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M. Smolik, D. Jadczak, A. Główczyk
68
Assessment of morphological and genetic variability in chosen Nepeta
accessions
Miłosz Smolik1*, Dorota Jadczak2, Aleksandra Główczyk1
Department of Horticultural Plant Breeding
Department of Vegetable Crops
Agricultural University
Janosika 8
71-424 Szczecin, Poland
1
2
*corresponding author: phone: +4891 4220851 ext. 356, fax: +4891 4232417,
e-mail: [email protected]
Summary
The aim of this study was to determine the morphological and genetic variability of
four Nepeta genotypes: N. cataria var. citriodora, N. × faassenii, N. mussinii, and N. nuda
from the Collection of Medical Plants in Dołuje, Vegetable Faculty, Agricultural University
of Szczecin, Poland. Morphological variation among the examined catnip forms was
determined using biometric measurements which included the plants height, the width of
a leaf pair, the length and width of a leaf blade, the length of an inflorescence, the weights
of fresh and dried green parts and the essential oils content. The DNA polymorphism within
microsatellite sequences was determined using the ISSR-PCR technique. The conducted
biometric measurements proved that the examined Nepeta plants differed significantly
in all analysed features. Twenty microsatellite primers were used in ISSR reactions; clear
products were generated in reactions with six of them. In general, 72 loci were amplified
(110 amplicons); five of them (7%) turned out to be monomorphic, 20 (28%) polymorphic
and 47 (65%) were specific for the examined accessions. Unique loci were amplified for each
examined Nepeta genotype. A phylogenetic tree was constructed on the basis of a UPGMA
cluster algorithm. It consisted of one similarity group ‘a’, in which N. × faassenii, N. mussinii
and N. nuda were included.
Key words: Nepeta sp., phenetic variability, genetic variability, ISSR markers
Assessment of morphological and genetic variability in chosen Nepeta accessions
69
Introduction
The Nepeta (catnip) genus includes over 200 species of perrenial plants and
some annuals [1]. They contain, depending on the species, up to 1% of essential
oil as well as nepetalactone, iridoid, bitter principles, tannins and minerals. Nepeta
essential oil is mainly composed of citral, citronellal, geraniol, carvacol, nepetol,
thymol, pulegon, actinidine and monoterpene alkaloid [2-5].
The active substances of catnip plants have been widely used in medicine, food
industry and perfumery. They are also used in folk medicine as a remedy for bronchitis, flu, cold and other illnesses. They have sudorfic properties, reduce fever
and help treat stomach-aches [1-3, 5].
In order to produce new, interesting garden plants, many species of catnip
have been crossbred repeatedly. One of the outcomes of these experiments is,
for example, the hybrid Nepeta × faassenii, which has been produced by crossbreeding N. mussinii × N. nepetella.
The aim of the breeding processes is not only creating plants of beautiful look,
but also producing larger amounts of biologically active compounds.
Molecular biology techniques are widely used in such actions. They allow precise characterization of parental components used in crossbreeding (genus or variety), which are interesting with respect to some of their particular properties.
Thanks to this, the selection of plants with particular properties in the breeding
process is much easier.
Along these lines, the objective of this study is to determine the phenotypic
and genotypic variability of four species from the Nepeta genus by means of the
ISSR-PCR technique. Specifically, we identify genotype-specific ISSR loci and determine the range of variability among them.
MATERIALS AND METHODS
The research material consisted of four species of catnip: Nepeta cataria var.
citriodora, Nepeta × faassenii, Nepeta mussinii and Nepeta nuda. All plants came from
the collection of the Vegetable Faculty, Agricultural University of Szczecin. Phenotypic variability of the examined species of catnip was determined in a field
experiment conducted in the Vegetable Research Station in Dołuje. Genotypic
variability was determined by means of the ISSR-PCR technique in the Department
of horticular Plant Breeding, Agricultural University of Szczecin.
