Morphological and genetic diversity among peppermint (Mentha × piperita L.) cultivars

Abstract

The study determined the morphological and genetic diversity among nine cultivars of peppermint (Mentha
× piperita L.): ‘Almira’, ‘Asia’, ‘Chocolate’, ‘Citaro’, ‘Granada’, ‘Grapefruit’, ‘Multimentha’, ‘Swiss’ and
‘Variegata’. The leaves of the peppermint cultivars were characterized by substantial variation in morphol-ogy and size. The leaves of ‘Multimenth’, ‘Grapefruit‘ and ‘Swiss’ were largest, and those of ‘Swiss’ were
considerably elongated. The ‘Almira’ cultivar had the smallest leaves. Although similar leaf morphology
was observed in ‘Asia’, ‘Citaro’ and ‘Chocolate’, in ‘Grapefruit’ and ‘Multimentha’ and in ‘Swiss’ and
‘Variegata’, no two cultivars were the same in this respect. Differentiation of tested peppermint cultivars
were also confirmed at genetic level. Genetic diversity among tested cultivars ranged from 0.388 to 0.846.
The most different were cultivars Almira and Citaro.

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Acta Sci. Pol. Hortorum Cultus, 16(3) 2017, 151–161
ISSN 1644-0692 DOI: 10.24326/asphc.2017.3.15
O R I G I NA L P AP ER
Accepted: 14.02.2017
MORPHOLOGICAL AND GENETIC DIVERSITY AMONG PEPPERMINT
(Mentha × piperita L.) CULTIVARS
Anna Kiełtyka-Dadasiewicz, Sylwia Okoń

, Tomasz Ociepa, Beata Król
University of Life Science in Lublin, Akademicka 15, 20-950 Lublin, Poland
ABSTRACT
The study determined the morphological and genetic diversity among nine cultivars of peppermint (Mentha
× piperita L.): ‘Almira’, ‘Asia’, ‘Chocolate’, ‘Citaro’, ‘Granada’, ‘Grapefruit’, ‘Multimentha’, ‘Swiss’ and
‘Variegata’. The leaves of the peppermint cultivars were characterized by substantial variation in morphol-
ogy and size. The leaves of ‘Multimenth’, ‘Grapefruit‘ and ‘Swiss’ were largest, and those of ‘Swiss’ were
considerably elongated. The ‘Almira’ cultivar had the smallest leaves. Although similar leaf morphology
was observed in ‘Asia’, ‘Citaro’ and ‘Chocolate’, in ‘Grapefruit’ and ‘Multimentha’ and in ‘Swiss’ and
‘Variegata’, no two cultivars were the same in this respect. Differentiation of tested peppermint cultivars
were also confirmed at genetic level. Genetic diversity among tested cultivars ranged from 0.388 to 0.846.
The most different were cultivars Almira and Citaro.
Key words: DNA polymorphism, leaf morphology, RAPD
INTRODUCTION
Peppermint (Mentha × piperita L.) is a perennial
aromatic herb native to Europe, cultivated in the
northern USA, Canada, Asia, and many other parts of
the world. A hybrid of spearmint (M. spicata L.) and
water mint (M. aquatica L.), peppermint grows par-
ticularly well in areas with soil of high water-holding
capacity. It is best known for its flavour and fra-
grance properties; peppermint leaves (fresh and
dried) and the essential oil extracted from the leaves
are used in many cosmetic, pharmaceutical and food
products [Işcan et al. 2002, McKay and Blumberg
2006, Kiełtyka-Dadasiewicz et al. 2016]. As a phar-
maceutical raw material peppermint leaves have been
described in the European Pharmacopoeia and
pharmacopoeias in many countries, but none of these
specify which varieties or cultivars may be used [Eu-
ropean Pharmacopoeia 2014, Pharmacopoeia Po-
lonica 2014, British Pharmacopoeia 2015]. Yet phy-
tochemical analysis indicates significant heterogenei-
ty between peppermint cultivars [Ludwiczuk et al.
