J. Phycol.
35, 884–888 (1999)
RIBOSOMAL RNA HETEROGENEITY AND IDENTIFICATION OF TOXIC DINOFLAGELLATE
CULTURES BY HETERODUPLEX MOBILITY ASSAY 1
Paulina Uribe C.2, Benjamı́n A. Suárez-Isla
Laboratorio de Toxinas Marinas, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Casilla 70005,
Santiago 6530499, Chile
and
Romilio T. Espejo
Laboratorio de Bioingenierı́a, Instituto de Nutrición y Tecnologı́a de los Alimentos, Universidad de Chile, Macul 5540, Santiago, Chile
The heteroduplex mobility assay (HMA) reveals
sequence dissimilarity between DNA by measuring
the retarded migration of the hybrid or heterodu-
plex using polyacrylamide gel electrophoresis. Het-
erogeneity in some cultures of toxic dinoflagellates
of the genus Alexandrium (Halim) Balech was ob-
served during comparison of the amplified D1–D2
region of the large subunit rRNA gene (rDNA) using
this method. HMA also allowed grouping of clones
obtained from toxic bloom events in the Chilean,
southernmost Pacific within the Asian Southern Pa-
cific lineage of A. catenella (Whedon et Kofoid) Bal-
ech. The applied methodology provides a rapid and
simple tool for use in assessing heterogeneity as well
as for molecular grouping of strains among the ge-
nus Alexandrium.
Key index words: Alexandrium; heteroduplex mobil-
ity assay; large subunit; Pacific; PCR; rDNA
Abbreviations: HMA, heteroduplex mobility assay;
LSU, large subunit; PSP, paralytic shellfish poison-
ing; SSU, small subunit
Marine dinoflagellates of the genus Alexandrium
(Halim) Balech inspire special economic and public
health concern, because they include a number of
species that produce saxitoxin and its derivatives,
highly toxic compounds that cause paralytic shell-
fish poisoning (PSP)(Taylor 1984, 1993). Toxic spe-
cies of the genus Alexandrium are widespread in
many regions of the world (Shumway 1990, Halle-
graeff 1995, Richardson 1997), and their rapid and
unequivocal identification has become an important
aim in toxic dinoflagellate research. An increasing
effort has been made to differentiate between or-
ganisms at species and strain levels using morpho-
logical and subcellular criteria (Oshima et al. 1982,
Cembella et al. 1987, Hayhome et al. 1989, Sako et
al. 1990, 1993). However, members of A. catenella/
tamarense/fundyense (the ‘‘tamarensis complex’’),
which include most toxic species, are not distin-
1Received 17 June 1998. Accepted 23 April 1999.
2Author for reprint requests; e-mail: puribe@machi.med.
uchile.cl.
884
guished as three distinct groups using the same cri-
teria (Scholin et al. 1994a). Based on nuclear-en-
coded, small- and large-subunit ribosomal RNA
(SSU and LSU rRNA) gene sequences, a number of
Alexandrium species are recognizable as distinct
groups, as defined morphologically (Destombe et al.
1992, Scholin and Anderson 1993, Scholin et al.
1994a, Adachi et al. 1994, 1996, Zardoya et al. 1995).
By the same genetic criteria, the tamarensis complex
is divided into at least five geographically distinct
lineages. These lineages do not reflect the devel-
opment of new morphotypes but rather reveal a
monophyletic radiation and independent evolution
of geographically isolated regional populations
(Scholin et al. 1995). Isolates of A. catenella that are
indistinguishable by morphological criteria can be
genetically grouped in different lineages that cor-
respond to their geographical origin (Scholin et al.
1994b).
