ISSN 1022-7954, Russian Journal of Genetics, 2009, Vol. 45, No. 5, pp. 571–574. © Pleiades Publishing, Inc., 2009.
Original Russian Text © S.V. Boronnikova, 2009, published in Genetika, 2009, Vol. 45, No. 5, pp. 652–655.
Conservation and mobilization of the gene pools of
rare plant species require preserving their morphologi-
cal, functional, and adaptive diversity as well as the
richness of intraspecies variation at both the intrapopu-
lation and interpopulation levels. The populations display-
ing the most pronounced variation of the traits are of the
highest interest as carriers of genetic information .
(L.) DC., ladybell lilyleaf
(family Campanulaceae Juss.), is a rare decorative and
medicinal plant of the Ural region. This species is
ascribed to category 3 (R) of the regional list of the
plants requiring preservation . In Perm region,
is at the edge of its range. Over the last 15 years,
the range of this plant in Perm region reduced by 25%
due to agricultural activities on the territory.
The main goal of this work was to perform a molec-
ular genetic analysis of the DNA polymorphism in four
populations of Perm region aiming to deter-
mine the level of their genetic diversity, one of the cri-
teria for assessing the state of populations.
population (P1) grew in a grass pin-
ery on the territory of the historical and natural monument,
the mountain Podkamennaya; the second (P2), in a birch
woodland located 2 km to the north of the village of Ena-
paevo; the third (P3), in a mixed forest 2 km to the north-
west of the village of Ust’-Turka; and the fourth (P4), in a
coniferous–deciduous forest on the territory of the park
Kyz’minka, Il’insky raion, which is a historical and natu-
ral reserve . Two of the populations selected for analy-
sis (P2 and P3) represent the northernmost unique region
of the Kungur forest steppe and are located at a distance of
63 km from one another; the fourth population is located
155 km to the north of the ﬁrst population.
In each population, leaves were harvested from
30 randomly selected plants growing at a distance of
30–50 m from one another. DNA was isolated accord-
ing to Torres et al.  with minor modiﬁcations. Poly-
morphism was analyzed in 720 DNA samples by poly-
merase chain reaction (PCR) using ISSR (Inter Simple
Sequence Repeats), or intermicrosatellite, markers .
The reaction mixture for PCR (25
l) contained 2 U of
polymerase (Sileks M), 2.5
l of standard 10
buffer (Sileks M), 25 pM primer, 2.5 mM
0.25 mM dNTP.
DNA was ampliﬁed in a
Tertsik (DNK-Tekhnologiya, Moscow) thermocycler
using the ISSR primers synthesized at the closed com-
panies Sintol and Evrogen (Moscow) according to the
following program: denaturation for 2 min at
5 cycles of 20 s at
10 s at the annealing tempera-
ture; and 10 s at
; 35 cycles of 5 sequence at
5 s at the annealing temperature, and 5 s at
ﬁnal elongation for 2 min at
. Depending on the
G/C composition, the annealing temperature varied
from 46 to
. The experiment was performed at
least in duplicate to test the reliability of DNA patterns.
As a negative control (C
) for testing the purity of
reagents, the reaction mixture was supplemented with
l of deionized water instead of DNA. The ampliﬁca-
tion products were separated by electrophoresis in
1.7% agarose gel in 1
TBE buffer, stained with ethid-
ium bromide, and photographed in transmitted ultravi-
olet light. The lengths of DNA fragments were deter-
mined with the help of a 100 bp + 1.5 + 3 kb DNA Lad-
der (OOO-SibEnzim-M, Moscow) marker using the
Quantity One program in a Gel Doc XR (Bio-Rad,
United States) gel documentation system.
For quantiﬁcation of the degrees of DNA polymor-
phism and divergence between the studied populations,
the data were represented as a matrix of binary traits.
Cluster analysis was performed by unweighted pair
group method with arithmetic averages (UPGMA). The
dendrogram reﬂecting the degree of similarity between
the studied populations according to ISSR patterns was
Genetic Variation in Ural Populations of the Rare Plant Species
(L.) DC. on the Basis of Analysis
of Polymorphism of ISSR Markers
S. V. Boronnikova
Department of Botany and Plant Genetics, Perm State University, Perm, 614990 Russia;
Received June 25, 2008
—The genetic variation in four populations of
(L.) DC., a rare plant species of
the Perm region, was analyzed using 56 ISSR markers. The characteristics of DNA polymorphism and popula-
tion genetic diversity were determined. These data demonstrate a high level of DNA polymorphism
= 82.14%). The studied
populations are weakly differentiated; the intrapopulation variation is
the main contributor to the genetic variation.