ASSESSMENT OF GENETIC DIVERSITY IN SOME WILD PLANTS OF ASTERACEAE FAMILY BY RIBOSOMAL DNA SE-QUENCE

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Egypt. J. Genet. Cytol., 41: 195-208, July, 2012
ASSESSMENT OF GENETIC DIVERSITY IN SOME WILD
PLANTS OF ASTERACEAE FAMILY BY RIBOSOMAL DNA SE-
QUENCE
M. H. AMAR1, A. H. M. HASSAN1 AND ESRAA A. M. EL SHERBENY2
1. Egyptian Deserts Gene Bank, Desert Research Center, North Sinai, Egypt
2. Department of Plant Genetic Resources, Desert Research Center, Cairo, Egypt
he Asteraceae (Compositae, alter-
nate name) with its approximately
1,620 genera and more than 23,600 spe-
cies is the largest family of flowering
plants. The family is distributed world-
wide except for Antarctica and is especial-
ly diverse in the tropical and subtropical
regions of North America, the Andes,
eastern Brazil, Southern Africa, the Medi-
terranean region, central Asia, and south-
western China. In Egypt, family
Asteraceae is represented by 98 genera
and 228 species, annuals, perennials, wide
spread in all its phytogeographical re-
gions. Many of these genera have eco-
nomic potentialities, e.g. folk medicine
(Bolous, 2002).
With the rapid development of mo-
lecular biology studies of Asteraceae
germplasm identification and genetic di-
versity offer numerous reliable molecular
marker information by means of Random
Amplified Polymorphic DNA (RAPD)
(Badr et al., 2012), Restriction Fragment
Length Polymorphism (RFLP) (Ito et al.,
2000), Inter-simple sequence repeat
(ISSR) (Gharibi et al., 2011), Simple Se-
quence Repeat (SSR) (Simko, 2009),
RAPD, ISSR and RFLP (Abd El-Tawab et
al., 2010), amplified fragment length pol-
ymorphisms (AFLP) (Czarnecki et al.,
2008) etc. Nevertheless, DNA-labeling
techniques still have many problems par-
ticularly the wild medicinal herbs such as
low repeatability, high subjectivity of ex-
perimental results and unshared study data
from different laboratories (Wang et al.,
1999). Thus, molecular sequence markers
have become a significant tool in species
classification, dependent on sequencing
the differences in genome directly without
environmental effects (Li et al., 2010).
Accordingly, advances in DNA se-
quencing techniques have allowed the
extensive use of short DNA fragments,
especially of the ribosomal DNA (rDNA).
The utility of genes coding for ribosomal
RNA (rDNAs) is found in the ubiquitous
presence and relative conservation of
many regions of their nucleotide sequenc-
es. A cluster of rDNA (Fig. 1) consists of
the gene coding for 18S rRNA (small sub-
unit ribosomal RNA, SSU rRNA), two
internal transcribed spacers (ITS1 and
ITS2) separated by the 5.8S rRNA gene,
and the gene coding for the 28S rRNA
(large subunit of ribosomal RNA, (LSU
rRNA) (Hillis and Dixon, 1991). Addi-
tional two external transcribed spacers
(ETS) are located upstream of the 18S
T

M. H. AMAR et al.
196
rDNA and downstream of the 28S rDNA.
A nontranscribed spacer (NTS) separates
adjacent copies of the rDNA repeat unit.
Both spacers (ETS and NTS) are also
called intergenic spacers (IGS) (Dabert et
al., 2006).The major concerns with the use
of the rDNA locus in taxonomic and phy-
logenetic analyses are the existence of
polymorphisms among repeated units,
which may cause extensive differentiation
even within a single individual and pro-
vide useful tools for phylogenic studies
(Wei et al., 2010). Accordingly, sequence
comparison of the rDNA region is widely
used in taxonomy and molecular phyloge-
ny. It has typically been most constructive
for variation between species, populations
and even individuals (or inbred lines) as in
tomato (Jo et al., 2009), rice (Chang et al.,
2010), apple (Giaretta et al., 2010), and
Compositae, Anthemideae (Sonboli et al.,
2012).
