5 Egypt. J. Bot., Vol. 55, No.1 pp.

61- 78 (2015)

Genetic Diversity Among Populations of the

Medicinal Plant Achillea fragrantissima (Asteraceae)
in Egypt

A.Badr*1, H.H. El-Shazly2, H.I. S. Ahmed3, M. Hamouda3,

E. El-Khateeb3 and M. Sakr4
1
Botany and Microbiology Department, Faculty of Science,
Helwan University, 11790 Cairo, 2Biological and Geological
Sciences Department, Faculty of Education, Ain Shams University,
11341 Cairo, 3Botany Department, Faculty of Science, Tanta
University, 31527 Tanta and 4Genetic Engineering and
Biotechnology Division, National Research Center, Giza, Egypt.

UBSTANTIAL variation, in morphological traits was observed

S among 20 populations of A. fragrantissima in Egypt. Such variation
was reflected in the clustering of the examined populations as major
groups, one representing populations in the mountainous area of South
Sinai and the other populations growing at lower elevations in the middle
of Sinai and the desert west of the Suez canal from Suez in the east to
Cairo in the west. Five populations in the eastern part of Sinai near
Nuwieba and Taba on the Gulf of Aqaba were loosely assigned to the
first group. The clustering of A. fragrantissima populations based on
ISSR markers also showed two major groups more or less similar to the
groups obtained from the analysis of morphological traits. The
populations growing at high elevations in South Sinai, under lower
temperature and higher humidity, were characterized by high number of
total and polymorphic ISSR markers compared to other populations.
Unique ISSR markers were observed in the fingerprinting of seven
populations including five populations growing in the high mountains
of Saint Catherine area in South Sinai and two populations growing at
low elevation South east of Cairo. A noteworthy observation is that
unique bands are found in populations that possess traits associated
with plant size and seed yield as well as better vigor. These are
important criteria for selection of populations for conservation and
commercial use of A. fragrantissima.

Keywords: Achillea fragrantissima, Genetic diversity, Egypt, ISSR

markers, Conservation

In many countries traditional medicine is wide spread and most of the medicinal
plants are harvested from the wild. Consequently up to 10 000 medicinal plant
species of the estimated 50 000 medicinal species might be endangered (Akerele
‫ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬
*
Corresponding author, [email protected].
62 A.BADR et al .

et al., 1991 and Edwards 2004). In Egypt and other arid countries, the medicinal
plants are threatened due to weak regeneration under frequent environmental
stresses such as drought and salinity which do not support viable populations
(Batanouny, 1999). In addition, heavy overuse by overgrazing and uncontrolled
collection, uncontrolled tourism, mining and quarrying and other human activities
resulted in habitat destruction and fragmentation; a problem that has been
exacerbated by the lack of knowledge and awareness, the paucity of research, and
the diminishing number of competent plant systematists (Ayyad , 2003 and Badr
et al., 2014a). In Egypt, a medicinal plants conservation project that was conducted
in the last decade recommended conservation of threatened medicinal plant species
for sustainable development (MPCP unpublished report, 2008). However, few
studies are available on the conservation genetics of these plants.

Referring to the general agreement on tariffs and trade (GATT), it is

important to assess the value of medicinal plants as important biological
resources. The conservation strategies should integrated into the development
plans of the Egyptian economy and ensure their sustainable use (Badr et al.,
2014a). Saving rare, threatened, and endangered medicinal plant species requires
diversity studies that should include extensive phenomic measurements and
genetic finger-printing of threatened populations using molecular technologies
and bioinformatics approaches for estimating the genetic diversity.
Morphological measurements provide quantifying genetic variation while
simultaneously assessing genotype performance under relevant growing
environments (Fufa et al., 2005).