Sequence heterogeneity in cultures of Alexan-
drium isolates has previously been observed in LSU
rDNA (Scholin et al. 1994, Zardoya et al. 1995, Ada-
chi et al. 1996), and this may hinder straightforward
identification by sequence comparison. Ribotypes
and subribotypes of Alexandrium have been defined
on the basis of the most prevalent class of LSU mol-
ecule cloned from the organisms and also on the
basis of unique combinations of heterogeneous clas-
ses of rDNA (Scholin et al. 1994b). RFLP analysis of
the whole PCR products more recently employed to
assess the heterogeneity among members of the
North American and temperate Asian ribotypes has
suggested that the heterogeneity is larger than pre-
viously observed through cloning and sequencing
(Scholin and Anderson 1996).
The first documented toxic bloom in Chile was
reported in 1972 in Magallanes (Guzmán and Cam-
podónico 1975). Since then, the dominant toxic di-
noflagellate species in Chilean southern coastal ar-
eas and estuaries during toxic bloom events has
been identified by morphological criteria to be A.
catenella (Guzmán and Campodónico 1978). How-
ever, the association of these strains with one of the
different geographical ribotypes requires sequence
comparison of the LSU rDNA.
 DINOFLAGELLATE GENOTYPING BY HMA
Here we report the assessment of intraculture se-
quence heterogeneity of the D1–D2 region of the
LSU rDNA in strains of Alexandrium species complex
using the heteroduplex mobility assay (HMA). This
assay is based on the decreased electrophoretic mi-
gration, caused by the formation of heteroduplexes,
of PCR-amplified D1–D2 regions of the rDNA in
polyacrylamide gels (Delwart et al. 1993). By using
this technique to perform sequence comparisons,
we also determined the genetic association between
the four Chilean isolates obtained from blooms that
occurred in Aysén in 1994 and in Magallanes in
1997 and the Asian Southern Pacific A. catenella ri-
botype.
MATERIALS AND METHODS
Cultures and strains. Clonal cultures of A. catenella ACC07 and
ACC01 from Aysén (458329 S, 738349 W) and MACBN8 and
MACBB1 from Magallanes (538559 S, 678099 W) were obtained
from the laboratories of the Instituto de Fomento Pesquero,
Puerto Montt, and the Universidad de Magallanes, respectively.
A. catenella strain OF101, A. tamarense strains PEIV, and A. fun-
dyense strain GtCA29 and A. lusitanicum GtPort were kindly pro-
vided by Dave Kulis from the Woods Hole Oceanographic Insti-
tution, Woods Hole, MA. Cultures were grown in f/2 medium at
158 C under a 16:8 h LD cycle (Guillard 1975, 1995).
DNA Extraction. DNA was extracted by a modification of the
method described by Adachi et al. (1994). Approximately 106 ex-
ponential phase cultured cells were harvested by centrifugation
for 10 min at 5000 3 g. Cell pellets were washed with f/2 medium,
submerged in N2, powdered with a mortar and pestle, and mixed
with three volumes of extraction buffer (1.0% Sarkosyl, 0.25%
sucrose, 50 mM NaCl, 20 mM EDTA, 50 mM Tris-HCl [pH 8.0],
and 10 mM 2-mercaptoethanol). After incubation at 658 C for 30
min, the solution was extracted with phenol/chloroform (Sam-
brook et al. 1989). The aqueous phase was treated with proteinase
K (100 mg·mL21; Sigma Chemical Co., St. Louis, MO) for 1 h at
378 C and was precipitated with 2-isopropanol. The precipitated
DNA was dissolved in 50 mM Tris-HCl (pH 8.0) and 10 mM
EDTA, and was subsequently incubated with RNAse A (100
mg·mL21; Sigma) at 378 C for 1 h. After a second extraction with
phenol/chloroform, the DNA was precipitated twice with ethanol
and resuspended in buffer TE (10 mM Tris-HCl [pH 8.0] and 1
mM EDTA).
