Genetic Description of Acacia Species Based on Different Markers

INTRODUCTION
Genetic markers represent genetic differences between individuals or
species. There are three major types of genetic markers: (1) morphological
(classical or visible) markers which themselves are phenotypic traits or characters;
(2) biochemical markers, which include allelic variants of enzymes called isozymes;
and (3) DNA or molecular markers, which reveal sites of variation in DNA (Jones,
et al., 1997).
Morphological markers are usually visually characterized phenotypic
characters such as flower colour, seed shape, growth habits or pigmentation.
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Isozyme markers are differences in enzymes that are detected by electrophoresis
using specific stains. The major disadvantages of morphological and biochemical
markers are that they may be limited in number and are influenced by
environmental factors or the developmental stage of the plant (Winter and Kahl,
1995). DNA markers are widely accepted as potentially valuable tools for crop
improvement in rice (Mackill, et al., 1999), Wheat (Koebner and Summers, 2003),
maize (Tuberosa, et al., 2003) and oilseeds (Snowdon and Friedt, 2004.
Genetic markers are of great value in genetic research and practical
breeding programs, since they reflect the genetic variation among individuals.
Morphological markers, or mutations in morphological markers, or mutations in
genes with visible consequences, have been used in genetic studies since early in
the twentieth century (Hussium, et al., 2000).Isozymes loci are excellent
biochemical markers since they are usually co-dominantly inherited do show
pleiotropic effects, rarely exhibit epistasis and are not affected by the
environment. Isozymes have been used very successlly in certain aspects of
plant breeding and genetics as nearly neutral genetic markers (Tanksley, et al.,
1989). Unfortunately, the number of genetic markers provided by isozyme
assays is insufficient for most applications in plant breeding. As a result, even
with the use of isozymes as biochemical markers, the fill potential of genetic
mapping in plant breeding have not been achieved (Tanksley, 1983).
Proline is the only organic cytosolute which able to make the major
contribution or osmotic adjustment at sever salinity in roots, while in shoots and
spikes the contribution of Proline in osmoregulation might be reduced. We
concluded that there is no stable situation in usage of organic or inorganic
soluble components in osmotic adjustment in the cultivars and lines on different
salinity levels. This is happened not only in different cultivars but also in
different organs which conferring the contrasting opinions about the
physiological significance of Proline which has remained controversial among
physiologists. Many reports have pointed out that Proline is mostly
accumulated when plants growth ceased (Joly, et al., 2000).
RAPD is one of the widely used molecular markers, where it was applied in
determination of paternity, gene mapping, identification of markers linked to traits
of interest without the necessity for mapping the entire genome, plant and animal
breeding, to understand the complexity of the transmission cycles of insects
vectors and population and evolutionary genetics (Marcili et al., 2009 and Sharma
et al., 2009). This wide range of applications due to that tiny amounts of DNA are
sufficient for the amplification, no prior knowledge of a DNA sequence is required,
commercially primers kits are available, simple, no expertise is required, low cost
of the unit of assay, relatively quick, produce high number of fragments and able to
distinguish between closely related individuals (Hadrys, et al., 1992 and Bardakci,
2000).
Acacia is the common name for plants of the genus Acacia in the bean
family, Leguminosae. This genus consists of approximately 1,100-1,200 species
primarily of trees, but also including some shrubs and climbers. Globally
distributed, acacia species are located in Asia, Madagascar, the Caribbean and
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Pacic islands, the Americas, and most prominently in Australia and Africa in arid
and semiarid tropical zones. Those tropical regions that have long, dry winters and
short wet summers often support shrubby vegetation known as thorn scrub or
savanna. Acacia trees constitute much of the woody vegetation in such plant
communities (Ross, 1981). Because of the wide distribution of Acacias in the arid
lands, and their multiple uses that include fodder, fuel, medicine besides the
environmental values of soil fixation and fertility (Shaw, et al., 2002). Acacia is the
second largest genus in the family Leguminosae with about 1350 species
(Maslin, 2003 and Maslin et al., 2003).The current classification of Acacia
differentiates three subgenera (Ross,1979 and Maslin et al., 2003): Acacia,
Heterophylum and Aculeiferum, Acacia raddiana belongs to the acacia
subgenus. The base chromosome number in the genus Acacia is x=13 with
polyploidy occurring in several species, (Blakesley et al., 2002; Khatoon and
Ali, 2006). There are 129 Acacia species in Africa. They are intermediate in plant
succession and colonize degraded land. They restore soil fertility and can be
maintained indefinitely in agricultural systems. Despite their benefits, they are
disliked for their thorns and invasiveness (Barnes, 2001). Acacia are allelopathic,
and their toxic aqueous leachates are used to detect differences in the patterns of
expression of cytoplasmic root proteins in crop plants, indicative of biochemical
alterations at the cellular level (Bukhari, 2002).The main objective of the current
research is genetic description and phylogenetic relationships among Acacia
species in Egypt based on different markers by calculate the morphological
variation among Acacia species, assay peroxidase activity and proline content,
estimate the level of polymorphism via RABD-PCR markers.