DNA-based molecular markers are reliable sources of genetic diversity

because they provide unique genetic information for each species independent of
age, physiological conditions and environmental factors (Kalpana et al., 2004).
These markers have a great utility in the drug analysis and are widely used for the
characterization of medicinally important plant species (Tharachand et al., 2012).
Of the PCR based methods, the robustness repeatability makes the Inter-Simple
Sequence Repeats (ISSRs) less prone to changes in band patterns with changes
in DNA-template concentration. In ISSRs, the di- and tri-nucleotide repeat
types of microsatellite are specifically targeted, because these are widespread in
the nuclear genome. ISSR locus heritability has demonstrated an exceedingly
close approximation to classic Mendelian ratios and thus ISSR markers are
considered dominant markers and consequently effectively act as bi-allelic loci
(band presence vs. absence) (Zietkiewicz et al., 1994 and Godwin et al., 1997).

The distribution of Achillea fragrantissima (Forssk.) Sch. Bip., of the family

Asteraceae (Compositae), is restricted to the semiarid regions in North Africa and
the Middle East where it is often recorded as associated species in limestone soils.
In Egypt, its occurrence extends from Cairo southwards in the Wadis and sandy
plains of the Eastern desert eastwards to Suez, it is also encountered in Sinai and
some Oasis of the Western desert (Boulos, 2002). Achillea fragrantissima is a
fragrant, white woolly perennial herb, up to 1 m height, with a bitter taste. Leaves
Egypt. J. Bot., 55, No. 1 (2015)
 GENETIC DIVERSITY AMONG POPULATIONS OF … 63

small, 0.2-1 x 0.15-0.3 cm, sessile with serrate margins. Flowers yellow in
discoid head. Flowering and fruiting occur during late spring and summer. Plants
withstand the hot summer while keeping green leaves (Boulos, 2002).

Fresh or dry leaves and flowering shoots of A. fragrantissima are used for the
treatment of cough and as aromatic bitter stomachic, anthelmintic and
hypoglycaemic treatments as well as for treating diabetes, intestinal colic, lowering
blood cholesterol level and as a carminative, dysmenorrheal and various infections
(Boulos, 1983; Yaniv et al., 1987; Atayat, 1993 and Batanouny, 1999) . The aerial
parts contain compounds with therapeutic and pharmacologic uses and exert
biological activities against microorganisms, insects, animals and viruses. Essential
oil from A. fragrantissima exerts a bactericidic effect on several gram-positive and
gram-negative bacterial strains, as well as on Candida albicans (Barel and
Yashphe, 1989 &1991) and its extracts have antiviral activity against polio in a
concentration dependent manner at complete non-toxic concentration range
10–100 μg/ml (Soltan and Zaki, 2009)

The present study aimed at investigating the genetic diversity among

populations of A. fragrantissima in Egypt based on morphological variation and
ISSR markers profiling. The results should contribute to the selection and
conservation of useful germplasm of this important medicinal plant for future
sustainable exploitation.

Material and Methods

Twenty populations of A. fragrantissima were collected from sites covering

most areas where the species is distributed in Egypt as common populations
mostly from Sinai and Eastern desert from Suez in the east to Cairo in the west
(Fig. 1). Voucher specimens representing all populations have been deposited at
the Herbarium of Botany Department, Faculty of Science, Tanta University,
Tanta, Egypt (TANE). Detailed morphological criteria of a number of plants of
each population were scored based on careful examination of 28 traits including
18 quantitative traits and 10 qualitative traits. The average value of each
quantitative trait ± standard deviation was calculated and the state of the
qualitative traits was determined based on the deacription of the targeted
populations with reference to Boulos (2002). For data analysis, the morphological
traits were given codes ranging between 0 and 3 as given in the Supplementary
(Table 1.).

DNA extraction
DNA was extracted and purified from the germinated seedlings of individual
samples representing all populations using the Thermo Scientific Gene JET

Egypt. J. Bot., 55, No. 1 (2015)

 64 A.BADR et al .

Genomic DNA Purification Mini kit following the protocol of the manufacturer
istructions. However, some samples of DNA were extracted from material
collected from the field using the CTAB method with some modifications
(Saghai-Maroof et al., 1984).

THE

MEDITERRANEAN

SEA

Fig. 1. Map illustrating the areas and the sites of collection of the studied populations of A.
fragrantissima (F1 – F20) plotted as coded in Table 1.