PCR Amplification. Amplification was performed by a modification
of the method described by Zardoya et al. (1995). DNA was am-
plified in 50 mM KCl, 10 mM Tris-HCl [pH 8.0], 2.0 mM MgCl2,
0.1% Triton X-100, and deoxynucleoside triphosphate 0.2 mM,
using primers corresponding to the D1–D2 region of the LSU of
Prorocentrum micans (Prorocentroides, Dinophyceae, Phyrrophy-
ta)(Forward 59-CCCGCTGAATTTAAGCATATAAGTAAGCGG-39
at position 23; Reverse 59-GTTAGACTCCCTTGGTCCGTGTTT-
CAAGA-39 at position 741) at a concentration of 0.25 pmol·mL21
and with Taq polymerase (Promega) 0.08 U·mL21. Amplification
was performed with 35 cycles of the following steps: 948 C for 1
min, 608 C for 1 min, 728 C for 1 min, with a final cycle at 728 C
for 5 min. A HYBAID Omm-E thermal cycler (Hybaid Limited,
Teddington, Middlesex, United Kingdom) was used.
Heteroduplex analysis. Heteroduplexes were obtained by dena-
turation and renaturation of the mixed amplification products
from different dinoflagellate strains, performed using the follow-
ing procedure: Pairs of amplified D1–D2 from LSU rDNA were
mixed at a concentration of about 5 ng·mL21 in the renaturation
buffer described by Delwart et al. (1993)(0.1 M NaCl, 10 mM Tris-
HCl [pH 7.8], and 2 mM EDTA), denatured at 988 C for 7 min,
and subsequently renatured at 608 C for 40 min. Denaturation
and renaturation for analysis of single PCR amplification products
were performed in the same buffer used for amplification, follow-
ing the addition of EDTA to a final concentration of 4 mM, using
the same temperatures and times. Electrophoresis was performed
885
FIG. 1. (a) Polyacrylamide gel electrophoresis after amplifi-
cation of D1–D2 LSU rDNA of different Alexandrium strains, lane
L1; A. lusitanicum GtPort, C7; A. catenella ACC07, T1; A. tamarense
PEIV, M; 100-bp ladder (Promega). (b) Heteroduplex nature of
polymorphism observed in amplification product cultures of A.
catenella OF101 and A. fundyense GtCA29. C1, F1, and L1 show the
products of A. catenella, A. fundyense, and A. lusitanicum, respec-
tively. Lane A, amplified products; B, products obtained after a
single amplification cycle of samples in A performed after dilu-
tion to avoid annealing (five times the volume applied for elec-
trophoresis in A was used to compensate for the dilution per-
formed, assuming that the product was doubled after the ampli-
fication cycle); C, products obtained after denaturation and re-
naturation of the samples shown in B.
in 7% polyacrylamide gels (7 cm long 3 8 cm wide 3 0.15 cm
thick), with Tris-borate buffer (Sambrook et al. 1989), at 150 V
for 1 h 45 min. DNA was visualized with silver nitrate staining
(Espejo and Escanilla 1993). Sequence similarities among D1–D2
regions were calculated using those reported by Scholin et al.
(1995), which were aligned with the SSEARCH program (Smith
and Waterman 1981, Pearson 1991).
RESULTS AND DISCUSSION
Sequence heterogeneity among Alexandrium isolates. A
single product was observed after amplification of
the LSU rDNA D1–D2 region of DNA obtained
from the cultures of A. lusitanicum 6t Port, A. cate-
nella ACC07, and A. tamarense PEIV (Fig. 1a). The
lower relative migration of A. lusitanicum corre-
sponded with the shorter sequence reported for this
species (Scholin et al. 1994b, Scholin and Anderson
1996). In contrast, the product of both A. catenella
OF101 and A. fundyense GtCA29 contained addition-
al electrophoretic bands. As shown below, these
bands consisted of heteroduplexes formed during
the amplification reaction. When the amplification
products differ in nucleotide sequence, they may
form heteroduplexes by annealing of the single
strands when the temperature is lowered from the
denaturation to the annealing step (Jensen and
Straus 1993). The heteroduplex nature of these
bands was established by subjecting the product to
886 PAULINA URIBE ET AL.
an additional amplification cycle after appropriate
dilution in order to avoid annealing of the single
strands (Delwart et al. 1993). Under these condi-
tions, heteroduplexes disappeared, but they reap-
peared when this last product was subjected to de-
naturation and renaturation. Figure 1b shows the
results obtained after subjecting the heterogeneous
amplification products of A. catenella OF101 and A.
fundyense GtCA29 to these treatments. The homo-
geneous product of A. lusitanicum was included as a
control.
