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Genetic diversity analysis of Rose varieties
from Grandiflora group based on ISSR
molecular markers
Sophia S. Yudanova
1,*
, Svetlana A. Plugatar
2
, Zinaida K. Klimenko
2,
Vera K. Zykova
2
,
Olena L. Rubtsova
3
, and Olga Yu. Vasilyeva
1
1
Central Siberian Botanical Garden, SB RAS, 630090 Novosibirsk, Russia
2
Nikitsky Botanical Garden, National Scientific Center of the Russian Academy of Sciences, 298648
Yalta, Russia
3
M.M. Gryshko National botanical garden National Academy of Sciences of Ukraine, 01014 Kiev,
Ukraine
Abstract. Kinship and genetic diversity determination among six rose
varieties from the Grandiflora group were carried out using ISSR
thechnics. The studied varities were divided into 3 clades: I) ‘Lezginka’,
‘Queen Elizabeth’ and ‘Koralovy surpriz’ varieties; II) ‘Gurzuf’ and
‘Love’ varieties; III) ‘Komsomolsky ogonek’ variety formed a separate
branch. This division into clades was confirmed by a statistical comparison
of morphometric characters. On the basis of the obtained data, it can be
concluded that the analysis using the selected primer group is well-suited
for differentiation rose varieties into groups, which makes it possible, for
one thing, to determine the genetic distance between varieties, and for
another, to use these data in the future by certification of the varieties
promising by resistance characters to the continental climate conditions.
Introduction
Rose is the most popular and widely cultivated crop in the world ornamental gardening. It is
an ornamental crop including different species, interspecific hybrids in genus Rosa L. The
classification of roses approved by the World Federation of Rose Society (WFRS) for garden
groups is not always corelated with the botanical classification. Modern classification of
garden roses is based in most cases not on the origin, but on ornamental and morphological
characters.
Grandiflora is a rose group created in the middle of the 20th century by back crossing of
hybrid tea forms and floribundas. Obtained hybrids did not match any of the groups that
existed at that time. ‘Queen Elizabeth’ was the first representative of the Grandiflora group
and still remains the standard for new cultivars of this group. This cultivar is widely known
and popular all over the world due to its decorative value, resistance to diseases and adverse
conditions, which in turn has led to the repeated using of ‘Queen Elisabeth’ as parental
form when breeding new varieties. Grandiflora roses have large goblet flowers of a hybrid
Corresponding author: judanowa.sophia@yandex.ru
© The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons
Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/).
BIO Web of Conferences 38, 00140 (2021)
Northern Asia Plant Diversity 2021
https://doi.org/10.1051/bioconf/20213800140
*
tea type. They are superior to medium-sized floribunda cultivars in bush height, but inferior
in the flowers number in the inflorescence.
Genetic diversity estimation was carried out by ISSR-PCR technique. Inter simple
sequence repeat, ISSR marker technique was developed by E. Zietkiewicz [1]. This
technique based on the amplification of regions between DNA adjacent microsatellite
sequences via polymerase chain reaction (PCR). ISSR markers are well suited to genetic
diversity studies, phylogeny, gene tagging, genome mapping, and evolutionary biology due
to the high polymorphism [2-5].
The goal of the present investigation was to estimate the level of relationships and the
genetic diversity among six rose varieties from the Grandiflora group.
Material
The study was carried out on the base of the bioresource scientific collection “Collections of
living plants in outdoor and indoor”, UNU No. USU 44053 (Central Siberian Botanical
Garden, SB RAS). Six rose varieties belonging to the grandiflora group were used as a
material for investigation:
- ‘Gurzuf’ – has deep pink buds, cup-shaped, with a strong fragrance, pink flowers with
creamy touches and a creamy reverse side; flowers arranged singly or in inflorescences
up to 4 FL; leaves are dark green, glossy.
- ‘Lezginka’ - has dark red buds, goblet-shaped, fragrant, bright red flowers with a yellow
eye; flowers arranged singly or in inflorescences up to 5 FL; leaves are dark green with
a bronze tint.
- ‘Koralovy surpriz’ - has scarlet buds, goblet-shaped, fruity fragrant, coral pink flowers;
flowers arranged singly or in inflorescences up to 5 FL; leaves are dark green, leathery.
- ‘Queen Elizabeth’ – has fragrant pink flowers with a high center, flowers arranged
singly or in inflorescences up to 6 FL; leaves are dark green, leathery.