Egypt. J. Bot., 55, No. 1 (2015)

19

0, F11
F9

F2
F3, F4
 GENETIC DIVERSITY AMONG POPULATIONS OF … 65

TABLE 1. Codes, sites, GPS information and elevation of the sites from which A.
fragrantissima populations were collected.

Elevation
Code Site GPS location
(m)
F1 Al-Tarfa (35 Km west of Saint- 28º 40´ 29.00'' N
1154
Katherine) 33º 49´ 59.00'' E
F2 28º 39´ 05.00'' N
El-Sheikh Awad, (Wadi Gharba) 1138
33º 39´ 06.00'' E
F3 28º 32´ 45.00'' N
Wadi El-Arbaein, Saint Katherine 1684
33º 57´ 14.00'' E
F4 28º 32´ 25.00'' N
Wadi El-Shak, Saint Katherine 1831
33º 55´ 53.00'' E
F5 Wadi El-Faranga (24 Km east of Saint- 28º 45´ 55.00'' N
1281
Katherine) 33º 03´ 44.00'' E
F6 Wadi El-Shogyrate (35KM east of 28º 46´ 16.00'' N
1240
Saint-Katherine) 34º 04´ 24.00'' E
F7 Wadi Zigzaga (50 Km east of Saint- 28º 46´ 18.00'' N
1083
Katherine) 34º 07´ 37.00'' E
F8 Wadi Al-Nawamees (60 Km east of 28º 49´ 13.00'' N
791
Saint-Katherine) 34º 20´ 17.00'' E
F9 29º 20´ 47.00'' N
Nuwieba, Wadi Wateer 694
34º 32´ 05.00'' E
F10 29º 31´44.00'' N
Wadi Grafi, Near Nuwieba. 764
34º 38´ 09.00'' E
F11 29º 39´ 17.00'' N
Wadi Grafi, 20 Km south of Taba 693
34º 41´ 22.00'' E
F12 30º 18´ 48.00'' N
Nakhl-El-Hasna, 47 Km before Hasna 339
33º 45´ 32.00'' E
F13 30º 01´ 33.00'' N
Wadi El-Gabry (45 Km after Mitla pass) 467
33º 14´ 33.00'' E
F14 30º 00´ 51.00'' N
Mid Sinai Mitla Pass 444
32º 57´ 11.00'' E
F15 29º 59´ 48.00'' N
50 Km after Ahmed Hamdi tunnel 419
32º 54´ 14.00'' E
F16 29º 59´ 13.20'' N
Wadi Hagul, Cairo Suez Road 302
32º 05´ 49.40'' E
F17 29º 55´ 38.40'' N
Bir-Gindali, south of Qattamia 390
31º 48´ 21.60'' E
F18 Wadi Abo-Syaal, Qattamia-Sukhna 29º 44´ 43.80'' N
293
Road 31º 53´ 49.20'' E
F19 Wadi Om-Khourba, Qattamia-Sukhna 29º 43´ 39.60'' N
245
Road, south east of Cairo 31º 56´ 00.00'' E
F20 Wadi Hof, south of Cairo 29º 52´ 43.00'' N
132
31º 22´ 28.00'' E

Egypt. J. Bot., 55, No. 1 (2015)

66 A.BADR et al .

ISSR primers and ISSR finger-printing

Twenty ISSR primers (Operon Nippon EGT CO. LTD.) have been secured for
DNA fingerprinting. The name, sequence, annealing temperature and GC ratio of
the selected 20 ISSR primers are given in Table 2. Isolated genomic DNA was used
as template in the amplification reactions using Thermo scientific Maxima Hot start
PCR Master Mix (2X). A total of 25 µl reaction mix was prepared (12.5 µl Maxima
Hot Start PCR Master Mix (2X), 0.5 µl Primer, 0.5 µl Template DNA and 11.5
nuclease-free water-R0581). Amplification conditions were optimized using a
gradient Biometra Uno thermal cycler, Germany. After several experiments for
optimizing the best conditions, a program for polymerase chain reaction (PCR)
was standardized with following settings, initial denaturation at 95°C for 4 min,
followed by 35 cycles of 30 sec. at 95°C for denaturation, 30 sec. for annealing
according to each primer annealing temperature, and 1min at 72°C for extension
and a final extension of 5 min at 72°C and stored at 4°C till removal of PCR tubes
within 12 hr.

