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International Journal of Plant Biology 2016; volume 7:6307

Molecular identification of Rosa Molecular study on the genetic framework of


these plants requires large yield of DNA and Correspondence: Sayed Amer, Biology
x damascena growing in Taif therefore, method described by Ibrahim10 was Department, Faculty of Science, Taif University,
region (Saudi Arabia) used for this purpose with few modifications. Taif, 888, Saudi Arabia.
The inter-specific relationships within Rosa Tel.: +96.6559822001 - Fax: +96.6127343414.
Sayed Amer,1,2 Salih A. Basaid,1 E-mail:yasser92us@yahoo.com
remain controversial. Different names were
Esmat Ali1,3 given to Rosa species based on the morpholog- Key words: Maturase K; rose varieties; Rosa dam-
1Biology Department, Faculty of Science, ical variants and hybrids.11 The genetic studies ascene; Saudi Arabia; Taif-rose.
Taif University, Saudi Arabia; 2Faculty of on the genus were primarily based on
Science, Cairo University, Egypt; isozymes12 and random amplification of poly- Contributions: SA, laboratory setting and manu-
3Horticulture Department, Faculty of morphic DNA (RAPD)-PCR.13,14 Microsatellite script writing; SB and EF executing idea and
analyses for both wild and cultivated roses experimental design; KA reading and revising the
Agriculture, Assiut University, Egypt
were also conducted by Baydar and manuscript.
colleagues.15 The sequences of the internal
Conflict of interest: the authors declare no poten-
transcribed spacer (ITS) were used by several tial conflict of interest.
Abstract researchers.16-18 The chloroplast matK
gene16,19 and the atpB-rbcL intergenic spacer18 Received for publication: 13 November 2015.
were conducted for phylogenetic resolution of Revision received: 25 December 2015.
A fragment of 772 bp of the chloroplast mat-
the genus and its subgenera, however, the Accepted for publication: 25 December 2015.
urase K gene was amplified and sequenced for
Rosa x damascena trigintipetala variety grow- genetic controversial is still standing. These
This work is licensed under a Creative Commons
investigations explained partly the extremely

ly
ing in Taif region of Saudi Arabia. The data Attribution-NonCommercial 4.0 International
were aligned with their counterparts of other low levels of sequence divergence observed License (CC BY-NC 4.0).

on
varieties already found in the Genbank data- across the genus.18,19 On the other hand DNA-
base and were analyzed by maximum-parsimo- based molecular marker systems are efficient ©Copyright S. Amer et al., 2016
ny, neighbor-joining and maximum-likelihood and informative for genetic analysis of roses Licensee PAGEPress srl, Italy

e
because DNA polymorphism indicated by these International Journal of Plant Biology 20165; 7:6307
methods and a single rooted tree was execut-
doi:10.4081/pb.2016.6307
ed. R. x damascena trigintipetala was para-
phyletic where one sample [A] clustered with
all varieties while the second [B] was basal. R. i.e., Restriction
us
markers is not affected by environmental con-
ditions. Several molecular marker systems,
Fragment Length
al
x damascena was sister to R. x chinensis sem- Polymorphism (RFLP),20 RAPD,21,22 Simple
perflorens with the later being basal. R. x dam- Sequence Repeat (SSR)23 and Amplified
ci

ascena gori was basal for all taxa studied. R. Fragment Length Polymorphism (AFLP)15 have
Materials and Methods
moschata was inside the clade of R. x damasce-
er

been used for genetic analysis of rose species,


na. Hybridization could be possible among R. cultivars and rootstocks. Samples collection and DNA
damascena, R. chinensis and R. moschata. The
m

R. x damascena is a famous species within extraction


genetic distance and tree topology indicated this genus that possesses different varieties of Fresh young leafs of 8 samples of R. x dam-
m

that [A] variety could be originated from R. which R. d. trigintipetala and R. d. semperflo- ascena were collected from the Botanical
moshata while [B] could be originated from rens. R. x damascena trigintipetala (the so- Garden at Faculty of Science, Taif University,
co

gori or R. chinensis semperflorens. We, there- called Ward taifi) is highly cultivated in Taif Taif, Saudi Arabia (5 samples from Taif variety
fore, may consider that R. x damascena gori or governorate and there are different debates Rosa x damascena trigintipetala, 2 samples
R. chinensis could be the origin of all nowadays about its origin. This Taif-rose is the famous from Rosa x damascena gori and one sample
on