2015]. In recent years many new cultivars of pep-
permint have been created, with diverse morphologi-
cal, flavour and utility features [Kiełtyka-Dadasie-
wicz et al. 2016].
In many cases, due to the high similarity of geno-
types, it is difficult to distinguish cultivars using
morphological and physiological methods. Some-
times also isozyme analyses are insufficient for the
cultivars identification. [Reynders and Bollereau
1994]. In these cases molecular markers provide the
best way to estimate of genetic diversity. DNA mark-
ers are independent of the confounding effects of
environmental features, can be used at a very early
stage of plant development, they are cheap and easy
to apply [Waugh and Powell 1992, Kabir et al. 2014].
One of the most recently method used to estimate



sylwia.okon@up.lublin.pl
© Copyright by Wydawnictwo Uniwersytetu Przyrodniczego w Lublinie

Kiełtyka-Dadasiewicz, A., Okoń, S., Ociepa, T., Król B. (2017). Morphological and genetic diversity among peppermint (Mentha ×
piperita L.) cultivars. Acta Sci. Pol. Hortorum Cultus, 16(3), 151–161. DOI: 10.24326/asphc.2017.3.15
www.hortorumcultus.actapol.net
152
genetic diversity is RAPD (Randomly Amplified
Polymorphic DNA). This fingerprinting technique
provides an unlimited number of markers which can
be used for various purposes [Williams et al. 1990,
Gupta and Varshney 2013]. RAPD markers have
been widely used in diverse plant species for assess-
ment of genetic variation in populations and species,
fingerprinting, and the study of phylogenetic relation-
ships among species and subspecies [Gupta 1999].
This technique has become an increasingly popular
tool in genetic studies because it is technically
straightforward and inexpensive [Emadpour et. al.
2009]. Mentha species have been assessed for genetic
relationships and cultivar identification [Fenwick and
Ward 2000, Khanuja et al. 2000, Grisi et al. 2006,
Smolik et al. 2007, Šarić-Kundalić et al. 2009].
The aims of presented study were identification
and characterization of differences in leaf morpholo-
gy and estimation of genetic diversity among 9 pep-
permint (Mentha × piperita L.) cultivars character-
ised by different biochemical compounds [Lud-
wiczuk et al. 2016].
MATERIALS AND METHODS
Plant materials
The study was performed on 9 cultivars of Men-
tha × piperita L.: ‘Almira’, ‘Asia’, ‘Chocolate’, ‘Ci-
taro’, ‘Granada’, ‘Grapefruit’, ‘Multimentha’,
‘Swiss‘ and ‘Variegata’. Selected cultivars were pre-
viously characterised by Ludwiczuk et al. [2016] as
a different in respect of essential oils and antibacteri-
al activity against Staphylococcus epidermidis.
The plants were taken from the collection of the Gar-
den of Cosmetic Plants and Raw Materials, Research
and Science Innovation Centre, located in Wola Za-
dybska in the Lubelskie region of Poland (51°45'N,
21°51'E). The plants were grown on lessive soil
which was slightly acidic (pH
KCl
6.1).
Morphological analysis
Morphological analysis of leaves was conducted
in 2014 and 2015. The results are reported as the
arithmetic mean of the two years. Each year meas-
urements were made four times at weekly intervals
(twice before flowering, at the bud stage, and at the
beginning of the flowering stage) as replications.
On each occasion 4 middle leaves were taken from
10 plants of each cultivar. The length of the petiole
and the length and width of the leaf blade were
measured. The results were used to calculate the leaf
blade shape index. Morphological qualitative traits
(leaf shape, margin, bases and colour) were defined
according to Tsukaya [2006] and Leaf Architecture…
[1999]. The numerical results were analysed statisti-
cally by analysis of variance at a significance level of
0.05. The Pearson correlation coefficient between the
length and width of the leaf blade and the length of
petiole and blade were determined. Calculations were
carried out in Statistica 9.0 and Excel 7.0.
