No differences were found between 13 strains (KMMCC P-5, 11, 18, 22, 24, 27, 32, 35, 36, 37, 43, 56 and 58: group A) and
Tetraselmis striata
PLY 443 or among 5 strains (KMMCC P-2, 3, 8, 9 and 47: group C) and
Tetraselmis
sp. Tsbre in their acquired rDNA sequences. Thirteen sequence positions were different between group A and C
(Fig. 1)
. Four strains in group C were collected in May and June from the southern coast of Korea, and 13 strains in group A were gathered from diverse Korean coastal waters during all four seasons. In terms of the algal length, those of group A ranged from 8.75 μm to 13.75 μm were shorter than those of group C, which ranged from 12.5 μm to 17.5 μm.
The sequences of the other 35 strains were seperated into 3 groups; B, D and E. Of these, fourteen strains belonged to group B, which was very close to group A. Only 1-2 sequence positions differentiated between group A and B, excepting KMMCC P-57. Group D contained seven strains which were close to group C but the level of similarity between groups C and D was less so than between groups A and B. Different sequence positions of six strains of group D were 1-4 base pairs compared with group C, whereas KMMCC P-20 was the furtherest from it with 14 sequence differences. The different sequence position of KMMCC P-6 and P-12 in group E showed only one base pair. But group E differed slightly from both of group A and group C. It had 17 different sequence positions in KMMCC P-6 or 18 differences in KMMCC P-12 compare with group A. Eighteen or 19 base pairs differed between group C and KMMCC P-6 or P-12, respectively.
Variable positions of 18S rDNA sequence from multiple strains of the genus Tetraselmis with five different groups (A-E). Dot corresponds to nucleotide of group A which has identical sequence with Tetraselmis striata and different sequence indicates nucleotide. Dash denotes a missing nucleotide.
Mean size in length of each Tetraselmis strain. Forty-one strains were divided into 5 groups (A-E). Data is expressed as mean ± SD. Bars with dissimilar superscripts indicate different significance at the level of 5%.
Comparing the algal size with the 18S rDNA sequence analysis in each group, the relationship exhibited more clearly in the algal length than in the width (data not shown). The distribution of mean length is divided into; groups A and B, and groups C and D. The length of the strains from group B was similar to that of group A,
Tetraselmis striata
, but KMMCC P-21, P-48 and P-62 were larger than the others
(Fig. 2.)
. These three strains were closer to group C than group A. Although KMMCC P-6 and P-12 were considered as the same group E at the point of the gene sequence, their size was significantly different. Among the strains, KMMCC P-6 was the largest with 14.25 μm, and P-12 was the smallest with 7.42 μm. This result means that relationships between 18S rDNA sequences and the size of the strains do not always coincide.
To analyze the relationship of 18S rDNA sequences among the strains of
Tetraselmis
, a phylogenetic tree was constructed using the NJ and MP methods with 2000 bootstrap values
(Fig. 3)
. KMMCC P-36 and P-2 were selected as representative strains from identical sequences of groups A and C, respectively. Most strains were contained within two clades although separation was supported with less than 50%; 38% and 43% bootstrap values from NJ and MP, respectively. Groups A and B were clustered with
Tetraselmis striata
and T. convolutae excepting the branch order for KMMCC P-31 and P- 57 strains from the clade with a 72% bootstrap value in NJ tree. They had two and five different sequence positions respectively. Other clades were groups C and D, which were comprised of
Tetraselmis chuii
and
Tetraselmis
sp. Tsbre. Group E, comprised of KMMCC P-6 and P-12 was separated from clades of groups C and D with 54% and 50% bootstrap support in NJ and MP tree, respectively. This corresponded with the results on different sequence positions.
Tetraselmis
currently contains about 30 species identified by morphological inspection from public microalgal culture collections (e.g., UTEX, CCMP, CCAP). However, only four species:
Tetraselmis striata
(Steinkotter
et al
. 1994),
T. convolutae
(de Jesus
et al
. 1995),
T. kochiensis
and
T. chuii
reported an 18S rDNA sequence. Other strains have not yet been identified as to the level of species.
Phylogenetic tree using the maximum parsimonymethod inferred from 18S ribosomal DNA sequences of thegenus Tetraselmis. P-36 and P-2 are indicated as a representativestrain for group A and group C, which have identicalsequences, respectively. Tree reliability is tested by 2000replications of bootstraps, which indicate numbers at thenodes from neighbor-joining (NJ) (left) and maximum-parsimony(MP) (right) analysis. Chlorella vulgaris is indicatedfor out group.
One of the objectives of this research on their rDNA sequences was to identify strains of
Tetraselmis
among culture collections. Variation of life-history characteristics such as the flagellate phase, non-motile phase and cyst stage of
Tetraselmis
depends on environmental factors (Norris
et al
. 1980). In our experience, morphological discernment of
Tetraselmis
under a light microscope was often confused with the genera Colacium and Chloromonas. Therefore, analysis of the 18S rDNA sequence is evaluated as a simple tool for distinguishing the genus
Tetraselmis
. In this study, 13 strains were identical with
Tetraselmis striata
PLY 443 and 5 strains with
Tetraselmis
sp. Tsbre. This was more than 43% of the total strains examined. Grouping of
Tetraselmis
strains based on the result of the 18S rDNA sequence showed a partially corresponding tendency with the mean size variation of the strains. To understand the characterization and discrimination among various strains of
Tetraselmis
, further detailed molecular studies on the ITS regions or rbc L gene reflecting higher evolutionary rate, which was used in the genus Nannochloropsis (Suda
et al
. 2002) and DNA hybridization analysis or amplified fragment length polymorphism used in
Chlorella vulgaris
(Despres
et al
. 2003; Muller
et al
. 2005), are needed.
Acknowledgements
This work was carried out with the financial support of the Fisheries Research and Development Program (R20800308H010000110) at the Ministry of Land, Transport and Maritime Affairs, and a grant (No. R21- 2005-000-10022-0) from the National Research Resource Bank Program of the Korea Science & Engineering Foundation at the Ministry of Science & Technology. The authors thank J. H. Bae and J. Y. Youn of Korea Marine Microalgae Culture Center for providing cultures of the microalgae.
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