Separation of ISSR amplification products

The amplification products were separated by mixing 20 µl of the PCR-
products of each primer and 2 µl of loading buffer and loading the mix into the
agarose wells. Electrophoresis was made in 1.7 % agarose gel prepared in 0.5 X
TAE buffer at 70 V for 3 hr. The ISSR fingerprinting was visualized using a Gel
Works 1D advanced gel documentation system (UVP, UK) and photographed
under UV light with Camera. The size of each band was estimated using 100 bp
DNA ladder (Fermentas) as a standard marker. In the meantime, the clear
unambiguous and reproducible ISSR bands were considered for scoring. For data
analysis, each ISSR band was considered a single locus and scored as 1 for its
presence and 0 for its absence.

Data analysis
The genetic diversity among the 20 populations of A. fragrantissima was
estimated based on variation in both morphological traits and molecular finger-
printting separately and in combination. The morplological and molecular data
were analyzed using two software programs; the Community Analysis Package
(CAP) version 4.0 (Seaby and Handerson, 2007) and the NTSYS-pc package
version 2.02 (Rohlf 2005). The Euclidean dissimilarity coefficient and distance
measures were calculated according to Legendre and Legendre (1983) using the
CAP software. The CAP software was also used to construct genetic distance
trees to illustrate the distance among the examined populations based on Ward
(1963). For tree construction, the agglomerative cluster analysis method in the
NTSYS-pc software was also used to construct trees elucidating the relationships
among the examined populations using the Neighbor Joining method (Saitou and
Nei, 1987) and the UPGMA method (Sokal and Michener, 1958).
Results

Morphological variation among populations of A. fragrantissima

Substantial variations, in the morphological quantitative traits have been
observed among the 20 populations and are detailed in Supplementary Table 2
Egypt. J. Bot., 55, No. 1 (2015)
 GENETIC DIVERSITY AMONG POPULATIONS OF … 67

(Available on request). The measured morphological traits indicated variation

between seven populations of A. fragrantissima (F1 to F7) growing in the Saint
Catherine area in South Sinai at eleveations ranginging between 1083 m and 1831 m
above sea level, four populations (F8 to F11) growing east of Saint Catherine and
extending to Taba and Nuwieba on the Gulf of Aqaba at elevations ranging
between 791 m asl and 693 m above sea level (Table 1 and Fig. 1) and nine
populations (F12 through F20) collected from localities in more dry and flat areas
in the middle of Sinai and the northern part of the eastern desert of Egypt from Suez
to Cairo at elevation ranging from 467 m above sea level at Wadi El-Gabry in the
middle of Sinai) to 132 m asl at Wadi Hof, south of Cairo (Fig. 2 and Table 1). The
populations growing in South Sinai area are growing at high elevations and
moderate temperatures, and have in general larger plant size compared to
populations at lower elevations in a more arid sites (Table 2).

NJ distance scale

Fig. 2. NJ tree showing the distance among the populations of A. fragrantissima

(F1-F20), based on the analysis of morphological traits using the NTSYS-pc
software (For populations site details see Table 1, Fig. 1) .

Egypt. J. Bot., 55, No. 1 (2015)

68 A.BADR et al .

TABLE 2. The name, sequence, annealing temperature and GC ratio and of the
selected 20 ISSR primers used for fingerprinting A. fragrantissima.