Phenotypic variability
The research material consisted of herb cuttings collected from mother plants
in April 2006 and included 24 pieces of each genus. The cuttings were rooted in
Vol. 54 No 4 2008
70
a seedling tray with 4 cm-diameter cells. In May 2006, ten plants from each genus
were planted in a field at 30 x 40 cm spacing. During their growth, the plants were
fertilized twice with ammonium saltpetre (10 g˙m-2). Nurturing included loosening the soil between rows of plants, removing weeds and watering. Biometric
measurements of the plants were made during the vegetation period (every 12–14
days), determining their height (cm), the width of a leaf pair (cm), the length and
width of a leaf blade (cm) and the length of inflorescences (cm). The herbs were picked during full blooming (August 2006). Sprouts were separated from leaves and
inflorescences, their weights were determined, and essential oils were extracted.
Genotypic variability
The total DNA from about 100 mg of fresh leaf material was extracted using the
A&A Biotechnology kit (DNA PrepPlus). The ISSR-PCR mixtures (25 µl) contained:
1.5 mM MgCl2, 100 mM KCl, 20 mM Tris-HCl pH 8.3, 0.1% Triton X-100, 0.2 µM
ISSR primer, 0.2 mM of each dNTP, 1.0 units of Taq DNA polymerase (Fermentas MBI) and 50 ng of template genomic DNA. The DNA was amplified using a
Mastecycler (Eppendorf) thermal cycler [6]. The amplified products were analyzed
and photographed (Polaroid DS-34) by electrophoresis on a 1.5 % agarose gel and
visualized with ethidium bromide (0.5 µg · ml-1) on an UV-21 transilluminator
(Fotodyne). O’RangeRuler 200bp DNA Ladder (Fermentas MBI) was used as a size
marker (3000–200 bp). Each fragment amplified with use of ISSR primers (UBC
– University of British Columbia primer sets) was coded in a binary form by ‘0’ and
‘1’ for absence or presence in each species, respectively. In order to infer phylogenetic relationships, the 0/1 matrix was used to calculate genetic similarity and
then employed to construct an unweighted pair-group method with arithmetic
means - UPGMA – dendrogram using Diversity one 1.3 (Pharmacia LKB) software
packages [7, 8]. The robustness of the tree topology was assessed by 1,000 bootstrap resamplings [8, 9].
RESULTS
Assessment of morphological variability
The experiment quantified selected morphological features of the plants, i.e.,
their height, the length of inflorescences and three biometric parameters of leaves: the width of a leaf pair, the length of a leaf blade and the width of individual leaves (fig. 1). Analysis of biometric measurements showed that N. citriodora
plants were the tallest (64.5 cm) and that N. mussinii were the shortest (27.3 cm).
They also formed the largest and the smallest leaves, respectively. Compared to
N. citriodora and N. mussinii, N. × faassenii and N. nuda, plants had average heights
71
and leaf parameters (fig. 1). It was also found that N. nuda plants did not differ
significantly from N. citriodora in any of features measured. However, both of them
produced the longest inflorescences (14.4 cm), whereas the shortest were produced by N. mussinii (fig. 1).
80
70
60
50
40
30
20
10
0
1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4
18
16
14
12
10
8
6
4
2
0
19.05.06 02.06.06 14.06.06 29.06.06 12.07.06 27.07.06 08.08.06 16.08.06
1 - N. citriodora, 2 - N. x faassenii, 3 - N. nuda, 4 - N. mussinii
Mmin
max
aaverage
7
6
5
4
3
2
1
0
1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4
19.05.06 02.06.06 14.06.06 29.06.06 12.07.06 27.07.06 08.08.06 16.08.06
Mmin
max
aaverage
1
2
6
5
4
3
2
1
0
1 2 3 4 12 3 4 12 3 4 12 3 4 12 3 4 12 3 4 12 3 4 1 2 3 4
19.05.0602.06.06 14.06.0629.06.0612.07.0627.07.0608.08.0616.08.06
Mmin
max
aaverage
3
1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4
19.05.06 02.06.06 14.06.06 29.06.06 12.07.06 27.07.06 08.08.06 16.08.06
Mmin
max
aaverage
4
1 - The average height of the longest sprout
2 - The average width of the twain leaves
3 - The average length of the blade leaves
4 - The average width of the leaves
5 - The average length of the inflorescences
25
20
15
10
5
0
1 2 3 4
29.06.06
1 2 3 4
12.07.06
1 2 3 4
27.07.06
1 2 3 4
08.08.06
1 2 3 4
18.08.06
Mmin
max
aaverage
5
Figure 1. Growth dynamics and the averages for sprout length, width of a leaf pair, and the length and
Figure
1. width
Growth
dynamics and the averages for sprout length, width of a leaf pair, and the length
of leaves as determined for species from the Nepeta genus
and width of leaves as determined for species from the Nepeta genus
It was found that neither leaf parameters nor the plants height influenced their
fresh mass production. N. × faassenii were observed to have the largest amount
of fresh mass (217.8 g), whereas N. mussinii had the smallest (119.2 g) (fig. 2). The
analysis of the share of individual parts of the plants in whole fresh mass proved
that the share of leaves, sprouts and inflorescences differed depending on the
examined plant. The results of the measurements are shown in fig. 2.