DNA preparation
Genomic DNA was extracted from fresh and
young tissue using GeneMATRIX Plant & Fungi
DNA Purification Kit (EURx). The quantity and
quality of the isolated DNA was assessed using
NanoDrop 2000 Spectrophotometer. Additionally
DNA concentration was determined by electrophore-
sis on a 1.5% agarose gel by comparison with a mo-
lecular weight standard MassRuler™ DNA Ladder
(Thermo Fisher Scientific). The samples were then
adjusted to equal DNA concentrations of 20 ng/ml.
RAPD analysis
PCR reactions were performed according to the
RAPD method described by Williams et al. [1990]
with modifications. Reaction mixtures contained 1 x
PCR Buffer (10 mM Tris pH 8.8, 50 mM KCl,
0.08% Nonidet P40) (Thermo Fisher Scientific), 160
µM of each dNTP, 530 pM oligonucleotide primer,
1.5 mM MgCl
2
, 70 ng of template DNA and 0.5 U
Taq DNA Polymerase (Thermo Fisher Scientific) in
a final reaction mixture of 15 µl. Amplification was
carried out in a Biometra T1 thermal cycler pro-
grammed for 3 min at 94°C of initial denaturation,
44 cycles: 94°C – 45 s, 37°C – 45 s and 72°C – 45 s,
with a final extension at 72°C for 7 min. A negative
control was added in each run. To verify reproduci-
bility, the primers were tested twice on the same
sample.

Kiełtyka-Dadasiewicz, A., Okoń, S., Ociepa, T., Król B. (2017). Morphological and genetic diversity among peppermint (Mentha ×
piperita L.) cultivars. Acta Sci. Pol. Hortorum Cultus, 16(3), 151–161. DOI: 10.24326/asphc.2017.3.15
www.hortorumcultus.actapol.net
153
Amplification products were separated by electro-
phoresis on 1.5% agarose gels containing 0.1% EtBr
(1.5 h, 120 V). Fragments were visualized under
a UV transilluminator and photographed using the
PolyDoc System. GeneRuler
TM
100bp DNA Ladder
Plus (Thermo Fisher Scientific) was used to establish
the molecular weights of the amplification products.
Data analysis
RAPD products were scored as present (1) or ab-
sent (0) from the photographs. Only bright and repro-
ducible products were scored. The level of polymor-
phism of the primer (polymorphic products/ total
products) and relative frequency of polymorphic
products (genotypes where polymorphic products
were present/ total number of genotypes) [Belaj et al.
2001] were calculated. The resolving power of the
primer was calculated using the following formula:
Resolving power (Rp) = Σ Ib (band informativeness).
Band informativeness was calculated individually for
each band scored by the primer: Ib = 1-[2(0.5-p)],
where p is the proportion of occurrence of bands in
the genotypes out of the total number of genotypes
[Prevost and Wilkinson 1999]. Polymorphic infor-
mation content (PIC) was calculated by applying the
following simplified formula [Anderson et al. 1993]:
PIC = 2fi(1-fi), where fi is the percentage of the i
th
amplified band present.
Genetic pairwise similarities (SI-similarity index)
between genotypes were evaluated according to
Dice’s formula after Nei and Li [1979]. A cluster
analysis was conducted using the distance method
UPGMA (Unweighted Pair-Group Method with
Arithmetic Mean), and clustering was verified by
bootstrapping (1000 rep.). PCA analysis was per-
formed using PAST software. Statistical analysis was
performed in PAST software [Hammer et al. 2001].
RESULTS AND DISCUSSION
In the case of plants producing oil glands, espe-
cially on the leaves, leaf area (which depends on leaf
length and width) is an important factor in the
productivity of essential oil [Maffei et al. 1994].
The results of our study indicate substantial biometric
variation in the leaves of the peppermint cultivars
analysed (tab. 1 and fig. 1).
