Genetic Distance of Two Halyloxon salicornicum Populations As
Revealed By RAPD Markers
Fahd H. Al-Qurainy
Department of Botany and Microbiology
College of Science, King Saud University
P. O. Box 2455, Riyadh 11451
Saudi Arabia
Abstract: Genetic distance within and between two natural populations
of Halyloxon salicornicum Saudi Arabia was investigated at the DNA
level by analysis of RAPD fragments. Out of 60 random primers, which
were initially screened, 5 generated highly reproducible RAPD fragments
which were then used for further population analysis. With these primers,
24 discernible DNA fragments were produced and 10 (41.7 %) were
polymorphic. In addition, Dahna population showed greater
polymorphism than those from Thumama. The majority of RAPD
variation in Halyloxon salicornicum was found within rather than
between populations and Dahna has more variation than Thumama as
estimated by genetic distance.
Key Words: Halyloxon salicornicum, RAPD, Genetic Distance,
POPGENE
Introduction
Haloxylon salicornicum ( synonymous: Hammade eleganes; rimth in
Arabic ) a member of the chenopodiaceae is a succulent undershurb. The
species is present in almost all of Migahid`s (1978) phytogeographical
regions of Saudi Arabia. H. elegance is an effective sand-binder and is
widespread in a variety of desert habitates:wadi-terraces.Sandy
plains,gravel desert,etc.
Haloxylon salicornicum is one of the most common plant species in
sandy habitats. It is a palatable plant and has the ability to resist
overgrazing. This species has been considered as one of the most
promising species for reseeding deteriorating desert range vegetation and
for sand dune fixation.
The genetic structure of plant populations reflects the interactions of
different processes including long-term evolutionary history of the
species (shifts in distribution, habitat fragmentation, and population
isolation), mutation, genetic drift, mating system, gene flow, and selection
(Slatkin, 1987 ; Schaal et al., 1998 ). All these factors can lead to
complex genetic structuring within populations, which is often difficult to
resolve. Nevertheless, the development of a number of different DNA
markers has provided powerful tools for the investigation of genetic
variation within a species and can facilitate understanding of such
complexities (Mitton, 1994
Recent technological advances in DNA analysis, such as random
amplified polymorphic of DNA(RAPD), which is described by Williams
et al 1990, have greatly increased the understanding of the genetic make
up in a wide range of plant species.
Random amplified polymorphic DNA (RAPD) technology via the
polymerase chain reaction (PCR) has fast become a means of
investigating genetic diversity within and between populations and has
been applied to many plant species including Digitalis (Nebauer, et al
2000 ).
The technical simplicity of the RAPD technique has facilitated its use
in the analysis of phlogenetic relationships in several plants genera; e.ge
Hordeium (Gonzales and Ferrer 1993), Allium ( Wilkie et al. 1993) and
Populus( Castiglione et al.1993) Furthermore,RAPD markers can be used
to detect genetic variation between closely related cultivares (Kresovich
et al 1992), Brassica (Jain et al., 1994), Lens
(Abo-elwafa et al. 1995), Petunia (Cerny et al. 1996) and Pisum
(Hoey et al . 1996). Additionally, this type of markers are extremely
useful to estimate genetic distances, mainly due to their
productivity in terms of number of markers per essay, sensibility,
quickness, and possibility of automation (Laucou et al. 1998)..
To my knowledge, there is no report on the application of molecular
markers to study the population structure of Haloxylon salicornicum.
Thus, an understanding of the extent and distribution of genetic variation
within Haloxylon salicornicum populations is also essential for devising
sampling strategies, which efficiently capture genetic diversity for
selection trials and subsequent use of material that fulfils the dual aim of
high genetic variation and reasonable performance
Materials and Methods
Plant material
Leaf material was collected from 10 trees from 2 populations namely
Thumama and Dahna. Trees were sampled along a linear transect within
each population and the minimum distance between sampled trees was set
as 100 m, to reduce the chance of sampling closely related individuals.
DNA extraction
Total celluler DNA was extracted based on a modified CTAB
procedure (Doyle and Doyle, 1990). DNA purity and concentration was
checked by electrophoresis in 1.4 % agarose gel containing ethidium
bromide in 1X TBE buffer and by measuring absorbency ratio
OD260 \ OD280 (Williams et al.1990).