MATERIALS AND METHODS
A- Morphological Marker:
Leaves of five Acacia species: Acacia Tortilis ssp. radiana, Acacia
farnesiana, Acacia stenophylla, Acacia sclerosperma and Acacia saligna were
collected completely random from 20 individual Acacia trees natural habitats along
different localities in Egypt i.e. Abis Station Farm, Faculty of Agriculture Saba
Basha., Borg Al-Arab, Marsa Matroh City and EL-Gara (Siwa Oasis). Leaf lengths
(cm), pinna length (cm), leaflet length (mm), spine length (mm) were measured. In
addition to, some qualitative characters recorded such as leaves type, growth form,
crown shape, stem number and spine shape.
B- Biochemical Marker:
Leaves from each species were grounded separately, using a cooled mortar
with a pestle, and adding 0.23 M Tris-acetate, pH 5.0. Homogenate was extracted
by the solution containing Tris (27.7 g) and citric acid (11.0 g) in 1L volume
adjusted with distilled water. Electrophoresis was carried out by the prescriptions
recommending 1% agar-starch-polyvinyl-pyrrolidone gel and Tris-orate or Trisacetate
separation buffers. Electrophoresis was conducted at 270 v, 4ºC for 100
min. 100 ml of0.01 M acetate buffer pH 5.0, containing 0.1% benzidine and 0.5%
hydrogen peroxide (H2O2) were layered over the gel immediately before staining.
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Proline was determined according to the method of Bates et al. (1973) by 3%
Aqueous Sulfosalicylic Acid, Acid Ninhydrin:1.25 g Ninhydrin ,30 ml glacial acetic
acid,20 ml 6M phosphoric acid.
C- Molecular Marker:
RAPD has been developed, in which DNA is amplified by the polymerase
chain reaction (PCR) using arbitrary short (10 nucleotides) primers (Williams et al.
1990). DNA extracted from 50 mg samples of leaves using either the DNeasy Plant
System. RAPD analyze was carried out using 5 oligonucleotide primers (Table 1)
that were selected from the Operon Kit (Operon Technologies Inc., Alabameda,
CA). The polymerase chain reaction mixture (25μl) consisted of 0.8U of Taq DNA
polymerase; 25pmol dNTPs; 25pmol of primer and 50ng of genomic DNA. PCR
amplification was performed in a Biometra T1 gradient thermal cycler for 40 cycles
after initial denaturation for 3 min at 94°C. Each cycle consisted of denaturation at
94°C for 1min; annealing at 36°C for 1min; extension at 72°C for 2min and final
extension at 72°C for 10min (Williames, et al. 1990). Amplification products were
separated on 1% Agarose gels at 100 volts for 1.30 hrs with 1 x TBE buffer. To
detect ethidium bromide/DNA complex, Agarose gels were examined on ultraviolet
transilluminator (302nm wavelength) and photographed.
Table 1. Primers name and their oligonucleotide sequences used in the study
primer number primer Code Sequence 3/---5/
1 OPA-18 AGGTGACCGT
2 OPB-03 CATCCCCCTG
3 OPC-02 GTGAGGCGTC
4 OPD-03 GTCGCCGTCA
5 OPE-12 TTATCGCCCC
RESULTS AND DISCUSSION
A- Morphological Markers:
The morphological variations among the five Acacia species were calculated
using; spine, pinna, leaf and leaflet length. The results were recorded in Table (2).