R. x damascena varieties. rose cultivated in different areas of Taif city at from Rosa x damascena French variety).
the western region of Saudi Arabia and is also Approximately, 300 mg of the leaf tissues were
N

known as Damask rose. There are two vari- ground separately to a fine powder under liq-
eties of Damask Rose, such as Autumn uid nitrogen with the use of pre-chilled mortar
Introduction and pestle. The method detailed by Ibrahim10
Damask rose (R. x damascena semperflorens)
and Damask rose Kazanlik (Rosa x damascena (CTAB-PVP- βME) was used to extract DNA
Rosa is a genus of an ornamental plant con- form the plant tissues with some modifica-
trigintipetala). It is known that Kazanlik in
taining hundreds of species and is one of the tions. These modifications were that the DNA
Bulgaria and Isparta in Turkey are the main
most cultivated plants in the world.1 Roses are precipitate was dissolved in 100 µL bi-distilled
areas of origin of R. x damascena (references
economically valuable as they contain essen- water. Finally, spin column (BioFlux, Tokyo,
tial oils which are a source of perfumes and cited in El-Assal and colleagues).24 Moreover,
Taif-rose has been suggested to be brought to Japan) was used to purify the extracted DNA
scents.2 The researchers have identified sig-
Taif from Balkans by Turks, who occupied this according to the manufacturer instructions.
nificant pharmacological properties of rose
extracts as it was used in radioprotective3 and area in the 14th century. However, El-Assal and
anti-inflammatory4 purposes. Compounds like colleagues24 indicated that Taif-rose has closed Polymerase chain reaction experi-
polysaccharides, polyphenols and other sec- genetic relations to the gory rose-Syrian culti- ments
ondary metabolites are found with high level vated in Syria. It was therefore necessary to PCR was conducted in a final volume of
in rose plants.5 They tightly bind with DNA and conduct PCR amplification and sequencing for 25 µL containing 2 µL DNA template, 0.2 µL of
inhibit its isolation.6 They co-precipitate with matK gene in order to identify genetically Taif- 10 Pmolar a forward primer (RosaL: 5`-CACT-
the DNA during its extraction and making it rose and construct its relationship to other TATCTTTCGGGAGT-3`), 0.2 µL of 10 Pmolar a
more viscous and also inhibit the polymerase varieties. reverse primer (RosaH: 5`-GAGTATCTTTTGAT-
chain reaction (PCR) enzymatic reaction.7-9 GCCAGA-3`), 12.5 µL PCR master mix

[International Journal of Plant Biology 2016; 7:6307] [page 7]


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(Promega Corporation, Madison, WI) and 10.3 Phylogenetic analysis replications of 10000 to confirm the robustness
µL autoclaved deionized water. PCR was car- The sequenced matK gene fragment of R. x of the ML tree topology. The distance option of
ried out using a PeX 0.5 thermal Cycler with damascena trigintipetala and R. damascena Tamura-Nei was adjusted for executing the
the cycle sequence at 94°C for 5 min one cycle, gori was aligned manually with its counter- neighbor-joining analysis.
followed by 35 cycles each of which consisted parts of other related taxa collected from the
of denaturation at 94°C for one min, annealing database (see their accession numbers on the
at 50°C for one min and extension at 72°C for tree) and the aligned data were used for phylo-
one min. A final strand elongation for one cycle genetic analysis with a total length of 772 bp. Results
at 72°C was done for additional 5 min. The The same sequence for R. californica was used
PCR products were analyzed in 1% agarose gel for rooting the tree. The phylogenetic analyses No morphological characteristics were
electrophoresis in TAE buffer (40 mm Tris, were done primarily by maximum-likelihood observed to differentiate between any of R. x
40mM acetic acid and 1 mM EDTA) with ethid- (ML) method with PAUP* 4.0b1025 by heuristic damascena samples and all specimens from
ium bromide staining. A 100-bp DNA ladder searches with the nearest-neighbor inter- each variety were identical. The extracted DNA
(Biolabs) was used as a molecular marker. change (NNI) branch swapping and 10 random of the studied samples was tested by elec-
PCR products were visualized under UV light taxon additions. The general reversible model trophoreses using 1% agarose and all samples
and photographed (Figure 1). The products (GTR+I) and parameters optimized by gave positive results (data not shown). The
were then purified using spin column Modeltest 3.026 were used. Bootstrapping repli- amplified fragments of the matK gene were
(BioFlux, Tokyo) according to the manufactur- cates were set to 300. Maximum-parsimony also examined on the agarose gel elec-
er instructions. (MP) and neighbor joining (NJ) were also con- trophoreses and the PCR profile was shown in
ducted with PAUP* 4.0b10 under bootstrapping Figure 2.
Unambiguous nucleotides of 772 bp of matK