By weighing the green parts and collected dried material it was determined
that N. × faassenii had the largest combined amount of green and dried material by
weight, whereas N. mussinii had the smallest. The largest amount of dried material
was obtained from N. citriodora while the smallest amount was obtained from N.
× faassenii (fig. 2).
Vol. 54 No 4 2008
72
1
3
250
200
150
100
50
0
Fresh herb
Nepeta
citriodora
20.15
20.10
20.05
20.00
19.95
19.90
19.85
19.80
19.75
Nepeta
citriodora
Leaf
Nepeta
x faassenii
Nepeta
x faassenii
Shoot
Nepeta
anuda
Nepeta
anuda
Inflorescence
Nepeta
mussinii
Nepeta
mussinii
2
4
120
100
80
60
40
20
0
Nepeta
citriodora
Fresh green parts
2.00
1.50
0.50
0
Nepeta
citriodora
Nepeta
x faassenii
Nepeta
x faassenii
Nepeta
nuda
Dried material
Nepeta
anuda
Figure
2. Values
of selected
features studied in thefeatures
experimentstudied
of the fourin
accessions
of Nepeta
Figure
2. Values
ofmorphometric
selected morphometric
the experiment
of Nepeta
1 - The weight ofaccessions
fresh green parts
and individual parts of the plant constituting it.
2 - The weight of fresh green parts and dried material.
Nepeta
mussinii
Nepeta
mussinii
of the four
The weight of fresh green parts and individual parts of the plant constituting it.
31 - -The
content
of
dry
matter.
4 - -The
content
of essential
oils. green parts and dried material.
2
The
weight
of fresh
3 - The content of dry matter.
4 - The content of essential oils.
73
The largest and smallest amounts of essential oils were extracted from N. citriodora (1.5 %) and N. × faassenii i N. nuda (0.5 %), respectively. The amount of essential
oils in N. mussinii totalled 1% (fig. 2).
Assessment of genetic variability
Twenty microsatellite primers were used in the ISSR-PCR reaction, and clear
products of the reaction were generated from 6 of them (tab. 1). For the examined
species of catnip, 72 loci were ISSR amplified generating 110 amplicons. Their
length varied from ~220 bp (primer 812) to ~3020 bp (primer 839) (tab. 1). Monomorphic, polymorphic and genotype-specific ISSR loci were amplified in a reaction
with 6 primers. Among 72 amplified loci, only 5 ISSR loci were monomorphic, 20
(48%) were polymorphic, and 47 (52%) were determined to be genotype-specific.
(tab. 1, fig. 3). One single primer used in the ISSR reaction amplified an average
of 12 loci, where 5 loci were amplified for N. cataria var. citriodora and N. mussinii,
and 4 loci were amplified for N. × faassenii and N. nuda (tab. 1). Six polymorphic
loci were amplified in a reaction with primer 804, and 4 loci were amplified in a
reaction with primers 812 and 819. The smallest numbers of loci were 3, 2 and 1
in reactions with primers 839, 824 i 826, respectively (tab. 2, fig. 3).
Ta b l e 1 .