Analysis of the variance showed high significant differences among the different
species concerning the marker measured. As for spine length (mm) data , it
indicates clearly highly significant variations among different species. The highest
values were recorded in Acacia Tortilis ssp. Radiana collected from(Siwa Oasis),
followed by Acacia Tortilis ssp. Radiana (collected from Borg Al-Arab city) in
means 28.75 and 19.25 mm, respectively. The lowest mean value was recorded in
Acacia farnesiana, the mean 6.50 mm with LSD=1.36. No spines were observed in
the other three species. Data showed inverse relationship between the spine and
pinna length, epically in the desert localities, these species grew in very hard
conditions such as drought, salt etc. the plants try to make modification in
increasing the spine length and vice versa decrease the pinna length to subject the
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biotic and Abiotic stress. The results showed that Acacia Tortilis ssp. Radiana
collected from Siwa and Borg Al-Arab, have the lowest values of pinna length (0.68
and 0.98cm), respectively, compared with the highest value of (4.28 cm) in Acacia
farnesiana.
For Leaf length (cm), data in Table 2 showed that Acacia sclerosperma and
Acacia saligna have the highest mean values (19.25and 26.08 cm), in respect.
While the lowest leaf length was recorded in Acacia Tortilis ssp. Radiana collected
from Siwa(2.85 cm), flowered by Acacia Tortilis ssp. Radiana collected from Borg
Al-Arab by mean (3.15 cm) and finally Acacia farnesiana with mean (3.80 cm),
while, no significant differences were observed among other species, the means
ranged from (2.0 to 2.50 mm) in the Acacia Tortilis ssp. Radiana collected
from(Siwa=2mm), followed by Acacia Tortilis ssp. Radiana collected from Borg Al-
Arab=2.50) and Acacia farnesiana=2.25mm.
High similarities were found between Acacia farnesiana and Acacia Tortilis
ssp. Radiana in leaves type it were pinnately compound. On the other hand,
Acacia saligna, Acacia sclerosperma and Acacia stenophylla had simple leaves
(Table 3).The same trend for growth form Shrub/small tree compared with other
spices were shrub or tree. For stem number, the highest number recorded for
Acacia farnesiana (2-5 stems) forward by both Acacia Tortilis ssp by (1-4 stems)
and finally Acacia stenophylla usually one stem. Concerning to spine shape, data
in Table 3 showed that, spine shape was small in Acacia farnesiana compared with
long white straight in both Acacia Tortilis ssp which collected from Siwa and Borg
EL-Arab.
Table 2. Morphological variations of Acacia species: spine length, pinna
length, leaf length and leaflet length
Leaflet
length (mm)
Leaf length
(cm)
Pinna length
(cm)
Spine
length(mm)
Species
Acacia farnesiana *6.50c 4.28a 3.80d 2.25a
Acacia Tortilis ssp.Radiana 19.25b 0.98b 3.15e 2.50a
(Borg)
Acacia Tortilis ssp. Radiana 28.75a 0.68c 2.85e 2.00a
(Siwa)
Acacia saligna - - 23.28b -
Acacia sclerosperma - - 26.08a -
Acacia stenophylla - - 19.25c -
L.S.D 0.05 1.36 0.252 0.508 0.705
*Mean followed by the same letter is not significantly different at 0.05 levels (-) not found
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Table 3 . Qualitative description of some Acacia species in Egypt
Spine
shape
Number
of stems
Crown
shape
Growth
form
Leaves
type
Species
often 2-5 small
spread
Shrub/small
tree
Pinnately
compound
Acacia farnesiana
Long white
straight
irregular/ 1-4
round
Shrub/small
tree
Pinnately
compound
Acacia Tortilis ssp.
Radiana (Borg Al-
Arab)
Long white
straight
irregular/ 2-4
round
Shrub/small
tree
Pinnately
compound
Acacia Tortilis ssp.
