1022-7954/01/3704- $25.00 © 2001
Russian Journal of Genetics, Vol. 37, No. 4, 2001, pp. 464–466. Translated from Genetika, Vol. 37, No. 4, 2001, pp. 574–576.
Original Russian Text Copyright © 2001 by Koveza, Kokaeva, Gostimsky, Petrova, Osipova.
RAPD (Randomly Ampliﬁed Polymorphic DNA)
procedure is one of the most widely used methods of
plant polymorphism analysis and search for molecular
markers. This method is based on the polymerase chain
reaction (PCR) with short arbitrary primers [1–3].
Wide application of the procedure is mainly con-
nected with its simplicity and rapid performance. In
addition, RAPD analysis may be effectively used for
high-resolution gene mapping in plants and rapid detec-
tion of markers linked to the genes of interest [4–7].
However, the RAPD procedure has several disad-
vantages. One of the main limitations of the method is
high sensitivity to reaction conditions that reduces
reproducibility of the results obtained in different labo-
SCAR markers (Sequence Characterized Ampliﬁed
Region) [5, 8, 9] derived from the polymorphic RAPD
markers lack most of their limitations and may be suc-
cessfully used in various studies.
The goal of this work was to create SCAR markers
in pea. Different lines and cultivars of
from the collection of the Department of Genetics and
Breeding (Moscow State University) were used.
Among them, six cultivars (Nord, Rannii zelenyi,
Raport, Pioner, Kapital, and Filbi), two translocation
lines (L-111 and L-25), one marker line (L-1238), and
seven pea mutants (R-64, Chl-42, Chl-7, Chl-13, Chl-18,
W-2, and Khameleon) were employed.
To reveal RAPD markers, DNA polymorphism in
these lines and cultivars was analyzed with the B-474
Fourteen microliters of the PCR buffer (DNK-Tekh-
nologiya NPF) and 1
l of mixture A were applied to a
0.5-ml ampliﬁcation tube, then 25
l of liquid parafﬁn
were added. After solidiﬁcation of parafﬁn, 10.5
the PCR buffer, 2.5 units of
l of pea DNA were added. To prevent evapora-
tion of the ampliﬁcation mixture, 25
of mineral oil
(Sigma) were added to the reaction mixture.
Mixture A contained: dATP, dGTP, dCTP, dTTP
(6 mM each), and 18.7 pM primer.
Ampliﬁcation was carried out in a MS2 thermocy-
cler (DNK-Tekhnologiya) in a Precise regime as fol-
First step: 1 min 30 s at 94
C; second step: 20 s at
C; 5 s at 42
C; 10 s at 71
C (5 cycles), and third step:
1 s at 94
C; 5 s at 42
C; 10 s at 71
C (35 cycles).
The ampliﬁcation products were separated accord-
ing to molecular weights by a standard electrophoretic
procedure in Tris-borate buffer. In line L-111 and the
Nord cultivar, a 750-bp fragment missing from other
lines and cultivars was discovered (Fig. 1).
To evaluate the reproducibility of results, different
plants of the Nord cultivar and line L-111 were ana-
lyzed using RAPD. Both the cultivar and line were
monomorphic. DNA obtained from all plants of the
Nord cultivar and line L-111 produced similar electro-
phoretic patterns of the ampliﬁcation products and pos-
sessed the 750-bp fragment that indicated reproducibil-
ity of the results obtained by RAPD analysis.
The polymorphic RAPD fragment of line L-111 was
excised, eluted from the agarose gel using
Gel Extraction Kit (QIAGEN), and cloned into the
pGEM-T vector (Promega).
The terminal regions of the cloned fragment were
sequenced. On the basis of this sequence, primers
(primer 1 and primer 2) of 23 bp in length were selected
using the OLIGO computer program and synthesized to
obtain a SCAR marker.
Primer 1: 5'-CGCCAAGAACCAATCAAGACAGC-3'
Primer 2: 5'-GGGCATAGGGATGACCATCAGAC-3'
All lines and cultivars, which were preliminarily
screened with the B-474 primer, and the pGEM-T vec-
tor containing the cloned polymorphic fragment of line
L-111 were tested with these SCAR primers.
For ampliﬁcation with speciﬁc 23-bp primers, 14
of the PCR buffer (DNK-Tekhnologiya) and 1.24
mixture A were added to a 0.5-ml tube. Then 25
Creation of a SCAR Marker in Pea
Using RAPD Analysis
O. V. Koveza
, Z. G. Kokaeva
, S. A. Gostimsky
, T. V. Petrova
, and E. S. Osipova
Department of Genetic and Breeding, Moscow State University, Moscow, 119899 Russia; fax: (095) 939-31-81
Timiryazev Institute of Plant Physiology, Russian Academy of Sciences, Moscow, 127276 Russia; fax: (095) 977-94-45
Received November 21, 2000
—A polymorphic 750-bp fragment, RAPD marker, speciﬁc to particular pea genotypes (line L-111
and the Nord cultivar) was identiﬁed. Using this RAPD marker, SCAR was obtained. SCAR inheritance in the
ﬁrst and second generations was studied and its dominant character was shown.