- ‘Komsomolsky ogonek’ - has red-orange buds, cupp-shaped, weak fragrant, blood-red
flowers – bright and velvety, with a white-yellow eye, flowers arranged singly or in
inflorescences up to 5 FL; leaves are dark green, glossy.
- ‘Love’ – has red buds, goblet-shaped, weak fragrant, bright red flowers with a silvery-white
reverse side of petals, flowers arranged mainly singly; leaves are dark green, leathery.
Methods
DNA isolation from dried leaves was carried out using CTAB procedure with some
modifications [6]. The CTAB buffer included CTAB, sodium chloride (NaCl), and ethylene-
diaminetetraacetic acid (EDTA) Tris2-amino-2-hydroxymethyl-1,3-propanediol (TRIS) and β
mercaptoethanol. Сrude DNA samples were purified twice with an equal volume of
chloroform : isoamyl alcohol (24:1) and precipitated by using isopropanol.
The study used 6 six primers characterized by a clear, reproducible, polymorphic
pattern: 17899B, HB-12, M-2, UBC 807, UBC 855, UBC 834 (table 1). Polymerase chain
reaction (PCR) was performed on C-1000 thermocycler (BioRad, USA). PCR reactions
were carried out in 25 µL volume reaction mixture containing 2 µL of genomic DNA
template, 2.5 µL 10X Taq buffer without Mg2+, 1 µM dNTPs, 2.3 µM MgCl2, 2 µL
primer, 0,2 µL Taq polymerase (5 u/µl) and 15 µL purified water (ddH
2
O). The PCR
amplification conditions were: initial extended step of DNA denaturation at 95°C for 2 min,
followed by 38 cycles of denaturation at 94°C for 20 sec, primer annealing for 30 sec
(annealing temperatures and nucleotide sequences are shown in Tab. 1), elongation – 1.5
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https://doi.org/10.1051/bioconf/20213800140
min and final elongation – 7 min. Reaction products were mixed with 2 µL Sybr Green I, an
intercalating dye specific for double-stranded DNA.
Electrophoretic separation of amplification products was done in a 1-1.5% agarose gel
with 1x TBE-buffer. Electrophoresis was done at 80 V for about 2 h. The polymorphism
quantitative estimation of applied markers and divergence level determination between the
studied simples was based on the state matrices of binary characters. The presence or
absence of the target sequence of a certain fragment length, i.e. the same size PCR fragment
was designated as states 1 or 0.
Statistical evaluation of obtained data was carried out using software package TreeCon
(version 1.3b) [7]. Genetic distances were calculated by the formula:
GDxy = 1 - 2Nxy / (Nx + Ny) (1)
where Nxy – is a common fragments number for x and y samples, Nx and Ny – are numbers
of fragments for x and y samples respectively [8].
Dendrograms were created using the Neighbor-Joining (NJ) algorithm with 100
bootstrap pseudo-replicas. The polymorphism percentage (P, %) of each primer was
calculated with the formula: P = 100 × Np/N (2)
where Np – is a polymorphic fragment number, N – is a total fragment number.
To determine a significant difference between morphometric characters of studied
varieties Student's t-test was used [9]. T-value was calculated with the formula:
21 2
2
2
1
MM
mmt
−
+=
(3)
where М – an average mean and m – a standard error.
Results and discution
Microsatellite DNA regions of the studied samples were analyzed using 6 ISSR primers
(Tab. 1). Each amplified fragment (DNA band) was defined as an independent character,
being designated as present - 1 or absent - 0, resulting a binary data matrix which was used to
compute primer banding characteristics such as: total number of bands, number of
polymorphic bands, percentage of polymorphic bands.
122 amplified fragments (bands) from 250 to 3000 bp in length were identified, from
which 109 were polymorphic. The number of fragments amplified by a primer range from
18 (HB12 and UBC834) to 23 (17899В) (Tab. 1).
The polymorphism level detected by a single primer varied from 77.8 (amplified with
HB-12) to 94.4% (with UBC-857), and averaged is 91.42 %. An ISSR profile of the
samples obtained by amplification with HB12 primer is presented on the figure 1.
Table 1. Characteristics of the ISSR primers used for the detection of genetic polymorphism in 6
rose varieties from grandiflora rose group.