Radiana (Siwa)
Acacia saligna Simple shrub or tree spread 1 -
Acacia sclerosperma Simple shrub or tree spread 1 -
Acacia stenophylla Simple shrub or tree rounded usually 1 -
These results are in the line with Fatima, et al., (2011) who studied the
morphological variations on Acacia species in Morocco. The authors assessed the
variability in eight pod traits of 300 genotypes (mother-tree) of A. tortilis ssp.
raddiana (Savi) Brenan collected from southern regions of Morocco. The results
showed that, in the analysis of variance, that Acacia raddiana have significant
differences in all traits due to genotype within provenances, but only in pod length,
seed weight per pod, seed number per pod, infected seed number per pod and
100-seed weight due to provenances. Results showed that analyses of the three
traits showed significant species differences. Our study in agree with those
Boxshall and Jenkyn (2001) and Wasowski and Wasowski, (2003) which studied
the morphological variation in Acacia stenophylla, they proved whole description
for this species via morphological parameters such as spreading shrub or small
tree, tree tall, leaves, branches, leaf arrangement, leaf venation: pinnate, leaf
margin: entire leaf apex: acute, leaf base: oblique, size notes. These results is
alien with Boulos, (1999) and Orwa, et al.(2009) described that Acacia tortilis is a
small to medium-sized evergreen tree or shrub that grows up to 21 m tall. Leaves
glabrous to densely pubescent, glandular, short at 1.25-3.75 cm long; petiole 0.2-
0.9 cm long, with a gland; rachis 0.3-2 cm long.
B. Biochemical Markers:
- Peroxidase assay:
Peroxidase iso-enzyme assay was applied as the most appropriate
technique for the evaluation of wild acacia and domesticated acacia species .In
contrast, as shown in (Table4), Peroxidase isozymes exhibited a wide range of
variability among the different species at different localities. One cathode (Pex.C1)
were found as common band for all the species. While, five anodal (Pex.A1;
Pex.A2; Pex.A3, Pex.A4 and Pex.A5) bands were recorded for all species in
different molecular weight. (Pex.A2, A3 and A5) was recorded in Acacia
tortilisssp. radiana (Siwa), (Pex.A2,and A4) was recorded Acacia tortilis ssp.
radiana(Borg).While, Acacia stenophylla, and Acacia sclerosperma showed
(Pex.A3).and finally Acacia saligna showed Pex. A4. From the data it can be
conducted that the peroxidase patterns in the Acacia radiana (Siwa) wild and the
five domesticated Acacia plants leaves showed two kinds of banding profiles. First,
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it was evident that all plants expressed the (Pex.C1) and the five domesticated
plants exhibited the same banding profile containing the sesame loci. Indicated
that, these one common locus was consistently monomorphic expressed. Second,
the Acacia radiana (Siwa) wild types displayed one common locus (Pex.C1). The
banding pattern activity of Acacia displayed a unique marker band at(Pex.A5) locus
indicating that (Pex.A2, Pex.A3and Pex.A4) loci are polymorphic specifically.
Isozyme have been used as markers in a number of genetic studies, such
as genetic diversity in Brassica juncea Persson, et al., (2001). Peroxidase are
enzymes related to polymer synthesis in cell wall (Bowles, 1990), as well as in the
prevention of oxidative damage caused by environmental stress to the membrane
lipids (Kalir, et al., 1984). It was found that salt stress increased peroxidase bands
intensity. Acacia Tortilis ssp. radiana (Siwa) showed higher band intensity
compared with the other species. Plant peroxidase have been used as
biochemical markers for various types of biotic and Abiotic stresses due to their
role in very important physiological processes, like control of growth by
lignifications, cross linking of pectins and structural proteins in cell wall,
catabolism of auxins (Gaspar, et al., 1982).
Table 4 . Different loci of peroxidase activity among Acacia species
- Proline content (μmoles / g fresh weight)
Proline is an amino acid and compatible solute commonly accumulates
in many plants exposed to various stress conditions such as salinity. Under
stress condition, Proline is synthesized from glutamate due to loss of feedback
regulation in the Proline biosynthetic pathway (Boggess and Stewart, 1980).
