1022-7954/05/4111- © 2005 Pleiades Publishing, Inc.
Russian Journal of Genetics, Vol. 41, No. 11, 2005, pp. 1254–1261. Translated from Genetika, Vol. 41, No. 11, 2005, pp. 1522–1530.
Original Russian Text Copyright © 2005 by Koveza, Gostimsky.
Molecular markers have been widely employed in
recent years for research in various ﬁelds of genetics
. The most rapid and simple method for identifying
molecular markers is polymerase chain reaction (PCR).
In particular, RAPD (random ampliﬁed polymorphic
DNA) analysis, i.e., PCR with a short arbitrary primer
, is one of highly effective methods for developing
molecular markers. This simpler and less expensive
method allowed rapid detection of many markers scat-
tered throughout the genome. However, RAPD analysis
leads to the identiﬁcation of anonymous genome
regions whose nature has not been characterized well.
With the aim of identifying particular genomic
sequences, SCAR markers (sequence characterized
ampliﬁed region) were developed . The conversion
from RAPD to SCAR markers is accomplished by
cloning and sequencing of RAPD products. Results of
sequence analysis showed that two more extended
SCAR primers (usually 20–25 nucleotides in size), cor-
responding to the ends of the RAPD fragment and, as a
rule, containing the original RAPD primers, are synthe-
sized. SCAR primers with high speciﬁcity and repro-
ducibility generally tend to amplify one band rather
than the whole spectrum of fragments, as in the case of
RAPD method, and allow the identiﬁcation of particu-
lar DNA regions [3, 4].
This motivated a great deal of interest in using
RAPD markers that characterize particular pea lines
and cultivars or are linked to various mutations of the
pea genome, to develop SCAR markers. The search for
such RAPD markers was described in our previous
work . Such approach can be used not only for geno-
typing various pea lines and cultivars but also for iden-
tifying certain genome sequences adjacent to the most
interesting and physiologically signiﬁcant genes, which
is an important step toward the cloning these genes and
our understanding of pea genome structure.
MATERIALS AND METHODS
In this work, we used cultivars, lines, and mutants
L. from a collection of the
Department of Genetics, Moscow State University.
Extraction of genomic DNA and ampliﬁcation.
cedure for DNA extraction, conditions for conducting
RAPD analysis, and RAPD primers used in this work
are described in .
DNA ampliﬁcation with SCAR primers was con-
ducted in a reaction mixture (30
l) containing 5
DNA, 2.5 units of
polymerase (Sylex M), dATP,
dGTP, dCTP, and dTTP (each 0.2 mM), and SCAR
primers (each at a concentration of 0.3
M). The reac-
tion was run in 24.5
l of a 1
PCR buffer (DNA-Tech-
nologia Research and Production Company) containing
3.1 mM MgCl
. To ensure a “hot start,” 25
l of liq-
uid parafﬁn was used. Mineral oil (Sigma), amount-
ing to 25
l, was layered on top of the ampliﬁcation
DNA was ampliﬁed in an MC2 thermocycler (DNA-
Technologia) using the Precise PCR regime. The ampli-
ﬁcation program consisted of the following cycles: ﬁrst
C for 2 min; second step, 94
C for 10 s; t
for 5 s; 72
C, for 1 min, the reaction consisted of
5 cycles; third step, 93
C for 1 s; t
for 1 s; 72
1 min, the reaction consisted of
Development and Study of SCAR Markers
in Pea (
O. V. Koveza and S. A. Gostimsky
Moscow State University, Department of Genetics, Moscow, 119899 Russia;
fax: (095) 939-43-09; e-mail: kovezaO@mail.ru
Received December 22, 2004
—In order to develop more speciﬁc markers that characterize particular regions of the pea genome,
the data on nucleotide sequences of RAPD fragments were used for choosing more extended primers, which
may be helpful in amplifying a fragment corresponding to the particular DNA region. Of the 14 STS markers
obtained from 14 polymorphic RAPD fragments, 12 were polymorphic, i.e., they are SCAR markers that can
be used in genetic analysis. The transition from complex RAPD spectra to ampliﬁcation of a particular SCAR
marker substantially facilitates analysis of large samples for the presence or absence of the examined fragment.
Inheritance of the developed SCAR markers was studied in F
. SCAR markers were used to identify
various pea lines, cultivars, and mutants. It was established that the study of ampliﬁcation of STS markers in
various pea genotypes at varying temperatures of annealing and the comparison with ampliﬁcation of the orig-
inal RAPD fragments in the same genotypes provide an approach for analysis of RAPD polymorphism origin.