Study of genetic diversity in local rose varieties (Rosa spp.) using molecular markers

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Banats Journal of Biotechnology
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148
Study of genetic diversity in local rose varieties (Rosa spp.) using molecular
markers
DOI: 10.7904/2068–4738–VIII(16)–148
Abbas SAIDI1*, Yazdan EGHBALNEGAD1 and Zahra HAJIBARAT1
1Department of Plant Sciences and Biotechnology, Faculty of Life Sciences and Biotechnology,
Shahid Beheshti University, G.C, Tehran, IRAN
Corresponding author: abbas.saidi@gmail.com, Phone: +982129901964
Abstract. This study was undertaken to evaluate genetic diversity in a germplasm
consisting of rose varieties. Genetic distances were estimated using three different molecular
marker techniques including: start codon targeted (SCoT), conserved DNA–Derived Polymorphism
(CDDP) and directly amplified minisatellite DNA (DAMD). According to the results, the average
polymorphism information content was 0.37, 0.36, and 0.36 for SCoT, CDDP and DAMD markers,
respectively indicating that the studied marker types were equal in terms of assessing diversity.
Cluster analysis using SCoT and CDDP divided the varieties to four distinct clusters whereas
DAMD markers data, grouped the varieties into three clusters. There was a positive significant
correlation (r=0.92, p<0.01) between similarity matrix obtained by SCoT and CDDP. Results
suggested that the efficiency of SCOT, CDDP and DAMD markers had a relatively same efficiency
in fingerprinting of varieties. This is the first time that the efficiency of the three molecular markers
have been compared with each other in a set of rose samples. The results showed that the studied
markers had an appropriate polymorphism and thus were suitable for the study of genetic diversity
in rose.
Keyword: Fingerprinting, PIC, molecular marker, genetic distance, correlation.
Introduction
Rose (Rosa sp.) is one of the
world's most important commercial
flowers which are used as garden plant,
cut flower and source of essential oil [GUDIN
2000]. Rose breeding programs are mainly
based on the production of new hybrids
and evaluation of genetic diversity is an
essential tool for such programs.
Molecular markers are of the
methods used in the study of genetic
diversity both within and between species
[POWELL et al., 1996].
Amongst them, DNA markers are
the most important and useful marker
systems which are widely used.
Previously, genetic variation has
been assessed in rose genotypes using
some molecular markers such as SSR
[ZHANG et al., 2013; SAMIEI et al., 2009], RAPD [JAN and
BYRNE, 1999; AZEEM et al., 2012], and RFLPs
[HUBBARD et al., 1992]. However, these markers
have some weaknesses.
For example: need to high–quality
DNA, laborious, complex to automate,
need to radioactive labeling and
characterization of probe are of great
disadvantages of RFLPs.
Also, SSR marker system requires
sequence information and may not be
suitable across species. Similarly,
disadvantages of RAPD markers include
dominant, non–reproducibility and lack of
detection of allelic system [MIAH et al., 2013].
In recent years, new markers have
been developed which can be considered
as suitable alternatives for previous
markers [GUPTA and RUSTGI 2004].
These new markers such as CDDP
[COLLARD and MACKILL 2009a], SCoT [COLLARD and
MACKILL 2009b] and DAMD [JEFFREYS et al., 1985]
have been developed based on the
conserved regions of genes and can
easily generate functional markers related
to a given plant phenotype [POCZAI et al., 2013].
Molecular markers that are
extended in genome across different plant
species, such as SCoT and CDDP, and
have longer primers with higher annealing
temperature will be more trustworthy and
reproducible than arbitrary markers such
as RAPDs or ISSR.

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SCoT and CDDP markers have
been used in many crop plants such as
wheat [HAMIDI et al., 2014], and chickpea
[HAJIBARAT et al., 2015].
Here, we assessed genetic diversity
in rose varieties using SCoT, DAMD and
CDDP markers for first time.