Data in (Figure 1) indicated clearly that, Acacia Tortilis ssp. Radiana (Siwa) had the
highest value of proline content was 43.4 μmoles / g fresh weight compared with
the lowest one 7.6 μmoles / g fresh weight for Acacia sclerosperma. There were
highly significant variations among all species in relation to proline content and this
formula is gained by the environmental effects and conditions. Siwa oasis had
special conditions in addition to increase the level of salt soil compared with other
localities. Acacia Tortilis ssp. Radiana (Borg Al-Arab) had the second value in
proline content by 23.1 (μmoles / g fresh weight). The aforementioned results
supported the conclusion that proline was more accumulated in the salt, dry soil
genotype, and may be useful as a possible salt injury sensor in plants. This
variation of proline could be useful in selection for salt tolerance and used as a
marker of salt tolerant plants. Similar results were obtained by Shen and Shen,
(1992). They observed that under high NaCl concentrations, the percentage of free
Genotypes Pex.C1 Pex.A1 Pex.A2 Pex.A3 Pex.A4
◌
Pex.A5
Tortilis ssp.radiana(Siwa) 1 0 1 1 0 1
Tortilis ssp.radiana(Borg) 1 0 1 0 1 0
Acacia farnesiana 1 1 0 0 0 0
Acacia stenophylla 1 0 0 1 0 0
Acacia sclerosperma 1 0 0 1 0 0
Acacia saligna 1 0 0 0 1 0
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proline in total amino acids markedly increased in barley seedlings. Genotypic
variations in proline accumulation have been observed in many studies and
attempts were made to correlate its accumulation with tolerance of plants to
stress. This apparent correlation between proline accumulation and
environmental stress suggests that proline could have a protective function
(Ahmed and Hasan, 2011).
Figure 1. Proline content (μmoles/g fresh weight) in some Acacia species
C- Molecular Markers:
Plant molecular geneticists are currently used RAPD markers routinely to
identify genetic variations (Irwin et al., 1998 and Sun et al., 1998). RAPD markers
have been also used successfully in various taxonomic and phylogenetic studies
(Wilkie, et al., 1993). In addition, it locates regions of the genome linked to
agronomically important genes (Pillay and Kenny 1996). Furthermore, it facilitates
introgression of desirable genes into commercial accessions (Lavi et al. ,1994).
In a total of 156 fragments, DNA banding pattern OPA-18 primer is
presented in Table (5). Five fragments were produced in the six samples of Acacia
species with molecular weight ranging from 251 to 832 bp. Three monomorphic
and two polymorphic bands were recorded with polymorphism degree reach to
40%.While 18 fragments were observed with OPB-03 primer. Molecular weight
ranged from 469 to 2264 bp. one monomorphic DNA bands, eleven unique bands
and three polymorphic bands were recorded with polymorphism degree reach to
93.3%. Ten fragments with molecular weight 326 to 1503 bp were recorded for
OPC-02 primer and polymorphism degree reach to 70 %. Three monomorphic
DNA band, four unique DNA bands and three polymorphic bands were recorded.
The amplified DNA fragments of the studied of Acacia species with primer
OPD-03 are tabulated in Table (5). Eleven fragment with wide molecular weight
extended from 299 to 3178 bp. Three monomorphic bands, four unique bands and
four polymorphic bands were observed. This primer revealed polymorphism degree
reach to 72.7%. Finally, primer OPE-12 gave nine fragment bands with the six
samples of Acacia species their molecular weightwere 295 to 1600 bp. two
monomorphic bands, two unique bands and five polymorphic bands were recorded
with polymorphism level 77.8 %.
RAPDs are generated by applying the polymerase chain reaction (PCR)
to genomic DNA samples, using randomly constructed oligonucleotides as
primers. Since the technique is relatively easy to apply to a wide array of plant
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and animal taxa, and the number of loci that can be examined is essentially
unlimited, RAPDs are viewed as having several advantages over RFLPs and
DNA fingerprints. When the primers are of intermediate size (on the order of 10
base pairs), multiple amplifiable fragments (from different loci) are usually present
for each set of primers in each genome. The fragments can be separated by size
on a standard Agarose gel and visualized by ethidium bromide s t a i n i n g ,
elim inat ing the need for radio labeled probes. Since the primers consist of
random sequences, and do not discriminate between coding and nonbonding
regions, it is reasonable to expect the technique to sample the genome more
randomly than conventional methods.
Morphological and genetic diversity among Acacia aroma, A. macracantha,
A. caven, and A. furcatispina were studied with morphometric, isozymal, and
RAPD approaches by Paola et al. (2002). The analysis of seven isozyme systems
revealed 21 loci, and RAPD analysis showed 34 loci. Most of these loci allowed us
to differentiate the species, with the exception of A. aroma and A. macracantha,
the two most similar species. The levels of genetic variability estimated by
isozymes were higher than those obtained from RAPD analyses. Morphometric
characters showed highly significant differences among the species,
although A. aroma and A. macracantha are differentiated only by thorn length.
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Table 5. Polymorphism of the Acacia species amplified with different primers.
M.W. A.Tortilis
(wild)
A. Tortilis
(domes.)