RAPD assay
Decamer random primers were used for amplification of data palm
DNA. Primers which consistently revealed informative patterns through
out all the accessions were used to generate the basic data set, and was
used for analysis of the genetic polymorphism in the employed varities.
Eighty arbitrary 10-base primers ( Operon Technologies Inc.) were used
for amplification of targeted DNA based on the protocol of Williams et al
(1990) with minor modification. Amplifications was carried out in 25 ul
of reaction mixture containing 2 ul 10x PCR buffer, 2.5 ul (100mM)
dNTP`s, 4 ul (10ng /ul) Taq DNA polymerase 1 ul (25ng/ul) of genomic
DNA and 15 ul deionized water.
PCR program
DNA amplification was performed in a Perkin Elmer Thermal Cycler
480. A preliminary step at 94 for 5 min. followed by 35 cycles consisting
of a annealing step at 36 for 1min. and an extension step at 72 for 2 min,
ending in a final step at 72 for 10 min.
All the reactions were repeated at least twice, and only the consistently
reproducible bands were considered.
Gel electrophoresis.
TBE (Tris,Boric acid and EDTA) gel of 1.4% agarose was used. PCR
product was carefully placed in an eppendorf tubes and mixed with the
ethidium bromide ( 0.5 µg / ml ) and run at 100 volts for 2 hours. Stained
gel was visualized and photographed on UV transilluminator.
Data analysis:
Genetic distance (GD) among individualtrees in the study populations
was estimated according to the Nei ( 1978 ) formula :
GD = 1−[2Nxy / (Nx
Ny)], where Nx is the number of bands in
individual X, Ny is the number of bands in individual Y, and Nxy is the
number of RAPD bands present in both X and Y. The GD value was
calculated.
Popgene ( Yeh 1997 ) used to calculate the frequency of polymorphic
bands in each population.
Results and Discussions
the development of a number of different DNA markers has provided
powerful tools for the investigation of genetic variation within a species
and can facilitate understanding of such complexities (Mitton, 1994 ).
Recent technological advances in DNA analysis, such as random
amplified polymorphic of DNA(RAPD), which is described by Williams
et al 1990, have greatly increased the understanding of the genetic make
up in a wide range of plant species.
RAPD – PCR reactions were performed with 10 indivisibles from two
populations namely Thumama and Dahna.
To identify primers that detect polymorphism, 60 primers were screened
and five primers produced polymorphic RAPD banding profiles were
used (Tables 1 ). In total, 5 primers yielded a total of 24 bands for
Halyloxon salicornicum. On average 41.7 % of 24 fragments scored in
Halyloxon salicornicum were polymorphic for both populations
( Thumama and Dahna ). 21.9 % of scored bands were polymorphic for
Thumama population , and 39% for the Dahna populations. The number
of markers scored per primer ranged from 2 to 8. Primer OPA1 gave the
highest and primer OPB5 the lowest ( Fig. 1 ). The number of shared
bands between indivisuals were scored and used to calculate genetic
distance ( table 2 ).
The majority of RAPD variation in Halyloxon salicornicum was found
within rather than between populations and Dahna has more variation
than Thumama as estimated by genetic distance ( table 2 ). This low
variation within Thumama population could be due to the restricted gene
flow and small population size. The genetic distances are ranged from
0.030 – 0.260 and 0.022 – 0117 for Dahna and Thumama populations
respectively. That most genetic diversity existed within populations for
Halyloxon salicornicum is consistent with the general trend in other
outcrossing species (Huff et al., 1993; Nesbitt et al., 1995) based on
RAPD variation. Plant species differ markedly in the way genetic
diversity is partitioned between populations. The pattern of partitioning is
correlated with the mating system and life-history parameters (Hamrick
and Godt, 1989). Species that are primarily outcrossing and long-lived
have most of their genetic diversity partitioned within populations The
results probably correlated with the large population size of Dahna, which
contains more genetic variation.
More primers, populations and individuals are recommended to get more
understanding of genetic variations within and among populations of
Halyloxon salicornicum and factors influencing the variation.
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