Table 1. Leaf dimensions of analysed peppermint cultivars
Peppermint
cultivar
Length
of leaf blade
(mm)
Width
of leaf blade
(mm)
Petiole
length
(mm)
Shape
index
Correlation
coefficients
A B
‘Almira’ 25.5
f
±2.1 14.5
e
±0.9 26.4
g
±2.2 1.76
b
0.894* 0.235
‘Asia’ 43.1
e
±1.0 30.6
c
±1.6 70.4
d
±1.7 1.41
cd
0.755* –0.073
‘Citaro’ 56.4
c
±1.5
#
30.5
c
±1.2 81.6
bc
±2.2 1.85
b
0.913*
–0.296
‘Chocolate’ 41.8
e
±1.6 23.0
d
±0.9 89.4
b
±1.6 1.82
b
0.933* –0.255
‘Granada’ 48.3
de
±1.7 35.9
bc
±1.6 86.1
b
±3.6 1.35
d
0.847* 0.450
‘Grapefruit’ 66.1
b
±2.3 45.4
a
±3.0 53.6
e
±2.5 1.46
c
0.919* –0.282
‘Multimentha’ 64.8
b
±1.8 47.6
a
±2.0 73.9
cd
±3.8 1.36
d
0.954* 0.359
‘Swiss’ 75.5
a
±2.3 36.6
b
±1.5 41.8
f
±1.0 2.06
a
0.690* 0.548*
‘Variegata’ 51.8
cd
±1.1 23.5
d
±1.9 108.4
a
±1.7 2.21
a
0.859* 0.696*
Explanatory notes:
#
mean values ± standard deviation; values designated by different lowercase letters are significantly different (P < 0.05);
Pearson’s correlation coefficients between: A – length and width of leaf blade; B – petiole length and length of leaf blade; * significant
correlation

Kiełtyka-Dadasiewicz, A., Okoń, S., Ociepa, T., Król B. (2017). Morphological and genetic diversity among peppermint (Mentha ×
piperita L.) cultivars. Acta Sci. Pol. Hortorum Cultus, 16(3), 151–161. DOI: 10.24326/asphc.2017.3.15
www.hortorumcultus.actapol.net
154
Fig. 1. The leaves of peppermint (Mentha × piperita L.) cultivars: 1 – ‘Almira’, 2 – ‘Asia’, 3 – ‘Citaro’,
4 – ‘Chocolate’, 5 – ‘Granada’, 6 – ‘Grapefruit’, 7 – ‘Multimentha’, 8 – ‘Swiss’ (phot. M. Dadasiewicz)
Fig. 2. Examples of leaves of the peppermint cultivar ‘Variegata’
(phot. M. Dadasiewicz)
Table 2. Morphological leaf features of analysed peppermint cultivars
Peppermint
cultivar
Leaf margin Leaf shape Leaf base Leaf colour
type tooth shape teeth per cm
‘Almira’ serrate/irregular corrugated 2.4 triangular cordate green
‘Asia’ serrate CC/FL 2.7 ovate truncate dark green
‘Citaro’ serrulate ST/FL 3.1 ovate truncate green
‘Chocolate’ finely serrate ST/FL 2.9 ovate truncate dark green with purple
markings
‘Granada’ finely serrate ST/FL 2.6 oval rounded light green
‘Grapefruit’ serrate CV/FL 2.5 ovate cordate green
‘Multimentha’ serrate ST/FL 2.1 ovate cordate dark green
‘Swiss’ serrate CC/FL 2.2 elliptic convex green
‘Variegata’ serrate* ST/FL 2.6 elliptic convex irregular, bicolour white
and green
Explanatory notes: CC – concave, ST – straight, FL – flexuous, CV – convex; * sometimes 2 orders

Kiełtyka-Dadasiewicz, A., Okoń, S., Ociepa, T., Król B. (2017). Morphological and genetic diversity among peppermint (Mentha ×
piperita L.) cultivars. Acta Sci. Pol. Hortorum Cultus, 16(3), 151–161. DOI: 10.24326/asphc.2017.3.15
www.hortorumcultus.actapol.net
155
The longest leaf blades were noted for the ‘Swiss’
cultivar (75.5 mm) and the shortest for ‘Almira’
(25.5 mm). This cultivar also had the narrowest
blades. Peppermint leaves can be more or less elon-
gated, as indicated by the leaf shape index, i.e. the
length-to-width radio. The larger this parameter, the
more the leaf is elongated. The length-to-width ratio
in the leaves of the peppermint cultivars ranged from
1.35 to 2.21. The most elongated leaves were noted
for the cultivars ‘Variegata’ and ‘Swiss’, and the least
for ‘Granada’ and ‘Multimentha’. The leaves of
‘Multimentha’ and ‘Grapefruit’ were similar in size,
but the shape index was greater for ‘Grapefruit’.
