ISSN 1022-7954, Russian Journal of Genetics, 2008, Vol. 44, No. 3, pp. 312–319. © Pleiades Publishing, Inc., 2008.
Original Russian Text © E.V. Maslova, 2008, published in Genetika, 2008, Vol. 44, No. 3, pp. 366–373.
includes two morphologically
results in the inconsistency in the recognition of the
herbarium specimens, since the taxonomists can not
come to a consensus on the diagnostic value of individ-
ual characters [1, 2]. Some authors either suggest not to
separate these species , or point to the difference of
the overlapping characters, which requires further
investigation . A possible hybrid origin of the polyp-
was suggested by Müntz-
ing . Detailed morphological analysis of hemp net-
tles with identiﬁcation of the additional diagnostic charac-
ters was performed by Slavikova and
Rehorekˇ [6, 7], who
noted their high variability, as well as to the difﬁculties
in the species recognition.
At present, such problems are solved using ISSR
(Inter Simple Sequence Repeats) and RAPD (Random
Ampliﬁed Polymorphic DNA) markers. The ISSR
markers are used for the plant genome analysis based
on the investigation of inter microsatellite sequences
. This method provides determination of the levels of
genomic polymorphism at the interspeciﬁc level and
taxonomic afﬁliation, as well as the establishment of
phylogenetic relationships . RAPD analysis with the
use of the primers with random DNA sequence is
widely used for analysis of intergeneric genetic differ-
entiation [10, 11].
The present study was focused on evaluating the
species independence of
comparing genetic differentiation with the morphologic
MATERIALS AND METHODS
Plant material and DNA extraction.
The hemp net-
tle accessions for the DNA analysis were obtained from
natural populations of Belgorod and Moscow oblasts.
The accessions were selected with special concern to
equal representation of
seven accessions). Since the latter species is rarer in the
oblasts examined, some of the accessions were taken
from the Herbarium of the Main Botanical Garden of
the Russian Academy of Sciences (MHA), from Mos-
cow oblast (Gt_Mosk_5 and Gt_Mosk_6), Kaluga
oblast (Gt_Kalug_4), and Pskov oblast (Gt_Psk_28).
The accessions used and their characteristics are pre-
sented in Table 1.
Plant DNA was puriﬁed using a Nucleospin Plant
Extraction Kit (Macherey-Nagel, Germany), according
to the protocol of the manufacturer. Prior to the extrac-
tion, 20 mg of plant tissues were placed in a mortar and
grounded to a powder with 0.3 g of Al
Polymerase chain reaction was
performed using ﬁve ISSR and ten RAPD primers. The
reactions were run in 0.5-ml Eppendorf tubes in 20
of a reaction mixture containing 20 ng DNA, 60 mM
Tris–HCl (pH 8.5), 25 mM KCl, 1.5 mM MgCl
10 mM 2-mercaptoethanol, 200
M of each dNTP,
10 pM of each primer, and 2.5 units
merase. The reaction mixtures were overlaid with 20
of mineral oil.
Ampliﬁcation was carried out in a Tertsik MS-11
thermal cycler (DNK-Tekhnologiya, Russia) using the
program with the change of the annealing temperature
for each of the primers: initial denaturing for 3 min at
Differentiation of Two Hemp Nettle Species
Inferred from Morphological Characters and DNA Markers
E. V. Maslova
Belgorod State University, Department of Botany, Belgorod, 308015 Russia; e-mail: firstname.lastname@example.org
Received July 12, 2006; in ﬁnal form, March 26, 2007
—Two species of the genus
Boenn. (family Lamiaceae), are
problematic to distinguish often wrongly recognized, and treated by some taxonomists as a single species. Mor-
phological diagnostical characters of these species are variable and partly overlap. Species independence of
was evaluated and their diagnostic characters veriﬁed using ISSR and RAPD markers.
A total of 57 ISSR and 28 RAPD fragments were obtained providing distinct subdivision of the accessions exam-
ined into two groups. Analysis of molecular data using the neighbor-joining method showed that the accessions
studied fell into two clades in the same way as demonstrated by the analysis of 20 morphological characters using
single linkage method. These results suggest that
are distinct species. The morphological
characters were found to be more variable compared to the molecular markers, although the combined of these
characters provided differentiation of the species.