Sequence heterogeneity in cultures of Alexan-
drium isolates was observed previously in LSU rDNA
(Scholin et al. 1994b, Zardoya et al. 1995, Adachi et
al. 1996). It is likely that in strains with heteroge-
neous sequences, ribotypes and subribotypes have
been defined on the basis of the most prevalent
clone of the LSU molecule (Scholin and Anderson
1996). RFLP analysis of the whole PCR products was
employed in order to assess this heterogeneity in
members of the North American and temperate
Asian ribotypes. These results suggested that the het-
erogeneity is larger than previously observed
through cloning and sequencing (Scholin and An-
derson 1996). However, potential heteroduplexes
formed during PCR amplification, as shown here,
may generate nonannealed restriction sites that
would be resistant to the restriction enzyme and that
would generate additional bands that would suggest
a larger polymorphism.
HMA analysis of reference strain sequences and HMA
comparison of Chilean clones. Isolates from southern
Chilean coastal areas were identified by comparison
of the nucleotide sequences of the D1–D2 region of
their LSU rRNA with those of other Alexandrium
strains, including A. catenella. The sequence similar-
ity was assessed by the HMA of the amplified prod-
ucts of this region. Heteroduplexes were obtained
by denaturation and renaturation of the amplified
regions from different strains mixed in similar con-
centrations and were subsequently resolved by elec-
trophoresis in polyacrylamide gels (Fig. 2). The rel-
ative migration of the heteroduplex formed be-
tween strains of known nucleotide sequence was
roughly proportional to their sequence similarity.
Heteroduplexes between different strains (indicated
by dots) were usually resolved into two bands, which
probably correspond to the reciprocal hybrids
formed by each plus and minus complementary
strand of the amplified D1–D2 region. Although the
extent of dissimilarity in those hybrid pairs is iden-
tical, single-stranded regions in each hybrid may
form distinctive structural conformations, which
may result in variability in their mobility (Jensen and
Straus 1993). One of the Chilean isolates, A. catenella
ACC07, behaved identically to A. catenella OF101
and did not form a heteroduplex with this strain;
the faint band observed just above the homoduplex
corresponds to the heteroduplex formed after self-
annealing of the product in A. catenella OF101, as
FIG. 2. Polyacrylamide gel electrophoresis of the products ob-
tained after denaturation and renaturation of amplified D1–D2
LSU rDNA from different strains of the genus Alexandrium, in-
cluding the Chilean isolate A. catenella ACC07. The results ob-
tained with the amplification products of different strains are
shown in the following lanes (their sequence similarity is provided
between parentheses, when known). L1/T1, A. lusitanicum/A. ta-
marense PEIV (80.3%); L1/C7, A. lusitanicum/A. catenella ACC07;
L1/C1, A. lusitanicum/A. catenella OF101 (82.3%); T1/C7, A. ta-
marense PEIV/A. catenella ACC07; T1/C1, A. tamarense PEIV/A. ca-
tenella OF101 (90.2%); and C1/C7, A. catenella OF101/A. catenella
ACC07. The results obtained with the single strains A. catenella
OF101, A. catenella ACC07, A. tamarense PEIV, and A. lusitanicum
GtPort are shown in lanes C1, C7, T1, and L1, respectively. Het-
eroduplexes are indicated by dots. The darker bands above the
heteroduplexes correspond to single strands.
shown in Figure 1b and in lane C1 of Figure 2. Com-
parison of the other three Chilean clones using the
same procedure provided identical results (data not
shown). These observations indicate that the four
Chilean clones isolated to date belong to the Asian-
Pacific ribotype group of A. catenella.