ly
gene were sequenced for the collected sam-
ples. The base compositions of these data were

on
A=31.04%, C=17.50%, G=14.50% and
T=36.96%. Among the 772 sites included for
tree analysis, 766 were constant and 6 were

e
variables. Five variable sites were parsimony

us uninformative and only 1 was informative


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ci
er
m
m
co
on
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Figure 1. A neighbor-joining tree constructed from 722 bp matK


gene fragment sequenced in this study. The values at some nodes
refer to the bootstrapping of maximum-parsimony, neighbor- Figure 2. The amplified products of the chloroplast matK gene
joining and maximum-likelihood analyses. The numbers between for eight rose samples. R. x damascena trigintipetala (lanes 1-5),
brackets are the accession numbers of the sequences collected R. x damascena gori (lane 6) and R. x damascena Finish variety
from the Genbank database. (lanes 7-8). Lane 1 represents 100 bp DNA ladder (M).

Table 1. Pairwise genetic distances among the different varieties of R. damascena populations. The distances were calculated from matK
data sequenced in this study.
R. x damascena R. x damascena R. x damascena R. x damascena R. x chinensis
trigintipetala [A] trigintipetala [B] (other varieties) moschata simperflornes
R. x damascena trigintipetala [A] - - - - -
R. x damascena trigintipetala [B] 0.003 - - - -
R. x damascena (other varieties) 0.000 0.003 - - -
R. x damascena moschata 0.000 0.003 0.000 - -
R. x chinensis simperflornes 0.0013 0.0013 0.0013 0.0013 -
R. x damascena gori 0.0013 0.0013 0.0013 0.0013 0.000

[page 8] [International Journal of Plant Biology 2016; 7:6307]


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under parsimony criterion. The constructed ter. All varieties of R. x damascena showed a R. x damascena trigintipetala varieties [A]
parsimony tree showed consistency index sister relationship to R. x chinensis semperflo- could be originated from R. moshata while the
(CI=1.00), homology index (HI=0.00), reten- rens with the later being basal. R. x damascena other [B] could be originated from the gori or
tion index (RI=1.00) and rescaled consistency gori appeared to the basal for all R. x damasce- R. chinensis semperflorens. We, therefore, may
index (RC=1.00). na studied. R. moschata was found to be inside consider that R. x damascena trigintipetala the
The ML analysis exhibited a negative log the clade of R. x damascena. It seemed, there- so-called Ward taifi was not the origin of all
likelihood score –lnL=1049.6. Both MP and NJ fore, that there was a possible hybridization nowadays R. x damascena varieties but the
methods executed a similar tree topology to among the three taxa of R. damascena, R. chi- gori or R. chinensis could be.
that given by ML method. The three methods nensis and R. moschata. The sequenced matK fragment was aligned
gave the same bootstrap probabilities at the The pairwise genetic distances among the with the complete gene sequence (1512 bp)
node clustering all varieties (Figure 1). The studied varieties are recorded in Table 1. The obtained from the Genbank database. This
best-fit model that explained the datasets was distance showed an identity among R. moscha- fragment was located between base 75 and
GTR+I exhibiting model parameters as fol- ta, R. x damascena trigintipetala [A] and the base 846 with a length of 772 bp. The sequence
lows: substitution rate matrix R (a)=3.97; R other varieties of R. x damascena (D=0.000). showed only 2 base substitutions within R. x
(b)=2.75; R (c)=0.00; R (d)=5.62, R (e)=2.76 On the other hand, the second variety of R. x damascena trigintipetala [B]. One of the two
and R(f)= 1.00. The proportion of invariable damascena trigintipetala [B] was similarly dis- substitutions was a transition within pyrim-
sites (I) was 0.947. tant from the [A] variety, the other R. x dama- idines in the third position of the codon where
In the NJ tree (Figure 1), a clustering of all scena varieties and R. moshata with a distance C114 was substituted with T114. The second sub-
varieties in one clade was shown with R. x of 0.003. R. x damascena gori and R. chinensis stitution was a transversion, in the second
damascena trigintipetala being paraphyletic. semperflorens were distant from all studied position, of C220 to G220. Only the later substitu-
One sample of R. x damascena trigintipetala varieties with genetic a distance 0.0013. tion was non-synonymous recording a change
[A] clustered with all available varieties while The genetic distance together with the tree