Serial Primer name Primer Annealing GC ratio

sequence temperature (ºC)
01 17898A (CA)6AC 42ºC 50.0%
02 17898 B (CA)6GT 42ºC 50.0%
03 17899 A (CA)6AG 44ºC 57.1%
04 17899 B (CA)6GG 44ºC 57.1%
05 HB-8 (GA)6GG 44ºC 57.1%
06 HB-9 (GT)6GG 44ºC 57.1%
07 HB-10 (GA)6CC 44ºC 57.1%
08 HB-11 (GT)6CC 38ºC 72.7%
09 HB-12 (CAC)3GC 38ºC 72.7%
10 HB-13 (GAG)3GC 38ºC 72.7%
11 HB-14 (CTC)3GC 38ºC 72.7%
12 HB15 (GTG)3GC 29.9ºC 72.1%
13 807 (AG)8T 34.3ºC 47.2%
14 809 (AG)8G 35.7ºC 52.9%
15 814 (CT)8TG 35.7ºC 50.0%
16 825 (AC)8T 35.1ºC 47.2%
17 834 (AG)8YT 35.1ºC 47.2%
18 841 (GA)8YC 38.8ºC 52.9%
19 UBC-820 (GT)8C 41.0ºC 52.9%
20 UBC-827 (AC)8G 41.0ºC 52.9%

Genetic diversity based on morphological variation

The genetic distance among populations was similar in all genetic distance trees; a
neighbor-joining distance tree constructed using NTSYS-pc is shown in Fig. 3. In this
tree, the examined populations are divided into two main groups at a total distance of
8 distance; one comprising populations F1, through F11 representing populations that
were collected from Saint Catherine mountains in south Sinai and the populations
from east Sinai as well as population F14 from the Mitla pass in the middle of Sinai.
The site of the latter population is a location where hills surround a narrow Wadi and
the plants were flourishing on the Wadi sides that seem to have been disturbed by
road construction few years ago. The second group comprises populations F12
through F20 which is more or less corresponds to populations collected from
locations in middle Sinai, and the desert west of the Suez Canal from Suez in the east
to Cairo in the west.

In the first group, the seven populations F1, F2, F3, F4, F5, F6 and F7, growing in
Saint Catherine area of South Sinai, were separated as one cluster from other five
populations (F8, F9, F10, F11 and F14) that were recognized as a second cluster at a
distance of 7.0. In the former cluster, the population F7 was distinguished from the
populations F1 to F6 at a distance of 6.0; at a distance of 5.0; F3 and were
differentiated from F1, F2, F5 and F6. In the second cluster, F8 was clearly
Egypt. J. Bot., 55, No. 1 (2015)
 GENETIC DIVERSITY AMONG POPULATIONS OF … 69

distinguished the population F14 as well as populations F9, F10, F11. In the second
group, population F16 from Wadi Hagul west of Suez was clearly separated, at a
distance of 7.0, from the other seven populations that were then divided into two
culsters at a distance of 6.0; one comprised populations F19 and F20, and the other
comprised populations F12, F13, F15, F18 and F17 (Table 1 and Fig. 2).

E
Fig. 3. Examples of ISSR finger printing produced by five primers in the 20 A.
E
fragrantissima populations. A = Pr-17898B showing 3 unique bands in F3, F2
and F19, B = Pr-HB-09 showing one unique band in F20, C = Pr-8.9 showing
absence of a band in F20, D = Primer HB-08 showing the largest number of
bands and the highest number of polymorphic bands, E =Pr- HB-13 showing
the highest number of monomorphic bands; M =100 bp marker, -ve =
negative control and F1 to F20 are the codes of populations as given in Table
1 and as shown in Fig. 1, Arrows indicate unique bands.
Egypt. J. Bot., 55, No. 1 (2015)
70 A.BADR et al .

ISSR fingerprinting in Achillea fragrantissima populations

The ISSR fingerprinting of the 20 A. fragrantissima populations as revealed
by 20 ISSR primers was scored in supplementary Table 2 and associated photos
(Available on request). Examples representing fingerprinting profiles by five
primers are shown in Fig. 3. Based on the score of banding profiles for the 20 A.
fragrantissima populations, the total number of bands as well as the number of
monomorphic, polymorphic and unique bands and the percentage of
polymorphism were calculated and given in Table 3. The number of bands and
the percentage of polymorphism are generally higher in the populations growing
in the mountainous area of South Sinai and low in populations growing at lower
elevations west of the Gulf of Suez to Cairo. Ten unique bands were scored in
seven populations including five populations growing in the high mountains of
Saint Catherine area in South Sinai (F1 to F5) and two populations growing in
south east of Cairo (F19 & F20). Three bands were scored in F3 and two in F20
and only one band in each of the other five populations (Table 3). A band that
was revealed in the profiles of 19 populations by primer 809 was absent in the
fingerprinting of population F20.