Comparison between the above
markers in estimating genetic relations
among rose varieties was the other goal
of this study.
Material and methods
Plant material. A total of 20 local
rose varieties provided from the National
Institute of Ornamental Plants (NIOP),
Mahallat, Iran were studied (Table 1).
Genomic DNA extraction and
DAMD marker analysis. DNA was
extracted from 1 g of fresh leaves
collected from 14–day–old seedling using
the modified CTAB method [LASSNER et al.,
1989] with the modification described by
Torres and collab. [TORRES et al., 1993]. Table 1.
Names of local rose varieties evaluated in this research.
Name
Entry no.
Name
Entry no.
Vandenta
1
Musk rose
11
cool water
2
PV6
12
Rosa moyesii
3
Mo1
13
meinyature
4
Rose sp. ablgh
14
red s. meinyature
5
Black
15
Ablagh meinyature
6
PO1
16
R.vandenta
7
PY3
17
red meiyature
8
Rosa sp color
18
Marociya
9
P.R. vandenta
19
R.marociya
10
PY52
20
To produce DNA fingerprint profiles,
ten primers were screened of which eight
were selected based on GC content of
50–60 % and an annealing temperature of
49 °C (Table 2).
PCR amplification was performed in
25 μL reaction containing 1× PCR buffer,
30 ng sample DNA, 2.5 μM primer, 200
μM of each dNTP, 1.5–2.5 mM MgCl2 and
1.5 unit of Taq DNA polymerase. Table 2.
Primers used in DAMD, SCoT, and CDDP marker systems for study of genetic variation
among 20 local rose variteies.
Marker
system
Primer
Sequence 3′ to 5′
GC
(%)
Annealing
temperature
DAMD
UPR–2F
GTGT GC GA TC AG TT GC TG GG
60
49
UPR–4R
AGGA CT CG AT AA CA GG CT CC
55
49
UPR–6R
GGCA AG CT GG TG GG AG GT AC
65
49
UPR–13R
TACA TC GC AA GT GA CA CA GG
50
49
UPR–17R
AATG TG GG CA AG CT GG TG GT
55
49
UPR–25F
GATG TG TT CT TG GA GC CT GT
50
49
UPR–30F
GGAC AA GA AG AG GA TG TG GA
50
49
UPR–38F
AAGA GG CA TT CT AC CA CC AC
50
49
SCoT
SCOT–1
CAACAATGGCTACCACCA
50
48
SCOT–2
CAACAATGGCTACCACCC
55
48
SCOT–11
AAGCAATGGCTACCACCA
50
48
SCOT–13
ACGACATGGCGACCATCG
61
48
SCOT–22
TACATCGCAAGTGACACAGG
55
48
SCOT–28
CCATGGCTACCACCGCCA
66
48
SCOT–36
GCAACAATGGCTACCACC
55
48
CDDP
KNOX–1
AAGGGSAAGCTSCCSAAG
45
48
KNOX–2
CACTGGTGGGAGCTSCAC
61
48
KNOX–3
AAGCGSCACTGGAAGCC
55
48
WRKY– 1R
GTGGTTGTGCTTGCC
50
48
WRKY–2R
GCCCTCGTASGTSGT
45
48
WRKY–3R
GCASGTGTGCTCGCC
55
48
All amplifications were carried out in
an Eppendorf thermocycler as followed: 94 °C for 2 min, followed by 40 cycles of
45 s denaturation at 94 °C, 1 min

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annealing at 57 °C, and 2 min. elongation
at 72 °C. The obtained amplicons were
run on 1.5 % agarose gel, stained with
ethidium bromide.
SCoT and CDDP analysis
In this study, seven SCoT and six
CDDP primers designed by Collard and
Mackill [COLLARD and MACKILL 2009a, 2009b] were
applied (Table 2). These primers were
18–mer and their GC content ranged
between 50 and 72 %.