ly
of the amino acid number 74 from leucine into
the second sample [B] was basal to this clus- topology indicated, therefore, that one of both valine at C220 G220 (Figure 3). This mutation

on
makes R. x damascena trigintipetala variety
[B] close to both the gori and R. chinensis sem-
perflorens while the [A] variety is identical to

e
the other varieties of R. x damascena. The

us number below the letter referred to the corre-


sponding position of the nucleotide inside the
complete gene sequence.
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ci

Discussion and Conclusions


er

Using the protocol detailed by Ibrahim10


m

with some modifications described herein,


m

DNA necessary for molecular genetics and bio-


diversity applications were successfully
co

extracted from eight different Taif-rose sam-


ples containing high levels of ployphenolics
and secondary metabolites. According to the
on

protocol of CITAB mixed with PVP and ME fol-


lowed by DNA purification kit, a high pure
yield of DNA from fresh leaf tissue grinned
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with liquid nitrogen was obtained. The DNA


quantity and purity obtained herein was com-
parable to that obtained by other methods.6,7,9
Good DNA quality and quantity was clearly
shown. We did not measure the A/A ratio
(absorbance at 260/ absorbance at 280) and we
went directly to the PCR amplification to
ensure the little contamination of proteins,
polysaccharides or polyphenolics. This extrac-
tion procedure was found suitable for direct
PCR amplifications of nuclear or chloroplast
DNAs.
In the present procedure, β-mercap-
toethanol and high concentration of EDTA in
the extraction buffer made some temporary
Figure 3. The aligned amino acid sequences for the two varieties of R. x damascena trig- pores in the cytoplasm membrane, eliminating
intipetala [A] and [B] along with that of R. x damascena [C] found in Genbank database. some of soluble polysaccharides, polyphenols
The underlined letter showed the non-synonymous change in the variety [B]. Stars indi- and pigments improving the DNA yield of the
cate sequence identity. The missed data from both varieties A and B are denoted by X. rose plants and similar ornamental plants.5,6

[International Journal of Plant Biology 2016; 7:6307] [page 9]


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ascena trigintipetala was the common ancestor 1998;16:1-8. Genetic diversity in rosa as revealed by

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on
85.
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Huxely,29 who revealed that R. x damascena and essential oils. Plant Mol Biol Rep Molecular characterization of interspecific

e
originated partly as a hybrid from R. moschata. 1999;17:1-7. hybrids of scented roses using RAPD
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one variety of R. x damascena flowering during
autumn. Based on random amplified polymor-
9.
us
Khan IA, Awan FS, Ahmad A, Khan AA. A
modified mini-prep method for economi-
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23.
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Identification of cut rose (rosa hybrida)
al
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ci

sequence tagged microsatellite site mark-


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gory rose.24 These results are in agreement DNA extraction from young flower petals ers. Theor Applied Genet 2003;106:277-86.
er

with the present finding where R. x damascena of some medicinal plant species. Gene 24. El-Assal SE, El-Awady MA, El-Tarras A,
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m

Conser 2011;10:165-82.
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m

In conclusion, Taif-rose (Ward taifi) or R. x relationships in the genus rosa: new evi- (Rosa sp.) based on random amplified
damascena trigintipetala is not considered as dence from chloroplast DNA sequences polymorphic DNA, inter simple sequence
co

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on

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