Statistical analysis showed a positive correlation
between the length and width of the leaf blade for all
tested cultivars. Moreover, in the case of ‘Variegata’
and ‘Swiss’, a significantly positive correlation was
found between the length of the leaf blade and peti-
ole. Šarić-Kundalić et al. [2009] also observed con-
siderable morphological diversity between different
species, varieties and cultivars of mint. However, the
peppermint leaves in their study were smaller than in
most of the varieties we analysed (2.6–4.6 cm length
and 1.2–1.6 cm width of leaf blade) [Šarić-Kundalić
et al. 2009]
Leaves of cultivars of the same species may have
different morphological features [Marotti et al. 1996,
Klimko et al. 2015]. Table 2 presents the morpholog-
ical features of the leaf blade of the peppermint culti-
vars analysed (Mentha × piperita L.). Differences
were observed between cultivars in leaf margin, leaf
shape, leaf base and colour (tab. 2).
All of the cultivars analysed have leaf margins
with teeth, but the shape of the teeth was varied
(tab. 2). The serrate and finely serrate types of leaf
margin were most common. ‘Citaro’ had serrulate
leaf margins and its teeth were closest together
(3.1 per cm). Only ‘Almira’ had a significantly dif-
ferent tooth shape, which was difficult to define be-
cause the leaf margin was irregular, undulating and
corrugated, sometimes with spinose tooth apices
(fig. 1). Tooth apices were simple (except for ‘Almi-
ra’, which had spinose tooth apices) and of one order,
except for the ‘Variegata’ cultivar, with 2 orders in
some cases.
The cultivars can be classified as follows on the
basis of leaf blade shape and leaf base:
– ovate with truncate base (‘Asia’, ‘Citaro’ and
‘Chocolate’);
– ovate with cordate base (‘Grapefruit’ and ‘Mul-
timentha’); these also have the same type of leaf
margin, but different tooth shape;
– elliptic with convex base (‘Swiss’ and ‘Variega-
ta’); these also have the same type of leaf margin, but
different tooth shape.
The remaining cultivars are distinctive: ‘Almira’
has triangular leaves with a cordate base, and ‘Gra-
nada’ has oval leaves with a rounded base.
Table 3. Selected RAPD primers used in the study
No. Primer Primer sequence
1 C-05 GATGACCGCC
2 C-19 GTTGCCAGCC
3 E-04 GTGACATGCC
4 J-13 CCACACTACC
5 N-06 GAGACGCACA
6 T-01 GGGCCACTCA
7 T-05 GGGTTTGGCA
8 T-06 CAAGGGCAGA
9 T-07 GGCAGGCTGT
10 T-12 GGGTGTGTAG
11 T-13 AGGACTGCCA
12 T-14 AATGCCGCAG
13 T-15 GGATGCCACT
14 T-16 GGTGAACGCT
15 T-20 GACCAATGCC
Straumite et al. [2015] reported that the colour of
mint leaves depends on the content of chlorophyll
and carotenoids. Tarhan et al. [2010] demonstrated
that peppermint leaf colour changes on drying. The
colour of the fresh leaves of the peppermint cultivars
analysed ranged from light green (‘Granada’) to
green (‘Almira’, ‘Citaro’ ‘Grapefruit’ and ‘Swiss’) to
dark green (‘Asia’ and ‘Multimentha’), or dark green
with purple markings in the case of the ‘Chocolate’
cultivars. Similar colouring of mint leaves of differ-

Kiełtyka-Dadasiewicz, A., Okoń, S., Ociepa, T., Król B. (2017). Morphological and genetic diversity among peppermint (Mentha ×
piperita L.) cultivars. Acta Sci. Pol. Hortorum Cultus, 16(3), 151–161. DOI: 10.24326/asphc.2017.3.15
www.hortorumcultus.actapol.net
156
ent species and varieties was observed by Grisi et al.