Sequences were scanned for short
conserved amino acid regions with the
low permutations of possible codons.
Up to three degenerate nucleotides
were included in a single primer.
Since plant exons are typically
guanine–cytosine (GC) rich, some
degeneracies were incorporated into
primers corresponding to the third
nucleotide position of a codon (i.e., G or C
in the primer sequence was designed as
an “S”).
Primers were 15 to 18 nucleotides
in length and had >60 % GC content.
PCR proliferation was used in 25 μL
reaction containing 1× PCR buffer, 50 ng
sample DNA, 2.5 μM primer, 200 μM of
each dNTP, 3 mM MgCl2 and 1.5 unit of
Taq DNA polymerase (Cinnagen, Iran).
Thermal cycling (Eppendorf)
initiated with 95 °C for 3 min, followed by
40 cycles of denaturation at 94 °C for 1
min, annealing at 49 °C for 2 min, and
extension at 72 °C for 2 min.
A final elongation step of 8 min at
72 °C was added.
Amplified PCR products were
separated by gel electrophoresis on 1.5 %
agarose gels, stained with ethidium
bromide.
Data analysis
Polymorphic alleles were scored as
presence or absent (1/0). DARwin version
5.0 was applied for analyzing pairwise
genetic distances and for making the
distance matrix [PERRIER et al. 2003].
The produced genetic distance was
used to calculate the frequency of
dissimilarity and dendrogram analysis
using the unweighted neighbor–joining
method (UNJ) [GASCUEL, 1997]. The bootstrap
analysis running 1000 replication was
employed to determine a sampling
variance of the genetic similarities
calculated from the data sets gained of
different marker systems [PERRIER et al., 2003].
The Mantel test of importance
[MANTEL, 1967] was also applied to compare
each pair of similarity matrices created.
Almost of all methods were performed by
NTSYSpc version 2.0 [ROHLF, 1997].
Polymorphic information content
(PIC) values were calculated for each
primer according to the formula: PIC=1–
S(Pij)2; where Pij is the frequency of the
ith pattern showed by the jth primer
aggregated across all patterns revealed
by the primers [BOTSTEIN et al., 1980].
Results and discussion
The results of the banding pattern of
electrophoresis showed that the three
markers could demonstrate the high level
of diversity existing among the individuals
consequently; the markers were
functional for each of 20 local rose
varieties (Figure 1). PCR–based
molecular markers can play an important
role in the analysis of genetic diversity in
such species. Fingerprinting data
obtained using DAMD; SCoT and CDDP
markers were as below:
SCoT Analysis
The used Seven SCoT primers
generated 47 bands which were
polymorphic up to 98 %. The maximum
and a minimum number of amplified
bands with 11 and 2 bands belonged to
SCOT–13 and SCoT22, respectively.
The polymorphism value was varied
from 77 % to 100 %. PICs ranged from
0.45 to 0.25 for primers SCOT–36 and
SCoT22, respectively. Marker Index (MI)
ranged from 0 (SCoT23) to 8.066
(SCoT31).
Primer SCoT13 (4.82) had the
highest MI value while; Primer SCoT 22
(0.5) had the lowest. Cluster analysis
classified the varieties into three major
groups (Figure 2). Cluster I, II and III each
contained seven, four and seven varieties
while, PY52 and red melyature were not
included in any group.
CDDP Analysis
CDDP primers produced a total of
32 bands (Table 3). The average number
of polymorphic bands was 5.33 per primer

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ranged from 2 (Knox2) to 10 (WRKY–1R,
WRKY–2R). The polymorphism
percentage was 100 % with an average of
100 % showing a high polymorphism
level. The average value of PIC was 0.37
per locus ranged from 0.25 to 0.41 (Table
4). Neighbor–Net cluster analysis based
on CDDP divided local rose varieties into
four clusters (Figure 3).