Primer Sequence, 5’-3’ Annealing
temp., С°
°°
°
TNB* NBP* PPB*,
% Size range of
fragments, bp
HB 12 (CAC)
3
GC 42°C 18 14 77, 8 500-2400
17899 B (CA)
6
GG 42°C 23 21 91,3 250-2800
UBC 807 (AG)
8
T 52°C 21 19 90,5 500-2500
UBC 834 (AG)
8
YT 60°C 18 17 94,4 300-2000
UBC 855 (AC)
8
YT 50°C 22 20 90,9 700-2000
М2 (AC)
8
YG 50°C 20 18 90,0 300-2500
3
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* TNB – total number of bands, NPB – number of polymorphic bands, PPB – percentage of
polymorphic bands.
Fig. 1. Electrophoregram of PCR products obtained by DNA amplification with ISSR primer H12 –
(CAC)
3
GC. Track M – DNA marker, tracks with designation of samples:
9 – ‘Gursuf’, 11 – ‘Lezginka’, 12 – ‘Koralovy surpriz’, 14 – ‘Queen Elizabeth’,
16 – ‘Komsomolsky ogonek’, 45 – ‘Love’
On the basis of the obtained dendrogram (Fig. 2), the studied samples were divided into
3 clusters. The first cluster (I) included the ‘Lezginka’, ‘Queen Elizabeth’ and ‘Koralovy
surpriz’ varieties, the second – the ‘Gurzuf’ and ‘Love’ varieties (II). ‘Komsomolsky
ogonek’ variety was the farthest from other samples and formed a separate branch (III). The
‘Queen Elizabeth’ variety was used more likely as a parent form by ‘Lezginka’ variety
breeding, as evidenced by the genetic distance (0.1) with a high level of statistical support
1
(> 90).
Fig. 2. Dendrogram built with the Neighbor-Joining algorithm based on ISSR-PCR spectra data of
studied rose varieties. Node numbers indicate the level of statistical branch bootstrap.
Morphometric character data of studied varieties are presented in Table 2: flower
diameter, petal number, peduncle length, bush height. Statistical comparison of
morphometric characters using Student's t-test confirms the division of the studied varieties
into 3 clade. Comparison of the ‘Komsomolskiy Ogonek’ variety that distinguished as the
third separate clade with varieties from the first clade showed a statistically significant
1
bootstrap proportions >70 considered as high reliable (with greater than or equal to 95% probability).
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difference (T criterion, P> 0.95) in all characters with the ‘Queen Elizabeth’ and ‘Lezginka’
varieties, as well as in three characters with the ‘Koralovy surpriz’ variety (flower diameter,
petal number and bush height). Comparison of the ‘Komsomolsky Ogonek’ variety with the
‘Gurzuf’ and ‘Love’ varieties from the second clade showed a statistically significant
difference (T criterion, P> 0.95) in two characters (flower diameter, number of petals).
Table 2. Morphometric characteristr of studied rose varieties from grandiflora group.
Variety N Flower
diameter, cm Petals
number Peduncle
length, cm Bush
height, cm
Queen Elizabeth 20 8,75 ± 0,22 29,92 ± 0,71 41,33 ± 2,20 63,67 ± 1,36
Komsomolsky ogonek 20 7,92 ± 0,14 21,25 ± 0,64 50,17 ± 1,91 73,25 ± 2,68
Koralovy surpriz 20 9,27±0.18 24.75±0.81 45,25±2,37 65,95±1,33
Gurzuf 20 10,24±0,19 35,65±0,91 51,95±1,71 78,15±1,80
Lezginka 20 8,97±0,23 25,20±0,79 68,45±1,62 85,50±1,44
Love 20 9,42±0,20 32,85±0,84 53,50±1,38 70,20±1,92
On the basis of the obtained data, it can be concluded that the analysis using the selected
primer group is well-suited for differentiation rose varieties into groups, which makes it
possible, firstly, to determine the genetic distance between varieties, and secondly, to use
these data in the future when certifying of the varieties that are promising in terms of
complex resistance to the conditions of the continental climate.
Acknowledgements. The work was carried out with financial support from the budgetary project of
Central Siberian Botanical Garden, SB RAS "Analysis of biodiversity, conservation and restoration of
rare and resource plant species using experimental methods. (No.АААА-А21-121011290025-2) within the
framework of the State Assignment and with material of CSBG representing USU (Unique Scientific
Unit) “Collections of living plants indoors and outdoors”.
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