In this study, we examined the mo-
lecular divergence of complete rDNA
sequenced of ITS and IGS regions among
four wild species of Asteraceae
germplasm in Egypt. To our knowledge,
even now there has been no report about
the comparison of ITS and IGS sequences
and their efficiency and utility as molecu-
lar markers in Asteraceae family.
MATERIALS AND METHODS
A. Plant Materials
A total of four wild and endemic
species of Asteraceae family (Echinops
spinosus L., Achillea santolina L.,
Matricaria recutita L. and Artemisia
monosperma Delile) were collected from
different eco-geographical localities of the
natural habitat in Sinai protected area,
Egypt. In Egypt, these species is recorded
as rare, endemic and neglected wild me-
dicinal taxon. Five plants from each spe-
cies were collected together as bulk mate-
rials. The list of species and their collec-
tion sites (Latitude longitude and altitude)
are presented in Table (1).
B. Methods
1. DNA Isolation
Total genomic DNA was isolated
from fresh leaves following the procedure
as previously described by Pirttila et al.
(2001). Five DNA samples of each species
were dissolved together as bulk DNA. The
quality and concentration of the DNA
samples were checked in a UV-1601 spec-
trophotometer (Shimadzu, Japan) and a
portion of the DNA was diluted to 50
ng/μl for use in ITS and IGS analyses.
Both the stock and diluted portions were
stored at -20C.
2. PCR amplification and sequencing of
the ITS region
Complete ITS region of rDNA was
amplified with universal primers ITS-1 (5'
TCCGTAGGTGAACCTGCGG3') as
forward primer and ITS-4
(5'TCCTCCGCTTATTGATATGC 3') as
reverse primer as described by White et
al. (1990). Final reaction volumes of 25 μl
each contained 50 ng genomic DNA, 0.5
pmol of each primer, 0.2 mM dNTPs, 1U
Taq DNA polymerase (Fermentas, Shen-
zhen, China), 2 µl of 10x PCR buffer sup-

GENETIC DIVERSITY IN SOME WILD PLANTS OF ASTERACEAE FAMILY
197
plied by the manufacturer and about 2.5
mM MgCL2. The amplification pro-
grammed consisted of pre denaturation at
94C for 4 min; 35 cycles at 94C for 45
s, 55C for 60 s, 72C for 90 s, and a final
incubation at 72C for 7 min. Then the
PCR products were subjected to electro-
phoresis on a 1.5% agarose gel, stained
with ethidium bromide, and visualized
under ultraviolet (UV) light. The PCR
fragments of each sample were excised
and purified from the gels using
E.Z.N.A® Gel Extraction Kit (Omega
Bio-Tek, Inc., Norcross, USA). The puri-
fied products of the PCR were ligated to
pMD18-T Easy Vector using the appro-
priate kit (TaKaRa, Tokyo, Japan) and the
ligation products were transformed into
Escherichia coli DH5α competent cells.
The recombinant clones were selected on
Liquid Broth media plates containing am-
picillin.
3. PCR amplification and sequencing of
the IGS region
The primer pair 18S L (5’-
GAACGCCTCTAAGTCAGAATCC-3’)
and 28S R (5’-
ACTGGCAGAATCAACCAGGTA-3’)
was used to amplify across the IGS region
of ribosomal DNA (White et al., 1990).
The reaction mixture (25 μl) containing 2
µl of 10x PCR buffer, 0.2 mM dNTPs, 0.5
pmol of each primer, 1U Taq DNA poly-
merase (Fermentas, Shenzhen, China), and
50 ng genomic DNA template. The PCR
conditions were; 95C for 5 min for initial
genomic DNA denaturation; 35 cycles of
94C for 1 min, 57C for 45 s, 72C for 1
min, and final extension at 72C for 7
min. Then the PCR products of each sam-
ple were excised, purified and cloning
were separated and visualized with the
same procedure as for ITS. Three positive
colonies from each amplified amplicons
were selected for sequenced by the Uni-
Gene Company (Shanghai, China). The
Open Reading Frame Finder (http://
www.ncbi.nlm.nih.gov/gorf/gorf.) was
used to verify the credibility of the results
and their conformity. Sequence similarity
was analyzed using BLAST
(http://www.ncbi.nlm.nih.gov/BLAST/).