[2006] and Erum et al. [2012]. One of the cultivars
(‘Variegata’) had irregular, bi-colour green and white
leaves (fig. 2).
In the present study, nine M.× piperita cultivars
were analysed by the RAPD method. Initially three
randomly selected cultivars were used to screen
50 RAPD primers (Operon Technologies). Among
the primers tested only 15 amplified polymorphic
and repeatable fragments (tab. 3, phot. 1). In total,
the selected primers generated 120 fragments. The
number of amplicons ranged from 3 to 13, with an
average of 8 per primer, and 13.33 bands per geno-
type. Khanuja et al. [2000] analysed Mentha spe-
cies using 60 RAPD primers which produced 630
bands, of which 93.5% were polymorphic. The
number of fragments obtained with single primers
was higher than in the present study, ranging from
11 to 19, with an average of 10.5 bands per primer.
Kabir et al. [2014] used the RAPD method to assess
genetic diversity of Mentha species. The authors
selected 9 primers which generated 60 bands, on
average 6 amplicons per primer. Fenwick and Ward
[2001] also used RAPD markers to evaluate genetic
diversity of Mentha species. The authors used
24 RAPD primers which produced 133 amplicons,
of which 104 were polymorphic. The mean num-
bers of amplification products per primer obtained
by the authors were lower than in our study and
scored 5.5%. A high number of bands in Mentha
analysis by RAPD was obtained by Soheila et al.
[2006], who selected 31 RAPD primers, which
produced 617 bands. The number of bands generat-
ed by a single primer was higher than in the present
study, ranging from 1 to 32 with an average of
19.9 per primer.
Table 4. Characteristics of selected RAPD primers
No. Primer Number of products Primer
diversity
(%)
Frequency
of polymorphic
products
Resolving power
of the primer PIC
total polymorphic monomorphic
1 C05 12 11 1 91.67 0.4 9.56 0.28
2 C19 8 8 0 100 0.42 6.67 0.39
3 E04 6 4 2 66.67 0.70 8.44 0.30
4 J13 9 8 1 88,89 0.42 7.56 0.28
5 N06 7 6 1 85.71 0.40 5.56 0.32
6 T01 13 13 0 100 0.46 12.00 0.37
7 T05 9 9 0 100 0.52 9.33 0.35
8 T06 4 3 1 75 0.58 4.67 0.22
9 T07 10 10 0 100 0.39 7.78 0.41
10 T12 6 5 1 83.33 0.61 7.33 0.33
11 T13 9 9 0 100 0.46 8.22 0.35
12 T14 7 6 1 85.71 0.27 3.78 0.21
13 T15 9 8 1 88.89 0.43 7.78 0.35
14 T16 8 7 1 87.50 0.31 4.89 0.25
15 T20 3 2 1 66.67 0.52 3.11 0.26
Total 120 109 11 1320.04 6.89 106.68 4.67
average/ primer 8 7.27 0.73 0.31
average/ genotype
13.33 12.11 1.22 0.52

Kiełtyka-Dadasiewicz, A., Okoń, S., Ociepa, T., Król B. (2017). Morphological and genetic diversity among peppermint (Mentha ×
piperita L.) cultivars. Acta Sci. Pol. Hortorum Cultus, 16(3), 151–161. DOI: 10.24326/asphc.2017.3.15
www.hortorumcultus.actapol.net
157
Table 5. Unique RAPD markers identify for M. × piperita cultivars
Cultivar Unique markers
‘Citaro’ T16
400
T01
350
T05
850
C05
450
‘Granada’ T15
350
C05
700
C05
750
C05
1500
‘Multimentha’ T14
400
T16
400
J13
700
‘Variegata’ T15
2500
‘Chocolate’ T12
1700
‘Almira’ T14
350
T16
700
T13
400
J13
460
C05
300
C19
400
‘Grapefruit’ T14
450
T14
1300
T16
2000
J13
200
N06
350
‘Asia’ T01
550
Table 6. Similarity matrix for Dice coefficient of nine M. × piperita cultivars
Cultivar ‘Citaro’ ‘Granada’ ‘Multimentha’
‘Swiss’
‘Variegata’
‘Chocolate’
‘Almira’ ‘Grapefruit’
‘Asia’
‘Citaro’ 1.00