TABLE 3. Number of total, monomorphic, polymorphic and unique bands and the
percentage of polymorphism in 20 populations of A. fragrantissima.

Pop. Total number of Monomorphic Polymorphic Unique % of

Code bands bands bands bands polymorphism
F1 99 24 72 1 72.8%
F2 86 24 61 1 72.1%
F3 91 24 66 3 75.6%
F4 90 24 65 1 76.3%
F5 86 24 62 1 72.1%
F6 84 24 60 - 71.4%
F7 78 24 54 - 69.2%
F8 78 24 54 - 69.2%
F9 74 24 50 - 67.6%
F10 77 24 53 - 68.8%
F11 78 24 54 - 69.2%
F12 82 24 58 - 70.7%
F13 76 24 52 - 68.4%
F14 77 24 53 - 68.8%
F15 70 24 46 - 65.7%
F16 68 24 44 - 64.7%
F17 65 24 41 - 63.1%
F18 66 24 42 - 63.6%
F19 66 24 39 1 63.6%
F20 72 24 45 +2, -1 66.7%

Egypt. J. Bot., 55, No. 1 (2015)

 GENETIC DIVERSITY AMONG POPULATIONS OF … 71

Genetic diversity among A. fragrantissima based on ISSR markers

The genetic diversity among the examined populations of A. fragrantissima
based on ISSR markers, as expressed by a NJ tree constructed using the NTSYS
software, is illustrated in Fig. 4. In this tree, the examined populations are divided
as two main groups at a total distance of 8; one comprising populations F1, through
F11, which were collected from south and eastern Sinai and the other comprising
populations F12 through F20 which more or less corresponds to populations
collected from locations in middle of Sinai, and the desert from Suez to Cairo. The
topology of this tree shows that population F7 which was also differentiated from
populations F1 to F6 which represent populations growing at the mountainous area
of Saint Catherinein South Sinai. Populations F9 & F8 and populations F11 & F10
are clearly separated in this tree as two small clusters in group 1. These four
population were collected from sites in eastern part of Sinai extending from 60 km
east of Saint Catherine to Nuwieba and Taba on the Gulf of Aqaba. The second
group comprised populations F12 through F20 which is more or less correspond to
populations collected from locations in middle Sinai, and the desert west of the suez
canal from Suez in the east to Cairo in the west. In this group, F16 is clearly
differentiated from the other populations, which are divided in two clusters; one
composed of the populations F12, F13 and F14 and the other composed of
populations F15, F19 and F17 well as populations F18 and F20.

Fig.4. NJ tree showing the distance among the populations of A. fragrantissima (F1-
F20), based on the analysis of ISSR fingerprinting using the NTSYS-pc
software (For populations site details see Table 1, Fig. 1).
Egypt. J. Bot., 55, No. 1 (2015)
72 A.BADR et al .

Genetic diversity based on morphological variation and ISSR markers

The genetic diversity among the examined populations of A. fragrantissima
based on morphological variation and polymorphism in ISSR markers is
expressed by a Ward tree constructed using the CAP software (Fig. 5). In this
tree, the examined populations are divided into two main groups at a total
distance of 78.3; one comprising populations F1, through F11 including the
populations that were collected from south and east Sinai and the other
comprising populations F12 through F20 which more or less corresponds to
populations collected from locations in middle Sinai, and the west of the Suez
Canal from Suez in the east to Cairo in the west. The topology of this tree
resembles that of the tree based on analysis of ISSR markers but F 7 was
clustered with F8 & F9 was clustered with F11 & F10 in the group 1. In
additions, F14 from the Mitla pass in middle Sinia was grouped here in a cluster
of F12, F13 and F16 is at close distance with populations 14 & F12 and F13.
Both trees differ from the tree based on morphological variation where F14 was
grouped with populations F10, F11 and F9. However, the three agree in
differentiating populations of south and east Sinai from populations growing in
the middle of Sinai and west of the Suez Canal from Suez to cairo.