4. Sequence analysis
Vector sequences were cleaned and
the sequences were aligned using Clustal
X version 1.81 (Thompson et al., 1997)
with manual adjustments wherever neces-
sary. Gaps were positioned to minimize
nucleotide mismatches. The MEGA pro-
gram version 5.0 (Molecular Evolutionary
Genetics Analysis, Tamura et al., 2011)
was employed to estimate GC and AT
contents, nucleotide substitution, nucleo-
tide diversity (π), estimated values of tran-
sition/transversion bias (R), substitutions
(r) for each nucleotide pair, and cluster
analysis among the four Asteraceae
germplasm. We further computed Maxi-
mum Composite Likelihood (MCL) Esti-
mate of the pattern of nucleotide substitu-
tion according to Tamura et al. (2004).
5. Phylogenetic analysis
Pair-wise evolutionary distance
among four Asteraceae family was deter-
mined by Kimura 2-Parameter method

M. H. AMAR et al.
198
(K2P) (Kimura, 1980). The Maximum
likelihood (ML) tree phylogenetic tree
was conducted using MEGA version 5.
RESULTS AND DISCUSSION
1. Sequencing analysis for ITS and IGS
In this article, we used a compara-
tive analysis approach using several pa-
rameters like nucleotide frequency, nucle-
otide substitution (r), nucleotide diversity
(π), and the estimated values of transi-
tion/transversion bias (R) to provide better
understanding of the genetic diversity and
phylogenetic relations across the studied
genotypes of the Asteraceae family. The
results of the confrontation between DNA
sequence analysis of the isolates and
GenBank database ranged from 94 to 99%
similarity, through BLAST search (Table
1), supporting good credibility for ITS and
IGS. The length of variation for the entire
ITS (650 to 750 bp) and IGS (800 to 950
bp) regions showed very distinctive se-
quences for individual species. Similarly,
variation was observed in the nucleotide
composition of the ITS and IGS, which
may be due to the sequence length varia-
tion of the analyzed markers (Table 2).
With regard to ITS sequence diver-
gence among taxa, the averages of nucleo-
tide frequencies were A (25%), T (24%),
C (26%), and G (25%) with an average of
GC (51%) and AT (49%) contents (Table
2). The highest numbers of nucleotide
frequency for ITS sequence was observed
in Artemisia monosperma (729 bases),
whereas the lowest one was recorded in
Achillea santolina L. and Matricaria
recutita L. (712 base). The maximum nu-
cleotide percentage for GC content (53%)
was observed in Echinops spinosus L.
However, the lowest GC content (46%)
was recorded in Artemisia monosperma.
Within the analysis of IGS sequence di-
vergence among taxa (Table 2), the aver-
ages of nucleotide length were A (25.1%),
T (26.7%), C (26.1%), and G (22.1%)
with an averages of GC (48.2%) and AT
(51.8%) contents. The highest numbers of
nucleotides for IGS sequence were ob-
served in Achillea santolina L., Artemisia
monosperma, and Matricaria recutita L.,
(640 bases). Whereas, Echinops spinosus
L. recorded the minimum number of nu-
cleotide frequency (365 bases). The max-
imum nucleotide percentage for GC con-
tent (53.6%) was observed in Artemisia
monosperma. In contrast, the lowest GC
content (46.3%) was recorded in Achillea
santolina L. The Tajima's Neutrality test
(Tajima, 1989) was performed to calculate
the nucleotide diversity value (π). There
were a total of 754 and 667 positions
across the final dataset for ITS and IGS
sequences, respectively. The nucleotide
diversity rate (π) was observed higher in
IGS (0.60) as compared to ITS sequence
(0.49) (Table 3).