Characteristics of ISSR products for investigated Nepeta accessions
sprimer sequence
no
5’–3’
Loci amplified
amplicons number
length of ISSR
products
polymonoNepeta cataria Nepeta × Nepeta Nepeta
total
specific
(bp)
morphic morphic
var. citriodora faassenii mussinii nuda
total
804
(AC)8C
2630–770
15
6
1
8
4
5
10
6
25
812
(GA)8C
1480–220
15
4
1
10
5
3
7
7
22
819
(GT)8A
1420–390
13
4
3
6
7
6
8
7
28
824
(TC)8G
1500–480
6
2
0
4
2
3
1
2
8
826
(AC)8G
3000–970
5
1
0
4
3
3
0
0
6
839
(AG)8T
3020–350
18
3
0
15
8
3
6
4
21
total
72
20
5
47
29
23
32
26
110
average
12
3
1
8
5
4
5
4
18
Ta b l e 2 .
ISSR polymorphic products of investigated Nepeta accessions
accession
primer number, loci ISSR amplified (bp)
Nepeta cataria var. citriodora
Nepeta × faassenii
804(~820, ~1040, ~1240, ~1530, ~2360), 812(~220, ~450, ~670, ~1110),
Nepeta mussinii
819(~390, ~590, ~680, ~1350), 824(~1200, ~1260), 826(~1220)
Nepeta nuda
Vol. 54 No 4 2008
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In the experiment, 47 polymorphic ISSR loci that were also of genotype specific were
amplified in the four species of Nepeta examined (tab. 3, fig. 3). The largest number
of loci (15) was amplified in reactions with 6 primers for N. cataria var. citriodora. The
smallest number of loci (7) was amplified with five primers for N. × faassenii. For the
remaining two species, N. mussinii and N. nuda, 12 of them were amplified in reactions
with four and five primers, respectively (tab. 3). The lengths of ISSR loci amplified in each
of the individual species of catnip and the associated primers are shown in table 6.
Ta b l e 3 .
Accession-specific loci ISSR amplified for Nepeta accessions
accession
primer number and loci ISSR amplified (bp)
804(~850, ~1760), 812(~760, ~1050, ~1250), 819(~480, ~600), 824(~820),
826(~1160, ~3000), 839(~400, ~820, ~1070, ~2330, ~3020)
Nepeta × faassenii
812(~520), 819 (~1420), 824(~480, ~930), 826(~970, ~1100), 839(~350)
Nepeta mussinii
804(~770, ~840,~1600), 812(~790, ~1300, ~1480), 819(~740, ~970), 839(~610, ~810, ~1400, ~1600)
Nepeta nuda
804(~920, ~1010, ~2630,), 812(~430, ~900, ~990), 819(~510), 824(~480, ~1500), 839(~450, ~1020, ~1960)
MW 1
2
3
MW 1
4
3
3
4
MW 1
2
3
4
3000
3000
3000
3000
2000
1600
1400
1200
1000
2000
1600
1400
1200
1000
2000
1600
1400
1200
1000
2000
1600
1400
1200
1000
800
800
800
600
800
600
600
600
400
400
400
400
Primer 804
2
2
Primer 812
Primer 819
MW - molecular weight, 1 - Nepeta cataria var. citriodora, 2 - Nepeta x faassenii, 3 - Nep
MW 1 2 3 4
MW 1 2 3 4
nuda
4
3000
MW 1
2000
1600
1400
1200
1000
3000
2000
1600
1400
1200
1000
Figure 3. Electrophoregrams
of ISSR products amplified on DN
3000
MW
1
2
3
4
MW
1
2 3 4
MW 1 2 3 4
3
4
Nepeta accessions.
2000White arrows mark ISSR polymorphic products
2
1600
1400
1200
1000
3000
2000
1600
1400
1200
1000
800
600
800
600
800
800
400
600
Primer 812
400
600
400
400
Primer 804
Primer 819
Primer 812
3000
3000
2000
1600
1400
1200
1000
2000
1600
1400
1200
1000
800
800
600
600
400
Primer 824
400
Primer 819
Primer 824
cataria var. citriodora
, 2 - Nepeta
, 3 - var.
Nepeta
mussinii,
- Nepeta
MW - molecular
weight,x1faassenii
- Nepeta cataria
citriodora
, 2 - Nepeta 4
x faassenii
, 3 - Nepeta mussinii, 4 - Nepeta
nuda
Figure3. Electrophoregrams
3. Electrophoregrams
ISSR products
on DNA
templates
Figure
of ISSRofproducts
amplified amplified
on DNA templates
of four
Nepeta of four
ms of ISSR accessions.
products
amplified
on polymorphic
DNA
templates
ofproducts
four
Nepeta accessions.