Alexandrium fundyense was analyzed independently
because of its large intraculture sequence dissimilar-
ity in the D1–D2 region of the LSU rRNA. This high
intraculture dissimilarity caused the formation of
several heteroduplexes, which were clearly observed
when annealed with A. lusitanicum, which is 21.4%
dissimilar (Fig. 3, lane F1/L1). However, when it was
annealed with A. catenella OF101, which is 12.9% dis-
similar, the heteroduplexes migrated close to those
observed after self reannealing of A. fundyense, and
only a change of the relative intensity pattern was
noticeable (lane F1/C1). This change is probably
due to the heteroduplexes formed between F1 and
C1 or C7. The expected migration for these hetero-
duplexes is difficult to estimate, because the actual
differences in nucleotide sequence among the dif-
ferent LSU rRNAs in A. fundyense are unknown. The
dissimilarities indicated above were calculated from
the reported sequence for A. fundyense (Scholin et
al. 1995), which should correspond to only one of
the different LSU rRNA observed in this culture.
These LSU rRNA with dissimilar sequences could
show either greater or lesser similarity with the LSU
rRNA of other strains. According to the relative mi-
gration of the heteroduplexes formed by self-an-
nealing of A. fundyense, the intraculture dissimilarity
 DINOFLAGELLATE GENOTYPING BY HMA
FIG. 3. Polyacrylamide gel electrophoresis of the products ob-
tained after denaturation and renaturation of D1–D2 LSU rDNA
amplification product of A. fundyense alone (F1) or when com-
bined with those of A. lusitanicum GtPort (F1/L1), A. catenella
AC007 (F1/C7), or A. catenella OF101 (F1/C1). M, 100-bp ladder
(Promega). The darker bands above the heteroduplexes corre-
spond to single strands. Those heteroduplexes that could be un-
equivocally identified as having been formed by the interculture
dissimilarity are labeled with dots. Those formed by intraculture
dissimilarity are labeled with asterisks.
could be as high as 9.8%, that calculated between
A. tamarense PEIV/A. catenella OF101, which form
heteroduplexes of similar migration; this high intra-
culture dissimilarity causes the formation of several
heteroduplexes, with relative migrations impossible
to predict or to interpret (Fig. 3).
The HMA methodology allows for the detection
of fine-scale intraculture heterogeneity, in terms of
both sequence and length, determined directly
from the PCR products. HMA also seems to be a
simple technique to use to distinguish and group
dinoflagellate species of the tamarensis complex
group, without the use of cloning and sequencing.
According to the HMA, the amplified D1–D2 do-
mains of the LSU RNA gene (rDNA) of four A. ca-
tenella clones isolated in Chilean southern coasts—
ACC01, ACC07, MACBN8, and MACBB1—contain
the same nucleotide sequence present in A. catenella
strain OF101. This sequence is common in Alexan-
drium strains from the ‘‘Japanese temperate Asian’’
lineage (Scholin et al. 1995), and it is different from
those of other strains, morphologically defined as A.
catenella, that have been isolated from Northern Pa-
cific waters. Genetic identification of the species of
dinoflagellate involved in PSP intoxication events in
Chile constitutes a significant step in the develop-
ment of molecular probes for the characterization
of harmful algae of the southernmost Pacific waters.
HMA for the detection of different strains of bac-
887
teria in natural populations has been previously dis-
cussed (Espejo and Romero 1997), and it could be
applied to harmful algal blooms as well.
The authors thank Dr. Miriam Seguel, from the Instituto de Fom-
ento Pesquero, Mr. J. C. Uribe, from the Universidad de Magal-
lanes, and Dr. Jacob Larsen and Mr. Dave Kulis, who provided
clonal cultures for this study. This study was funded by grants
FONDEF-Conicyt 2-37 and Fondecyt 1990765.
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