Clusters I, II, III and IV contained
seven, four, seven members. Of these,
some clusters had a relatively similar
grouping pattern with those obtained by
means of SCoT and DAMD markers.
Figure 1. Amplification profile obtained with SCoT13 (a), URP13R (b) and WRKY–R1 (c)
primers.
DAMD analysis
PCR amplification was successful
for seven DAMD primer and produced 47
fragments (Table 3).
The number of proliferated alleles
ranged from 4 to 9 with a mean of 5.8
polymorphic bands per primer.
Averagely, the PIC value was 0.37
per locus (0.26 to 0. 43) (Table 3).
Cluster analysis grouped the
varieties into three distinct clusters
(Figure 4).
Cluster I contained 11 local varieties
and cluster II contained six local varieties.
Cluster II revealed similar grouping
pattern with those obtained by SCoT data.
Cluster III included three local
varieties.
Correlation among marker
systems
Estimated cophenetic correlation
coefficient (CCC) indicated a good fit of
data obtained by the three markers
(DAMD=0.84; SCoT=0.83 and
CDDP=0.89) representing consistent
results.
The CCC was notably high (0.92
between SCoT and CDDP, 0.89 between
DAMD and SCoT, and 0.85 between
CDDP and DAMD, P<0.01) (Table 4)
indicating a good relationship between
genetic distances obtained through all
marker techniques.
Also, high CCC between SCoT and
CDDP indicated a similarity in DNA
sequence variation at primer binding
b
a
c

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regions between the two markers which is
important in hybridization programs.
Comparison between the results
derived from this study with those
obtained using SSR [ZHANG et al., 2013], and
RAPD markers [JAN and Byrne, 1999] revealed
that the three studied marker systems had
a relatively higher polymorphism
percentage and PIC values.
Figure 2. Dendrogram of the 20 local rose varieties using SCoT markers based on
genetic distance.
Comparison among the studied
markers
In the present study, the average
amount of genetic distances obtained by
CDDP, SCoT, and DAMD markers were
0.36, 0.37, and 0.36, respectively.
There was a relatively significant
level of polymorphism within the varieties
which was in agreement with findings of
Saeed and collab. and Ghaffari and
collab. but was not consistent with reports
of Byrne, and Matsumoto and collab. who
reported a low level of genetic diversity
within rose germplasm [SAEED et al., 2011,
GHAFFARI et al., 2014, BYRNE, 1999, MATSUMOTO et al.,
1998]. In agreement with our results,
several authors reported that SCoT,
CDDP and DAMD marker techniques
were able to provide more dependable
diversity information compared to RAPD
or ISSR techniques [AMIRMORADI et al., 2012; LI et
al., 2013; POCZAI et al., 2013].
Based on the calculated PIC and
polymorphism percentage, the used
markers were highly efficient for
assessing diversity among studied
varieties.
A high level of polymorphism (125
polymorphic bands) was detected using
five CDDP, six DAMD, and seven SCoT
markers, with an average of 5.3, 5.9 and
6.6 bands per primer, respectively.
Although, the source of diversity
was different however, the rate of
diversity for the three marker techniques

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was approximately equal suggesting
primers could properly and similarly bind
to different regions of the genome.
Based on results, the average
genetic distance obtained by SCoT,
CDDP, and DAMD (0.44, 0.42 and 0.44,
respectively) was similarly high showing a
relatively high genetic dissimilarity among
studied varieties.
Table 3.
Total number of amplified bands (TB), polymorphism bands (PB), percentage of
polymorphism bands (PPB) and PIC values in rose varieties as revealed by DAMD, SCoT,
and CDDP markers.