A.
Farnesiana
A.
Stenophylla
A.
Sclerosperma
A.
Saligna Poly/Mon/Un.
OPA-18
832 1 0 0 0 1 0 polymorphic
783 0 1 1 1 0 1 polymorphic
524 1 1 1 1 1 1 Monomorphic
438 1 1 1 1 1 1 Monomorphic
251 1 1 1 1 1 1 Monomorphic
OPB-03
2264 0 0 0 1 0 0 Unique
2058 0 0 0 0 0 1 Unique
1706 0 1 0 1 0 0 Polymorphic
1616 1 0 0 0 0 0 Unique
1601 0 0 0 0 1 0 Unique
1479 0 0 0 0 0 1 Unique
1374 0 0 0 1 0 0 Unique
1218 0 0 0 0 0 1 Unique
1103 0 0 1 0 0 0 Unique
1029 1 1 0 1 1 1 Polymorphic
932 0 0 1 0 0 0 Unique
793 0 1 0 0 0 0 Unique
763 1 0 1 1 1 1 Polymorphic
609 0 0 1 0 0 0 Unique
469 1 1 1 1 1 1 Monomorphic
OPC-02
1503 0 1 0 1 1 1 Polymorphic
1240 0 1 0 1 1 1 Polymorphic
951 1 1 1 1 1 1 Monomorphic
759 1 1 1 1 1 1 Monomorphic
625 0 0 1 0 0 0 Unique
577 1 1 0 1 1 1 Polymorphic
542 0 0 1 0 0 0 Unique
483 0 0 1 0 0 0 Unique
428 1 1 1 1 1 1 Monomorphic
326 0 0 1 0 0 0 Unique
3178 1 0 0 0 0 0 Unique
3035 0 0 0 0 1 0 Unique
2150 1 1 1 0 1 0 Polymorphic
1595 1 1 1 0 1 0 Polymorphic
1123 1 1 1 0 1 0 Polymorphic
933 1 1 1 1 1 1 Monomorphic
833 0 1 0 0 0 0 Unique
732 1 1 1 0 0 0 Polymorphic
622 0 1 0 0 0 0 Unique
461 1 1 1 1 1 1 Monomorphic
OPD-03
299 1 1 1 1 1 1 Monomorphic
1600 1 0 0 0 0 0 Unique
1264 1 1 1 0 0 1 Polymorphic
763 1 1 1 0 1 1 Polymorphic
623 1 1 1 1 1 1 Monomorphic
488 0 0 0 0 1 1 Polymorphic
453 1 1 1 1 0 0 Polymorphic
392 1 1 1 1 1 1 Monomorphic
344 0 0 0 0 1 1 Polymorphic
295 0 0 0 0 1 0 Unique
The phonogram obtained from isozyme data is consistent with
morphological data. The RAPD phenogram based on allelic frequencies showed
agreement with morphological and isozymal approaches only at the intraspecific
levels, while the RAPD phenogram based on Nei and Li’s similarity measures
agreed with the phenograms constructed from isozyme and morphological data.
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High similarities and high indirect gene flow were found between A. aroma and A.
macracantha, results that call the relationship between them into question.
To study the genetic similarities and phylogenetic relationships among the
six tested samples of Acacia species were based RAPD-PCR. The obtained data
were subjected to cluster analysis with dice equation by using SPSS (ver.15)
computer program to calculate proximity matrix and design dendogram.Genetic
similarity values generated from RAPD marker varied between 0.60 and 0.78 with
an average of 0.69. Dendogram based on similarity values (Table 6) from RAPD
was constructed to reveal similarities between the five different Acacia species.
The dendogram (Figure 2) demonstrated that the sample of Acacia species fall into
two main groups. The first one was divided into two clusters containing 4 and the
second continue 5 and 6 in similarity from 73 to 77%. The second one divided into
two sub clusters. According to similarity, the first one contained 3 and the second
continue 1 and 2 in similarity from 71 to 74 %.
C A S E 0 5 10 15 20 25
Label Num +---------+---------+---------+---------+---------+
Tortilis (wild) 1─┬───────────────────────┐
Tortilis (domes.)2─┘ ├───────────────────────┐
Farnesiana 3 ─────────────────────────┘ │
Sclerosperma 5 ─────┬───────────────────┐ │
Saligna 6─────┘ ├───────────────────────┘
Stenophylla 4 ─────────────────────────┘
Figure 2. Dendrogram of similiratiy of different Acacia species based on 5
RAPD primers.