‘Granada’ 0.430 1.00
‘Multimentha’
0.411 0.700 1.00
‘Swiss’ 0.535 0.558 0.596 1.00
‘Variegata’ 0.543 0.614 0.632 0.846 1.00
‘Chocolate’ 0.484 0.645 0.758 0.659 0.735 1.00
‘Almira’ 0.426 0.535 0.551 0.418 0.436 0.450 1.00
‘Grapefruit’ 0.388 0.702 0.641 0.589 0.629 0.626 0.458 1.00
‘Asia’ 0.467 0.683 0.764 0.644 0.701 0.822 0.491 0.684 1.00
Among the 120 amplified products obtained in the
study using 15 RAPD primers, 109 (90.83%) were
polymorphic. The number of polymorphic bands
amplified by a single primer ranged from 2 to 13,
with an average of 7.27 per primer and 12.11 per
genotype (tab. 4). A similar percentage of polymor-
phic products, 93.5%, was obtained by Khanuja et al.
[2000]. Soheila et al. [2006] demonstrated that RAPD
primers amplified a high percentage of polymorphic
products (98.5%). In our study 5 of the 15 primers
used in the experiments showed 100% polymor-
phism. The lowest level of primer diversity was noted
for primers E04 and T20 (66.67%). In results ob-
tained by Soheila et al. [2006], 23 of 31 RAPD pri-
mers showed 100% polymorphism. Differences in
numbers of products obtained by different authors
may result from the use of different RAPD primers or
different Mentha species in the experiments. Among
analysed cultivars 8 could be identify by presence of
unique RAPD markers. 11 primers initiate amplifica-
tion of 25 unique markers. Cultivar Almira could be
identify by 7 specific amplicons, Grapefruit by 5,

Kiełtyka-Dadasiewicz, A., Okoń, S., Ociepa, T., Król B. (2017). Morphological and genetic diversity among peppermint (Mentha ×
piperita L.) cultivars. Acta Sci. Pol. Hortorum Cultus, 16(3), 151–161. DOI: 10.24326/asphc.2017.3.15
www.hortorumcultus.actapol.net
158
Fig. 3. UPGMA dendrogram of nine M. piperita cultivars based on RAPD primers
Citaro and Granada by 4 unique markers, cultivar
Multimentha by 3, and cultivars Chocolate, Asia and
Variegata by 1 specific primers (tab. 5).
The relative frequency of polymorphic products
and the resolving power of the primers were calculat-
ed in the study. The relative frequency of polymor-
phic bands ranged from 0.11 (polymorphic band pre-
sent in only one genotype of the 9 studied) to 0.89
(polymorphic band absent in only one genotype of the
9 studied). Overall the average frequency generated by
a single primer was high (0.46), varying from 0.27 to
0.70 (tab. 4). These results suggest that the material was
characterized by a high level of polymorphism.
The average frequency of polymorphic products has
also been calculated for tea [Chen et al. 2005], A mon-
tana [Okoń et al. 2014] and M. chamomilla [Okoń and
Surmacz-Magdziak 2011]. The average values of these
coefficients were similar to those obtained in the pre-
sent study: 0.47, 0.58 and 0.42 respectively.
Primers with high resolving power are used for
molecular diagnosis of a species from a mixed popu-
lation [Prevost and Wilkinson 1999]. In our study the
resolving power of the 15 RAPD primers ranged from
3.11 to 12.00 (tab. 4). Resolving power values ob-
tained for the RAPD primers were able to distinguish
all analysed cultivars, and could potentially be used to
identify them from any mixed population. A similar
approach has previously been used successfully for
molecular diagnosis of Rhus species [Prakash et al.