Marker type
Primer
TB
PB
PPB
PIC
MI
DAMD
UPR–2F
4
4
100
0.31
1.25
UPR–4R
8
8
100
0.43
3.42
UPR–6R
7
7
100
0.42
2.93
UPR–13R
9
9
100
0.43
3.9
UPR–17R
6
6
100
0.41
2.46
UPR–25F
4
4
100
0.26
1.03
UPR–30F
4
4
100
0.36
1.43
UPR–38F
5
5
100
0.37
1.84
SCoT
SCOT–1
3
3
100
0.33
0.98
SCOT–2
8
8
100
0.42
3.4
SCOT–11
4
4
100
0.34
1.37
SCOT–13
11
11
100
0.44
4.82
SCOT–22
3
2
77
0.25
0.5
SCOT–28
10
10
100
0.44
4.42
SCOT–36
8
8
100
0.45
3.59
CDDP
KNOX–1
4
4
100
0.37
1.48
KNOX–2
2
2
100
0.25
0.5
KNOX–3
6
6
100
0.39
2.32
WRKY–1R
7
7
100
0.4
2.82
WRKY–2R
7
7
100
0.37
2.78
WRKY–3R
6
6
100
0.41
2.46
Discordance between dendrograms
obtained by SCoT and DAMD with CDDP
could be explained by the different nature
of each technique, region coverage of
genome by each marker, level of
polymorphism and the number of loci
[SOUFRAMANIEN and GOPALAKRISHNA 2004; GORJI et al.,
2011]. Our results were in agreement with
the previous reports about clustering
varieties using different marker systems in
potato [GORJI et al., 2011], chickpea [HAJIBARAT et
al., 2015] and wheat [HAMIDI et al., 2014].
The MI, general rate of efficiency in
discovering polymorphism [KHODADADI et al.,
2011], was different in three marker
systems (Table 3).
On the other hand, an important
property of a suitable marker system is its
capacity to distinguish among different
accessions.
Our study revealed that the
resolving power of SCoT and DAMD
primers is higher than CDDP primers.
These results were in accordance
with previous studies [KHODADADI et al., 2011].
This study has implications not only
just for revealing the genetic diversity
within the genotypes used, but also for
the management of genetic resources
and their uses in applied breeding
programs.
Information about current genetic
diversity permits the classification of our
available germplasm into various heterotic

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groups, which is particularly important to
hybrid/cross–breeding programs in rose.
Up to now the classical breeding
programs for rose at National Institute of
Ornamental Plants (NIOP) has maily
relied on beed morphological traits.
The current study concluded the
importance of molecular studies (easy,
rapid and informative markers) in
detecting genetic variation among
varieties in selecting diverse parents to
carry out a new crossing program
successfully.
SCoT markers produced large
numbers of amplification products per
genotype.
Figure 3. Dendrogram of the 20 local rose varieties using CDDP markers based on
genetic distance.
Table 4.
Mantel test correlation coefficients among similarity matrices obtained using CDDP, SCoT
and DAMD markers.
SCoT marker is a simple, low cost,
and reproducible marker system
compared with other marker systems,
such as ISSR and SSR [GORJI et al., 2011].
We propose that SCoT marker be
used in combination with SSR or CDDP
genetic analysis.
CDDP
SCoT
DAMD
CDDP
1
SCoT
0.92**
1
DAMD
0.85**
0.89**
1

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Figure 4. Dendrogram of the 20 local rose varieties using DAMD markers based on
genetic distance.
Ornamental plants are
heterogeneous and contain numerous
groups of species.
Both genetic diversity and
fingerprinting studies are of useful tools
which enable plant breeders to make
better decisions regarding selection of
germplasm to be used in crossing plans
[MILBOURNE et al., 1997; RUSSEL et al., 1997].
Conclusions
Our findings showed that SCoT
marker analysis was successfully
performed to evaluate the genetic
relationships among the local rose
varieties.
High polymorphism revealed by
SCoT could be used for molecular
genetics study of the rose varieties,
providing high–valued information for the
management of germplasm, improvement
of the current breeding strategies, and
conservation of the genetic resources of
rose species.
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Received: April 11, 2017
Article in Press: October 27, 2017
Accepted: Last modified on: November 20, 2017