Fig. 5. A CAP Ward tree showing the relationships among the populations of A.
fragrantissima (F1-F20) based on the analysis of variation in morphological
traits and ISSR markers using the CAP software.

Egypt. J. Bot., 55, No. 1 (2015)

 GENETIC DIVERSITY AMONG POPULATIONS OF … 73

Discussion

The measurements of morphological traits for populations of A. fragrantissima in

the Saint Catherine area of South Sinai showed that these populations are more
similar to each other compared to populations growing in other parts of the study
area. In the South Sinai area, plant height is more than its height in the
populations in the middle of Sinai and to the west of Suez except for populations
F19 and F20. In that mountainous area, the temperature is much lower, the
humidity is higher in the non-mountainous areas in middle Sinai and west of
Suez. Gurevitch (1992) reported that environmental factors, such as temperature
and altitude can affect morphological characters, such as the size and
compactness of Achillea leaves.

The populations of A. fragrantissima in the mountains of South Sinai at

elevations ranging between 1154 m asl and 1831 m asl were clearly distinguished
from the populations F12 - F20 which grow at lower elevations in middle Sinai and
west of the Suez Canal from Suez to Cairo. The close genetic distance among
populations growing in that area is in agreement with variation in karyotype
features among populations of A. fragrantissima in Egypt as they have shorter
chromosomes (Sayed Ahmed et al., 2012) . In addition, populations F7, F8 and F16
that were distinguished based on morphological variation the two populations F10
and F11 were also differentiated based on ISSR markers are populations growing in
sites in the eastern part of Sinai extending from 60 km east of Saint Catherine and
extending east to Nuwieba and north to Taba on the Gulf of Aqaba. The results
generally indicate closer genetic affinities among geographically closer
populations.

Few studies have been conduct on Achillea, Rahimmalek et al. (2009) found
that the germplasm of A. santolina in Iran showed low genetic diversity, despite
the fact that the samples were collected from different geographical regions.
However, in A. santolina growing in Egypt, the morphological traits showed
much closer resemblance among populations compared to ISSR polymorphism
but agree with ISSR data in supporting the idea of a possible gene flow in
populations growing in close locations and limited gene flow among population
in geographically distant locations (Badr et al., 2014b). In A. fragrantissima,
(Rawashdeh et al., 2009, 2010) showed the existence of an association between
morphology and molecular analysis, especially in the populations of Shoubak and
Ma'an, which were recognized as separate groups. The results showed high
polymorphism indicating the presence of genetic variation among A.
fragrantissima populations. Morsy (2007) assessed the molecular variation of
five populations of A. fragrantissima in Sinai using RAPD and isozymes markers,
revealing that differences in locations were particularly reflected on DNA
fingerprints. Similar results were also found in Artemisia species in Egypt (Badr
et al., 2011) and Saudi Arabia (Badr et al., 2012).

Egypt. J. Bot., 55, No. 1 (2015)

74 A.BADR et al .

Population genetic diversity in a species is affected by a number of

evolutionary factors including mating system, gene flow, seed dispersal,
geographic range as well as natural selection (Hamrick and Godt, 1989). Of these
factors, the geographic range of a species appears to influence the levels of
genetic diversity of its populations. Representative population from the
geographical range of the species can help to ensure conservation of co-adapted
gene complexes (Beuselinck and Steiner, 1992). In the current study, the
grouping of populations growing in the mountainous area of South Sinai may be
attributed to differences in environmental variables particularly temperature,
humidity and soil due to the high altitude of that part of Sinai.