Table 6. DNA specific unique markers based on RAPD-PCR primers of
different Acacia species.
Species DNA specific unique marker Length (bp) Total
1-Tortilis ssp. radiana (wild ) 1600, 1616 and 3178 3
2-Tortilis ssp. radiana (domesticated) 793,833 and 622 3
3-Farnesiana 326, 483, 542, 609, 625, 932 and 1103 7
4-Stenophylla 1374 and 2264 2
5-Sclerosperma 295, 1601 and 3035 3
6-Saligna 1218, 1479 and 2085 3
Total 21
The characterization of a DNA sample by Random Amplified Polymorphic
DNA (RAPD) analysis, which is often referred to as DNA "fingerprinting", has
attracted considerable attention in the last ten years. RAPD is possibly the simplest
test of all recently applied DNA-based tests for date palm identification (Trifi et al.,
2000).RAPD as a molecular marker system has also been successfully applied in
cultivar identification. RAPD analysis is normally found to be easy to perform but
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has the major disadvantage that reproducibility is difficult to achieve between
different laboratories and often even between different people in the same
laboratory (Jones et al., 1997). Any diagnostic laboratory, which intends to use
RAPD analysis as a quality control tool, has, therefore; firstly to ensure constant
detection of identical DNA amplification products by several-fold repeated
experiments preferably by different people. Elimination of possible variation in both
DNA concentration and purity and assurance of consistent reaction conditions
maybe a first step to overcome difficulties with assay reproducibility (Williames et
al., 1990).
Fagg and Allison (2004) reported variation in chemical composition,
molecular as well as morphological characteristics between Ugandan and
Sudanese populations of A. senegal. Our results in a lien with Shrestha et al.
(2000) on A. raddiana populations and reported that there are a high degree of
polymorphism, contrary to the conventional expectation of small, isolated
populations. It is a maxim of conservation biology that the maintenance of genetic
variation is important because future evolutionary adaptation depends on the
existence of genetic variation.
Isozyme studies have also indicated that the West African provenances of
A. senegalvar. Senegal show little variation (Boer, 2002). Lower H values were
also obtained in four Argentinean species of Acacia by Casiva et al. (2002) using
isozymes and RAPD markers. Similar to the range of our H value, Playford et al.
(1993) found high levels of genetic diversity (0.208) in Acacia melanoxylon
population in association with a great genetic differentiation among geographic
areas.
The percent polymorphic loci (P) values obtained in this study were by far
higher than those observed in Acacia caven (29.4%) (Casiva et al., 2002), Acacia
anomala (43%) (Coates, 1988) and Faidherbia albida (42.7%) (Dangasuk and
Gudu, 2000). However, similar results were obtained in Haloxylon ammodendron
(74.9%) by Sheng et al. (2005) using ISSR markers, in Changium smyrnioides
(69%) by Fu, et al. (2003) using RAPD markers and in F. albida (90%) reported by
Joly, et al. (1992) using isozymes.
Several authors have studied the taxonomy of Acacia using morphological
characters (Vassal, 1972; Guinet and Vassal, 1978; Cialdella, 1984, 1997and
Pedley, 1986), in the last ten years some have used biochemical and molecular
markers instead (Playford, et al., 1992; Bukhari, 1997a and Clarke, et al., 2000).
Biochemical and molecular studies have been conducted on African and Australian
Acacia species to provide markers useful for plant breeding and conservation
programs (Moran, et al., 1989a, b; Muona, et al., 1991; Joly, et al., 1992; Sedgley,
et al., 1992; Playford, et al., 1993; Fagg, et al., 1997 and McGranahan, et al.,
1997). However, no population genetic studies have been carried out so far on
Argentinean species of Acacia.
Isozyme electrophoresis and random amplified polymorphic DNA (RAPD)
analysis are broadly used in plant population genetic studies (Soltis and Soltis,
1990; Avise, 1994; Soltis, et al., 1998 and Hollingsworth, et al., 1999). Mainly,
RAPD has allowed the resolution of complex taxonomic relationships (Voigt, et al.,
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1995; Comincini, et al., 1997; Cottrell, et al., 1997and Wolff and Richards, 1999).