1022-7954/01/3712- $25.00 © 2001
Russian Journal of Genetics, Vol. 37, No. 12, 2001, pp. 1448–1451. Translated from Genetika, Vol. 37, No. 12, 2001, pp. 1717–1720.
Original Russian Text Copyright © 2001 by Skurikhina, Kartavtsev, Chichvarkhin, Pan’kova.
In agreement with the latest taxonomic data, closely
Gould, 1850 and
Lamarck, 1819  occur in the
sublittoral of Peter the Great Bay of the Sea of Japan.
Although morphological criteria are available , iden-
tiﬁcation of mytilid species is difﬁcult even for experts
in morphology, in particular, due to the overlapping of
morphological characters of closely related species in
the same habitat .
The most promising approach to this problem is the
use of the polymerase chain reaction (PCR), which
allows one to analyze extremely low DNA amounts
typical of young bivalves [4, 5].
Recently, specimens of hybrids between
inhabiting the eastern coast of
the Japanese islands were revealed by PCR [6, 7].
In this work, we attempted to determine species
afﬁliation of closely related mussels inhabiting Peter
the Great Bay of the Sea of Japan and to identify hybrid
forms in their populations. The presence of hybrid
forms can be considered to be a strong argument for the
fact that a zone of hybridization between
exists in this region.
Gould, 1850 and
Lamarck, 1819 were collected in the
summer of 1999 (July–August). Of the analyzed 94
specimens, 67 were isolated at the collec-
tor of the Vostok Biological Station (Institute of Marine
Biology), Vostok Gulf (the eastern part of Peter the
Great Bay of the Sea of Japan); 15 were isolated in Pri-
boinaya bay of Vostok Gulf; and 12, on the Ostrovok
Fal’shivyi Peninsula (Posset Bay in the southwestern
part of Peter the Great Bay). All of the 49 specimens of
were collected from driftwood
found on Ostrovok Fal’shivyi. In addition, 12 speci-
collected in November
1999 on the southern coast of France (the town of Séte)
and 12 specimens of
from the North Sea
(Island Helgoland, Germany) were used as a control.
Species were identiﬁed according to diagnostic
morphological  and allozyme characters .
Total DNA was isolated either from the gills or from
the closing muscle ﬁxed in 95% ethyl alcohol. A piece
containing approximately 500 mg of tissue was homog-
enized and incubated for 1.5–2 h at 55
C in a buffer
solution of the following composition: 50 mM Tris–
HCl (pH 7.5), 10 mM EDTA, 0.5% SDS, and
200 mg/ml proteinase K. Chloroform deproteinization
was conducted, and DNA was precipitated with ethanol
and dissolved in TE (10 mM Tris–HCl and 1 mM
EDTA) [6, 10].
Ampliﬁcation was conducted in 25
l of the reaction
mixture containing 60 mM Tris–HCl (pH 8.5), 25 mM
KCl, 1.5 mM MgCl
, 10 mM 2-mercaptoethanol, 0.1%
Triton X-100, 2.5 mM of each of four deoxyribonucle-
otide triphosphates (dATP, dCTP, dGTP, and dTTP),
Study of Two Species of Mussels,
and Their Hybrids in Peter the Great Bay of the Sea of Japan
with the Use of PCR Markers
L. A. Skurikhina, Yu. F. Kartavtsev, A. Yu. Chichvarkhin, and M. V. Pan’kova
Institute of Marine Biology, Far East Division, Russian Academy of Sciences, Vladivostok, 690041 Russia;
fax: (4232) 31-09-00; e-mail: firstname.lastname@example.org
Received October 4, 2000; in ﬁnal form, February 19, 2001
—Bivalve mollusks of the genus
) occurring in Peter
the Great Bay of the Sea of Japan were ﬁrst studied in Russia. A region of nonrepetitive sequences of the gene
encoding the polyphenolic adhesive protein bissus was used as a species-speciﬁc genetic marker. After ampli-
ﬁcation using speciﬁc primers, a 126-bp fragment was found to amplify in all representatives of
collected from driftwood in the gulf Posset (the southwestern part of Peter the Great Bay).
specimens from the same region were shown to have a 168-bp fragment. In Vostok Gulf (the eastern part of
Peter the Great Bay), both artiﬁcially grown mussels and those from natural habitats contained a 168-bp frag-
ment or two fragments (126- and 168-bp) that corresponded to a hybrid form between the above species. The
possibility of using this genetic marker to identify closely related
strains and their hybrids in similar
habitats, near the Primorye coast in particular, was demonstrated. The presence of approximately 9% of hybrid
specimens conﬁrms that a zone of hybridization between
may exist in this