Within genomes, all organisms
have DNA sequences that code for ribo-
somal RNA (rRNA), an essential compo-
nent of cellular protein synthesis machin-
ery (Kollipara et al., 1997). Ribosomal
RNA typically accounts for about 40% of
all transcription within a cell, and riboso-
mal RNA makes up as much as 80% of
cellular RNA (Moss and Stefanovsky,

GENETIC DIVERSITY IN SOME WILD PLANTS OF ASTERACEAE FAMILY
199
1995). Owing to relatively rapid evolu-
tion, differences in sequence and/or length
of rDNA are possible between closely
related species of Asteraceae family (Zhao
et al., 2010). The IGS sequences, as an
intergenic region, may bear functional
sequences, such as promoter, enhancer,
transcription stop signals, and reproduc-
tion start signals (Dutta and Verma, 1990).
Meanwhile, the IGS sequences undergo
conversion and concerted evolution to
reach a homogenization within an array of
repeats. Subsequently, the intergenic
spacer of the rDNA cluster evolves quick-
ly and is highly polymorphic sequence,
providing a useful tool for assessing the
sequence phylogeny and genetic variabil-
ity studies (Singh et al., 2008).
2. Phylogenetic analysis
Based on the sequence data of the
flanking regions of ITS or IGS sequence,
a phylogenetic tree was constructed using
Maximum likelihood (ML) method
(Tamura et al., 2004) (Fig. 2 and 3). Max-
imum likelihood tree using Kimura two
parameter distances (K2P) was created
among the four Asteraceae germplasm, to
provide a combined graphic representation
of the patterns of divergences with ITS
rDNA sequence data (Fig. 2). Within the
group, two strongly supported clades were
clearly distinguished among the four spe-
cies of Asteraceae family. With regard to
the first clade, Achillea santolina L. and
Artemisia monosperma were grouped to-
gether in the first clade. Within the second
clade, Echinops spinosus L. and
Matricaria recutita L., were included in a
sister clade. With respect to IGS rDNA
sequence data (Fig. 3), Achillea santolina
L., was closely related to Matricaria
recutita L., in the first clade, while
Echinops spinosus L. shared individually
with the first clade. In contrast, Artemisia
monosperma was placed independently in
a separate clade (clade 4).
Taxonomic characterization lead-
ing to unambiguous identification of spe-
cies and varieties is critically important
for conservation and sustainable utiliza-
tion of the Asteraceae germplasm. In
Asteraceae family, molecular phylogeny
at various taxonomic levels has been ex-
amined in several earlier studies through
application of isozymes and RAPD (Ayers
and Ryan, 1999), AFLP (Huang et al.,
2009), ISSR and RFLP (Abd El-Twab et
al., 2010), SSR (Iqbal et al., 2011), as
well as chloroplast DNA and rDNA mark-
ers (Sonboli et al., 2012). In context, Dai
et al. (2008) found that some closely re-
lated cultivars with identical ITS sequenc-
es in rice could be clearly discriminated
based on the phylogenetic tree constructed
by IGS sequences. In subsequent studies,
(Plovanich and Panero, 2004; Dai et al.,
2008; Li et al., 2010) confirmed that IGS
sequences with the fastest rate of evolu-
tion could provide more hierarchical dis-
tinctions than ITS sequences. Therefore, it
was concluded that the IGS region could
be more suitable for measuring genetic
relationship in different cultivars of sub-
species, with more informational sites
than ITS sequences in the Asteraceae
germplasm.