White
mark ISSR
polymorphic
White
arrows
markarrows
ISSR
products
rrows mark ISSR polymorphic products
75
A phylogenetic tree was constructed on the basis of UPGMA cluster algorithm
for the species of Nepeta examined in this study. in this tree, N. mussinii, N. nuda
and N. × faassenii were grouped in one cluster (tab. 4, fig. 4). Phylogenetic similarity among the mentioned species varied from 0.30 between N. nuda and N. mussini to 0.16 between N. nuda and N. × faassenii. Phylogenetic similarity of 0.17, 0.19
and 0.26 was found between N. cataria var. citriodora and N. × faassenii, N. mussini
and N. nuda, respectively (fig. 4).
10%
Nepeta nuda
762
Nepeta mussinii
1000
Nepeta x faassenii
Figure 4. Dendrogram constructed from the results of iSSr analysis. numbers above branches
represent bootstrap support calculated with 1000 replicates
ta b l e 4 .
Matrix of phylogenetic similarity
Nepeta cataria var.
citriodora
Nepeta × fassenii
Nepeta mussinii
1.00
Nepeta × faassenii
0.26
1.00
Nepeta mussinii
0.17
0.30
1.00
Nepeta nuda
0.19
0.16
0.30
Nepeta nuda
1.00
dIScuSSIon
cultivation of herbs is one of the youngest fields of plant production. in Poland,
the development of herb cultivation has been brought about by the exhaustion
of natural resources and a demand for big, uniform batches of raw material. introducing herbal plants into cultivation and the growing demands concerning the
quality of raw material create an impetus to cultivate herbs with a view to creating
new varieties as well as to conducting studies in agricultural science, preservation
and mechanization [10-13].
Vol. 54 no 4 2008
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The work presented herein should support the cultivation of these species. The
morphological and genotypic variability of selected Nepeta species have been determined and could provide pertinent information for initiating their cultivation.
Wide morphological variability was determined in examined material. This variability was clear especially in the plants’ height, which ranged from 40.00 to 73.40
cm. The results of this examination are consistent with the results described by
Bown [4] for Nepeta cataria var. citriodora, and by Biggs et al. [14], Kvant and Palmstiern [15] and Broda and Mowszowicz [16] for Nepeta × faassenii.
Though other literature on this subject includes no suitable references regarding the other morphological features studied in this work, there are reports regarding the biologically active compounds produced by these plants. Héthelyi et
al. [17] determined the composition of essential oils for Nepeta cataria and Nepeta
cataria var. citriodora. They found that these oils contain 75–80% of citrals: 52–55%
of citral a and 20–24% of citral b. Morover, they observed the presence of nepetol
(32–33%) and geraniol (43–44%). According to Nowiński [2], Mackú and Krejcá [3]
as well as Senderski [5], catnip contains from 0.2 to 0.7% of essential oil, depending on the species. In this study, the largest amount of essential oil was found in
Nepeta cataria var. citriodora (1.5%), while 1% of oil was produced by Nepeta mussinii,
and 0.5% by both Nepeta × faassenii and Nepeta nuda.
The application of marker techniques, including the ISSR-PCR technique used
in finding loci or regions of the genome responsible for interesting features of the
examined species (e.g., the quantity and quality of the essential oils produced)
should significantly facilitate breeding projects [6].
Zietkiewicz et al. [6] presented a simple method of generating complex striated
images, making use of the polymorphic sections of DNA among microsatellite
sequences (ISSR). This method was also used to determine the genetic variability
and phylogenetic dependence among varieties of currant [18] and grapevine [19]
as well as in studies of similar characters in burnet [20] and mint [21].
On account of this method, the position of many immunity genes, abiotic stress
tolerance genes and loci determining qualitative features has been determined [22].
For the examined species of Nepeta, 72 loci were amplified in reactions, with 6
out of 20 microsatellite primers examined, from which 47 (65%) were determined
to be genotype-specific. Therefore, a specific, unique DNA profile was drawn for
each genotype, distinguishing it from other genotypes. When these DNA profiles
are analysed, conclusions can be drawn about the phylogenetic interdependence
among examined pieces of material. Polymorphic sequences can also be linked
to specific genotypes that have valuable features for cultivation, including, for
example, essential oil content. In addition, they can be used to select suitable
recombinants, using the MAS (Marker Assisted Selection) technique.
