1022-7954/05/4103- © 2005 Pleiades Publishing, Inc.
Russian Journal of Genetics, Vol. 41, No. 3, 2005, pp. 262–268. Translated from Genetika, Vol. 41, No. 3, 2005, pp. 341–348.
Original Russian Text Copyright © 2005 by Koveza, Kokaeva, Konovalov, Gostimsky.
A variety of methods for obtaining molecular mark-
ers were helpful in solving a problem of insufﬁcient
number of morphological markers and in constructing
detailed genetic maps of various plant species [1–3 and
Molecular markers are also used for precise rapid
identiﬁcation and characterization of various plant spe-
cies and cultivars, for studying the phylogenetic rela-
tionship and somaclonal variation .
RAPD analysis, i.e., polymerase chain reaction with
a short arbitrary primer , is one of methods for
obtaining molecular markers.
In this work, RAPD analysis was conducted for
studying polymorphism and mapping of the genome of
L. It was of interest to obtain new
RAPD fragments, which can be used for identiﬁcation
and characterization of pea lines and cultivars. In addi-
tion, the detection of new markers and determination of
the relative positions of these and classical markers cre-
ates important prerequisites for more detailed genetic
mapping of pea chromosome. Thus, the aim of this
study was to search for new RAPD markers linked to
genes of two new pea chlorophyll mutations (
) affecting the synthesis of chlorophyll and the
functioning of photosynthetic apparatus , since this
is the ﬁrst step toward precise mapping and cloning of
MATERIALS AND METHODS
In this work, we used cultivars, lines, and mutants of
L. from a collection of the Depart-
ment of Genetics, Moscow State University.
Extraction of genomic DNA.
Extraction of genomic
DNA from plant leaves was conducted by the method
, with minor modiﬁcations.
Ampliﬁcation of pea DNA.
DNA ampliﬁcation with
RAPD primers was conducted in a reaction mixture
l) that contained 5
l DNA, 2.5 units of
merase (Sylex M, Russia), dATP, dGTP, dCTP, and
dTTP (each 0.2 mM), and RAPD primer at a concentra-
tion of 0.6
M. The reaction was run in 24.5
l of a 1
PCR buffer (DNA–Tekhnologiya) containing 3.1 mM
. To ensure the “hot start”, 2.5
l of liquid paraf-
ﬁn was used. Mineral oil (Sigma) (25
l) was layered
on top of the ampliﬁcation mixture.
DNA was ampliﬁed in an MC2 thermocycler
(DNA–Tekhnologiya). We used the PCR regime Precise.
The ampliﬁcation program consisted of the following
cycles: ﬁrst step, 94
C for 1 min 30 s; second step, 94
for 20 s;
for 5 s; 71
C, for 10 s, the reaction con-
sisted of 5 cycles; third step, 94
C for 1 s;
for 5 s;
C, for 10 s, the reaction consisted of 35 cycles (
34, 36, 37, 38, 40, and 42
Primers used to conduct RAPD analysis are listed in
Each isolated RAPD marker was given the name
consisting of two parts: the ﬁrst part corresponded to
the designation of the primer used in ampliﬁcation
detecting the given fragment; the second part of the
name indicated an approximate size in bp.
To verify the absence of contamination, we ran con-
trol reactions without the addition of genomic DNA in
each series of ampliﬁcations.
PCR products were separated by electrophoresis in
a 2.5% agarose gel stained with ethidium bromide.
Electrophoresis was conducted at 5 V/cm in a 1
Identification and Mapping of Polymorphic RAPD Markers
of Pea (
O. V. Koveza, Z. G. Kokaeva, F. A. Konovalov, and S. A. Gostimsky
Department of Genetics, Moscow State University, Moscow, 119899 Russia;
fax: (095) 939-43-09; e-mail: kovezaO@mail.ru
Received November 6, 2003; in ﬁnal form, July 28, 2004
—Various pea cultivars, lines, and mutants were studied by the RAPD method. Polymorphic frag-
ments characteristic of certain pea genotypes and which can be used for identifying genotypes were detected.
Inheritance of some polymorphic RAPD fragments was studied. Mendelian inheritance of these fragments was
shown. By analyzing the data obtained in studies of RAPD polymorphism, genetic distances between different
pea cultivars, lines, and mutants were calculated and a genealogic dendogram showing a varying extent of dif-
ferences between RAPD patterns was constructed. Ten new RAPD markers linked to various pea genes were
detected. Genetic distances between RAPD markers and genes to which they are linked were calculated, and
the respective disposition of RAPD markers on chromosomes was established.