M. H. AMAR et al.
200
3. Transition and transversion
It is a well-known fact that during
DNA sequence evolution the rate of tran-
sitional changes differs from the rate of
transversional changes, with transitions
generally occurring more frequent than
transversions. This difference is often re-
ferred to as transition bias, and estimation
of the extent of transition bias may be of
interest (Cortey et al., 2011). In Table (4)
the substation pattern and rates were esti-
mated to compare the similarity matrix
under the Tamura-Nei 93 test model
(Tamura and Nei, 1993). The highest tran-
sition/ transversion rate ratios were rec-
orded among IGS sequence data (k1 =
38.28, purines), (K2= 12.58, pyrimidines),
respectively. Meanwhile, the lowest tran-
sition/ transversion rate ratios were ob-
served among ITS sequence data (k1 =
2.983, purines), (K2= 2.746, pyrimidines),
respectively. Moreover, the overall transi-
tion/transversion bias for IGS sequence
data (R = 12.10) was superior compared to
ITS sequence data (R = 1.43). This re-
flects that transitions are more dominant
than transversion in Asteraceae
germplasm across IGS sequence. This is
compatible with the results of Wetzer
(2001), who reported that transitions occur
more frequently than trans-versions, even
though for any given nucleotide position
twice as many possible transversions may
occur as transitions. In the context the
results of Wang et al. (2011) elucidate that
transitional substitutions at 3’UTR are
more common than transversions and
transitions are even more frequent than
transversions at CpG sites compared with
non-CpG sites. The Recent investigation
by Kruger et al. (2012) elucidated that in a
genome higher frequency of transition
occurred than transversions substitutions.
In the existing study, our result
from the IGS sequences confirmed the
feasibility of utilizing these sequences for
the study of species or intraspecies of
Asteraceae germplasm than ITS sequence.
Consequently, through previous results we
can confirm that IGS sequence divergence
seems to be the most appropriate regions
as a significant molecular marker for clas-
sification, taxonomic and identification at
the species level and beyond in
Asteraceae germplasm.
In conclusion, the assessment of
spacer length variation and rDNA poly-
morphisms in the rDNA genes in
Asteraceae germplasm provides new in-
sights in understanding the genetic varia-
bility among ecotypes and confirms that
this is a useful region for genetic variabil-
ity studies and phylogenetic relationships
in Asteraceae germplasm. Despite the fact
that Asteraceae germplasm is wild family,
which has not yet been cultivated, its nu-
tritional composition alone makes it an
important resource. Therefore, focused
research and development efforts are
needed if this wild species can be raised
from obscurity and improved sufficiently
to contribute to the food supply in Egypt.
SUMMARY
Ribosomal DNA genes are orga-
nized in clusters of tandem repeated units,
each of which consists of coding regions

GENETIC DIVERSITY IN SOME WILD PLANTS OF ASTERACEAE FAMILY
201
(18S, 5.8S and 28S) and two internal tran-
scribed spacers (ITS), in addition to
intergenic spacer (IGS) region. Accord-
ingly this article is focused on clarifying
the sequence divergence of complete
rDNA of ITS and IGS regions among four
wild and endemic species of Asteraceae
family in Egypt. Results indicated that
there were a total of 754 and 667 positions
across the final dataset for ITS and IGS
sequences, respectively. IGS regions were
superior compared to ITS region in sever-
al parameters like nucleotide diversity rate
(π = 0.60), the estimated values of transi-
tion/transversion rate ratios (k1 = 38.28,
purines), (K2 = 12.58, pyrimidines) and
the overall transition/transversion bias (R
= 12.10), respectively. This reflects that
transitions are more dominant than
transversion in Asteraceae germplasm
across IGS markers. Thus, it was conclud-
ed that the IGS region could be more suit-
able for measuring genetic relationship in
different subspecies of Asteraceae, with
more informative sites than ITS sequenc-
es. Generally ribosomal DNA particularly
intergenic spacer of the rDNA cluster
evolves quickly and is highly polymor-
phic, providing a useful tool for assessing
genetic diversity, taxonomic and phyloge-
netic studies in Asteraceae germplasm.
ACKNOWLEDGEMENTS
This work was financially sup-
ported by the open fund of the National
Key Laboratory of Crop Genetic Im-
provement and the Bioversity Interna-
tional (Rome, Italy). Special thanks are
given to Bioversity International for their
valuable support. Special thanks to Mr.
Wen-Fang Zeng, Mr. Nishawy, Mr. M.
Eweas and Mr. Xin-Jian Zhang for their
constructive comments and help.
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M. H. AMAR et al.
206
Table (1): The eco-geographical localities of the four natural habitats of Asteraceae family.