An analysis of the genetic profiles of selected Nepeta genotypes showed that
phylogenetic similarity varied from 0.30 between Nepeta nuda and Nepeta mussini
to 0.16 between Nepeta nuda and Nepeta × faassenii. A similarity of 0.17, 0.19 and
0.26 was found between Nepeta cataria var. citriodora and Nepeta × faassenii, Nepeta
mussini and Nepeta nuda, respectively.
77
CONCLUSIONS
1.The examined plants: Nepeta cataria var. citriodora, Nepeta × faassenii, Nepeta
mussinii, and Nepeta nuda are different morphologically with respect to their
height, the width of a leaf pair, the length and width of a leaf blade and essential oil content.
2.There are significant genotypic differences among them, determined at the
level of microsatellite DNA sequences. Among 72 amplified loci, 47 were determined to be genotype specific, distinguishing one examined genotype from
the other genotypes.
3.The results of this study are a source of valuable information on morphological
and genetic variability among plants from the Nepeta genus. They can also be
of significance in studies working to identify loci that determine essential oil
production as well as in work regarding the cultivation of these genus.
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of horticultural crops. Bydgoszczs 2007:167-78.
21. Smolik M, Jadczak D, Rzepka-Plevneš D, Sękowska A. Morphological and genetic variability of chosen
Mentha species. Herba Polonica 2007; 53: 90-7.
22. Pradeep Reddy M, Sarla N, Siddiq EA. Inter simple sequence repeat (ISSR) polymorphism and its
application in plant breeding. Euphytica 2002; 128:9-13.
OKREŚLENIE MORFOLOGICZNEGO I GENETYCZNEGO ZRÓŻNICOWANIA U WYBRANYCH
OBIEKTÓW NEPETA
Miłosz Smolik1*, Dorota Jadczak2, Aleksandra Główczyk1
Zakład Hodowli Roślin Ogrodniczych
Katedra Warzywnictwa
Akademia Rolnicza, ul. Janosika 8, 71-424 Szczecin
1
2
*autor do którego należy kierować korespondencję: tel.: +4891 4220851 wew. 356,
faks: +4891 4232417, e-mail: [email protected]
Streszczenie
Celem badań było określenie zmienności morfologicznej i genetycznej czterech
genotypów Nepeta: N. cataria var. citriodora, N. × faassenii, N. mussinii, N. nuda z Kolekcji
Roślin Leczniczych Katedry Warzywnictwa w Dołujach Akademii Rolniczej w Szczecinie.
Różnice morfologiczne między badanymi formami kocimiętki określono na podstawie
pomiarów biometrycznych, którymi objęto: wysokość roślin, szerokość pary liści, długość
i szerokość blaszki liściowej, długość kwiatostanu, masę świeżego i suszonego ziela oraz
zawartość olejków eterycznych. Polimorfizm DNA w obrębie sekwencji mikrosatelitarnych
określono przy pomocy techniki ISSR-PCR. Na podstawie przeprowadzonych pomiarów
biometrycznych stwierdzono, że wszystkie analizowane cechy badanych obiektów Nepeta
różniły się istotnie. Do reakcji ISSR zastosowano 20 mikrosatelitarnych starterów, spośród
nich jedynie w reakcjach z 6 generowano produkty. Ogółem amplifikowano 72 loci (110
amplikonów) spośród nich 5 (7%) okazało się monomorficznymi, 20 (28%) polimorficznymi,
47 (65%) specyficznymi dla badanych obiektów. Dla każdego z badanych genotypów Nepeta
amplifikowano unikatowe loci. Na podstawie algorytu skupień UPGMA wykreślono drzewo
podobieństwa filogenetycznego. Składało się ono z jednej grupy podobieństwa: ‘a’ do
której zaliczono: N. × faassenii, N. mussinii i N. nuda.
Słowa kluczowe: Nepeta sp., zmienność fenotypowa, zmienność genotypowa, markery ISSR

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