2007], Jatropha genotypes [Tatikonda et al. 2009],

Kiełtyka-Dadasiewicz, A., Okoń, S., Ociepa, T., Król B. (2017). Morphological and genetic diversity among peppermint (Mentha ×
piperita L.) cultivars. Acta Sci. Pol. Hortorum Cultus, 16(3), 151–161. DOI: 10.24326/asphc.2017.3.15
www.hortorumcultus.actapol.net
54
A. montana [Okoń et al. 2014] and M. chamomilla
[Okoń and Surmacz-Magdziak 2011].
Genetic similarity matrices were produced based
on RAPD using Dice’s coefficient. RAPD-based
genetic similarity was estimated between 0.388 for
the ‘Grapefruit’ and ‘Citaro’ cultivars and 0.846 for
‘Variegata’ and ‘Swiss’ (tab. 6). The mean genetic
similarity was calculated at 0.590. The lowest genetic
similarity to the remaining genotypes was calculated
for the ‘Almira’ (0.471) and ‘Citaro’ (0.460) culti-
vars, while the ‘Asia’ cultivar was the most similar to
the other genotypes (0.657).
Fig. 4. Principal component analysis (PCA) of M. × piperita cultivars
Phot. 1. PCR amplification using A – T12 and B – T15 RAPD primers. M- GeneRuler
TM
100bp DNA Ladder
Plus (Thermo Fisher Scientific), 1– Citaro, 2 – Granada, 3 – Multimentha, 4 – Swiss, 5 – Variegata, 6 – Choco-
late, 7 – Almira, 8 – Grapefruit, 9 – Asia
www.
hortorumcultus.actapol.net
159

Kiełtyka-Dadasiewicz, A., Okoń, S., Ociepa, T., Król B. (2017). Morphological and genetic diversity among peppermint (Mentha ×
piperita L.) cultivars. Acta Sci. Pol. Hortorum Cultus, 16(3), 151–161. DOI: 10.24326/asphc.2017.3.15
www.hortorumcultus.actapol.net
55
A genetic similarity matrix was used for cluster
analysis by the UPGMA method (fig. 3). The nine
M. × piperita cultivars were grouped into three major
groups based on bootstrapping. Group A contained
2 cultivars, ‘Granada’ and ‘Grapefruit’. In group B
the cultivars ‘Multimentha’, ‘Chocolate’ and ‘Asia’
were grouped together. Group C included ‘Swiss’
and ‘Variegata’. The ‘Almira’ and ‘Citaro’ cultivars
showed the lowest similarity to all other genotypes
and were located at the edge of the dendrogram.
Relationships between the M. piperita cultivars
were revealed by principal component analysis
(PCA) (fig. 4). PCA confirms the results obtained by
UPGMA clustering. The cultivars formed three dis-
tinct groups, which correspond to groups A, B and C
in the UPGMA dendrogram. The ‘Citaro’ and ‘Almi-
ra’ cultivars formed separate groups. For the RAPD
data, the first three principal components explained
79.3% of the total variation, with PC-1, PC-2 and
PC-3 accounting for 35.0, 32.0 and 12.3% of the total
variation, respectively.
CONCLUSIONS
1. The peppermint cultivars were characterized by
diversity of morphological features and leaf size. No
cultivars were found to have the same leaf shape and
size.
2. The morphology of the ‘Almira’ and ‘Granada’
was clearly distinct from the other cultivars.
3. The mint cultivars showed fairly high genetic
similarity. The highest genetic similarity was esti-
mated between ‘Variegata’ and ‘Swiss’, and the low-
est between the ‘Almira’ and ‘Citaro’ cultivars.
4. Our research shows that the peppermint culti-
vars tested are distinct, both morphologically and
genetically, which suggests the need for further re-
search into the chemical composition and pharmaco-
logical properties of the cultivars.
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