Meanwhile, unique ISSR markers characteristic for populations F19 and F20
south of Cairo may be regarded as molecular markers that differentiate these two
populations from the populations growing west of the Suez Canal and the middle
of Sinai taking into consideration the position of population F16. However, in the
current study, unique ISSR markers are mainly found in populations growing in
the mountainous area of Saint Catherine in South Sinai associated with traits of
plant size e.g. plant height and plant crown width and seed yield e.g. number of
florets in the head and weigh of 100 seeds as well as vigor estimated as the speed
of seed germination. This finding is in agreement with the view of Mittal and
Boora (2005) that unique markers are important criteria for selection of plant
populations for conservation. Unique markers may be regarded as markers for
genetic resources authentication and the establishment of property rights (Badr
et al., 2014a).

Acknowledgement: The authors are grateful to the Science and Technology

Development Fund (STDF) of Egypt for supporting the research work of this
article through funding the Project ID 4218 on Development of reference genetic
fingerprints, preservation of germplasm and biotechnology-based production of
pharmaceutically bioactive substances of some threatened Egyptian medicinal
plants.

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(Received 18/8/2014;
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‫‪78‬‬ ‫‪A.BADR et al .‬‬

‫التنوع الوراثيي بين عشائر نبات القيصوم العطري الطبي في مصر‬

‫‪3‬‬
‫عبد الفتاح بدر‪ ، 1‬هناء الشاذلي‪ ، 2‬وحنان إبراهيم سيد أحمد‪ ، 3‬مروة حمودة‬
‫‪4‬‬
‫ومحمود صقر‬
‫‪1‬قسم النبات والميكروبيولوجي ‒ كلية العلوم ‒ جامعة حلوان و قسم البيولوجي‬
‫‪2‬‬

‫والجيولوجي كلية التربية ‒ جامعة عين شمس و‪3‬قسم النبات ‒ كلية العلوم ‒ جامعة‬
‫طنطا و‪4‬قسم الهندسة الوراثية والتكنولوجيا الحيوية ‒ المركز القومي للبحوث ‒‬
‫مصر ‪.‬‬

‫تم تسجيل اختالفات جوهرية في صفات الشكل الظاهري بين ‪ 20‬عشيرة من نبات‬
‫القيصوم العطري في مصر‪ .‬وقد انعكس هذا التنوع بوضوح في شجرة النسب‬
‫للعش ائر التي تمت دراستها كمجمعاتين رئيسيتين‪ ،‬تمثل إحداهما العشائر التي تنمو‬
‫في المناطق الجبلية جنوب سيناء و تمثل األخرى العشائر التي تنمو في المناطق‬
‫األقل ارتفاعا في وسط سيناء والصحراء الشرقية غرب قناة السويس من مدينة‬
‫السويس الي القاهرة‪ .‬وقد أظهرت خمس عشائر في الجزء الشرقي من سيناء بالقرب‬
‫من نويبع و طابا في خليج العقبة ارتباطا بصورة ضعيفة بالمجموعة األولي‪ .‬كذلك‬
‫أظهرت شجرة النسب المبنية علي تباينات ‪ ISSR‬تمايز مجموعتين كبيرتين‬
‫تتشابهان نسبيا مع مجموعتي الشكل الظاهري‪ ،‬فالعشائر التي تنمو في في المناطق‬
‫المرتفعة بجنوب سيناء تحت درجات حرارة منخفضة ورطوبة تربة مرتفعة تميزت‬
‫بوجود أعداد كبيرة من حزم ‪ ISSR‬بالمقارنة بالعشائر األخري‪ .‬وقد لوحظت حزم‬
‫‪ ISSR‬فريدة في البص مة الوراثية لسبعة عشائر خمس منها تنمو في منطقة الجبال‬
‫المرتفعة بسانت كاترين بشمال سيناء واثنين في مناطق أكثر انخفاضا شرق القاهرة‪.‬‬
‫المالحظة الهامة األخرى هى أن هذه الحزم الفريدة موجودة في عشائر تتميز‬
‫بصفات ظاهرية مرتبطة بحجم النبات وإنتاج البذور وأيضا قوة النبات‪ ،‬وهذه صفات‬
‫مهمة الختيار عشائر القيصوم العطري التى يمكن اتخاذ تدابير لحمايتها وترشيد‬
‫استخداماتها التجارية‪.‬‬

‫)‪Egypt. J. Bot., 55, No. 1 (2015‬‬