Scientific name
Code
Location name
G. P. S. location
ITS similarity (%)
IGS similarity (%)
Latitude
Longitude
Altitude
Echinops spinosus L.
1
Rafah
31 15 57.1 N
034 08 57.7 E
270 F
AY538629 99
EU649670 96
Achillea santolina L.
2
El Gora
31 07 56.7 N
034 08 11.4 E
270 F
EU179212 99
AB359892 94
Matricaria recutita L.
3
Elsheik Zowaied
31 14 06.9 N
034 06 56.6 E
85 F
AY603253 99
GU818112 95
Artemisia monosperma L.
4
Elsheik Zowaied costal
31 13 59.8 N
034 06 55.3 E
97 F
GU724289 99
AB359871 94
Table (2): The evolutionary analyses using Tajima test among ITS and IGS sequences.
Scientific name
ITS nucleotide frequency
AT
CG
Scientific name
IGS nucleotide frequency
AT
CG
T(U)
A
G
C
Total
T(U)
A
G
C
Total
Achillea santolina L.
23.5
25.3
23.9
27.4
712
49.0
51.0
Achillea santolina L.
33.4
20.3
17.8
28.4
640
53.8
46.3
Artemisia monosperma L.
22.1
22.5
27.7
27.7
729
45.0
46.0
Artemisia monosperma L.
17.8
28.6
33.6
20.0
640
46.4
53.6
Avg.
24 %
25 %
25%
26 %
719
49 %
51 %
Avg.
26.7%
25.1%
22.1%
26.1%
571.3
51.8%
48.2%
Echinops spinosus L.
23.9
23.5
28.5
24.2
724
47.0
53.0
Echinops spinosus L.
21.9
31.5
19.2
27.4
365
53.4
46.6
Matricaria recutita L.
26.3
24.0
25.8
23.9
712
50.0
50.0
Matricaria recutita L.
33.6
20.0
17.8
28.6
640
53.6
46.4
Max.
26.3
25.3
28.5
27.7
729.0
50.0
53.0
33.6
31.5
33.6
28.6
640.0
53.8
53.6
Min.
22.1
22.5
23.9
23.9
712.0
45.0
46.0
17.8
20.0
17.8
20.0
365.0
46.4
46.3
Total
2877
2285

GENETIC DIVERSITY IN SOME WILD PLANTS OF ASTERACEAE FAMILY
207
Table (3): Nucleotide frequencies for ITS and IGS sequence among four natural habitats of
Asteraceae family.
Sequence
type
Number of
Sites (M)
Number of
positions (N)
Number of
Segregating (S)
Nucleotide
Diversity (π)
ITS
4
754
530
0.49
IGS
4
667
595
0.60
Table (4): Maximum composite likelihood estimate of the pattern of nucleotide substitution
matrix for combined data of ITS and IGS sequences. Each entry is the probability
of substitution (r) from one base (row) to another base (column). The rates of dif-
ferent transitional substitutions are shown in bold and those of transversional sub-
stitutions are shown in italics.
ITS
IGS
A
T
C
G
A
T
C
G
A
-
4.92
5.33
16.1
-
0.99
0.96
37.02
T
4.91
-
14.62
5.4
0.81
-
12.11
0.97
C
4.91
13.51
-
5.4
0.81
12.43
-
0.97
G
14.66
4.92
5.33
-
30.98
0.99
0.96
-
k1 =
2.983
k2 =
2.746
R =
1.43
k1 =
38.28
k2 =
12.58
R =
12.10
Over all mean distance = 286 SE = 8.15
Over all mean distance = 160
SE = 4.94
(k1) (purines) = Transition rate ratios, (K2) (pyrimidines) = Transversion rate ratios.
(R) = Transition/transversion bias.
Fig. (1): The elementary structure of (rDNA) of plants.
1 rDNA repeat unit
ITS 1 ITS 2

M. H. AMAR et al.
208
Fig. (2): ML tree generated among four Asteraceae genotypes based on ITS rDNA data.
Fig. (3): ML tree generated among four Asteraceae genotypes based on IGS rDNA data.