Quantitative and Qualitative Genetic Studies of Some Acacia Species Grown in Egypt

The objective of the current work is to study the genetic differentiation between Acacia species growing in Egypt as plant genetic resources based on morphological, biochemical, and molecular markers. The 20 replicates of Acacia tree collected from four localities from Egypt were A. tortilis ssp. raddiana and A. farnesiana (Siwa Oasis and Borg El-Arab City), A. stenophylla, A. sclerosperma (Marsa Matroh City), and A. saligna (Abis Station Farm, Alexandria). The results based on the previous markers indicated highly significant differences between Acacia species, confirming the hypothesis of the possibility of using morphological, biochemical, and molecular parameters in species identification. Qualitative characteristics results indicated some similarities and differences that are taxonomically important for comparing taxonomical grouping with morphological data for the genetic description of Acacia species. The activities of antioxidant enzymes have been studied intensively and the results provide strong similarities between the Acacia species (69%), between A. raddiana (Siwa and Borg Al-Arab) and A. saligna, followed by all Acacia species (50%). Finally, the molecular studies showed that a total of 563 amplification fragments, 190 fragments were monomorphic, and 373 fragments were polymorphic. The highest number of amplification fragments (21) was detected with OPB-20 primer, while OPA-20 showed seven amplification fragments; the average number was 13.09. The results indicated that Acacia species exhibit high genetic differentiation, helpful in the future for genetic improvement programs. The novelty of the current study is highlighting the importance of plant genetic resources in Egypt and using different techniques to measure the differentiation between these species.


Introduction
Egypt is well known for its expansive desert, as approximately 80% of the country is desert. This desert is largely composed of sand dunes, sheets, bed rock, sandstone, limestone, and salt marshes. In the western desert of Egypt, there are a series of oases stretching longitudinally from the north to the far south in a line almost parallel to the Nile valley. This represents natural depressions rich in natural springs and wells that can be used for agriculture. One of the advantages of this desert is the presence of wild flora adapted to biotic stresses such as high temperature, drought, and salinity.
Plants 2020, 9,243 3 of 18 approaches by Casiva et al. [22]. The analysis of seven isozyme systems revealed 21 loci, and RAPD analysis showed 34 loci. Most of these loci allowed us to differentiate species, except for A. aroma and A. macracantha, the two most similar species. The levels of genetic variability estimated by isozymes were higher than those determined from RAPD analyses. Morphometric characteristics were highly significantly different among the species, although A. aroma and A. macracantha were differentiated only by thorn length.
Proline is an amino acid and compatible solute that commonly accumulates in many plants exposed to various stress conditions, such as salinity. When a plant is subjected to stressful conditions, proline is synthesized from glutamate due to the loss of feedback regulation in the proline biosynthetic pathway [23]. The measurement of proline accumulation is an important criterion for determining plant tolerance to salt stress [24]. In salt-stressed plants, the osmotic potential of the vacuoles is reduced by proline accumulation [25]. It is believed that proline accumulation under environmental stress does not inhibit biochemical reactions and plays a protective role during osmotic stress [25]. It is suggested that low osmotic potential may cause proline accumulation in tissues [26].
Modern tools in molecular biology provide detailed information about the genetic structure of natural populations, which was previously not available. During domestication, genetic variation in crop plants decreased due to continuous selection pressure for traits such as great yield or disease resistance. It is necessary to compare the genetic composition of the germplasm of existing cultivars to their ancestors and related species. Genetic differentiation among populations is principally a function of gene flow via pollen and seed dispersal. Several authors have studied the taxonomy of Acacia using morphological characteristics [27]. In the last decade, some studies have alternatively used biochemical and molecular markers [28]. Biochemical and molecular studies have been conducted on African and Australian Acacia species to provide markers useful for plant breeding and conservation programs [29]. Isozyme electrophoresis and random amplified polymorphic DNA (RAPD) analysis are broadly used in plant population genetic studies [30]. RAPD has primarily enabled the resolution of complex taxonomic relationships [31]. Additionally, phylogenetic diversity is recognized as a relevant criterion for the conservation of species [32]. Analysis of genetic variation in different plant species was carried out by Attia and Al-Sodany [33], who studied the ecological distribution, and, in addition, genetic variations, of some Aloe species based on RAPD and SSR markers. Asaf et al. [34] detected the complete chloroplast genomes of Vachellia nilotica and Senegalia senegal and they recorded that this may help to elucidate the genome architecture of these species and evaluate the genetic diversity among species.
Acacia species are useful as livestock fodder [35]-Bedouin use Acacia leaves and pods for fuel, fodder, and medicine-but there are issues with the identification of species. Genetic descriptions and phylogenetic relationships among Acacia species are particularly valuable for the conservation management of evolutionarily diverse flora [36]. Genetic differentiations in plant growing in Egypt were studied based on horticulture, molecular and morphological markers [37]. Genetic variation and differentiation in many plants species were studied based on morphological, biochemical, and molecular markers such as tea (Camellia sinensis), and revealed by RAPD and AFLP variation by Wachira et al. [38]; Cornus florida L using Isozyme and morphological variation [39]; Cornus [40]; genetic markers and horticultural germplasm management [41]; genetic diversity of Salvia species [42]; micropropagation of A. chundra (Roxb.) [43]; genetic diversity and differentiation of invasive A. longifolia in Portugal [44]; microsatellite markers used for A. senegal [45]; phylogenetic analysis based on nuclear DNA and morphology definitions of eastern Australian species of Acacia [46]; phylogenetic connections of phyllodinous species of Acacia outside Australia [47]; assessment of the phenology of A. longifolia [48]; characterization of microsatellite markers used for tree legume A. koa [49]; genetic diversity of Australian acacias [50]; genetic diversity of A. senegal (L.) willd in Kenyan populations combined RAPD and ISSR markers [51]; genetic diversity and structure of A. senegal (L.) Willd. in Uganda [52,53]; molecular phylogeny of Acacia s.s. [54]; genetic consequences of anthropogenic disturbances and population fragmentation in A. senegal [55]; isolation and characterization of SSR markers in the East African tree, A. brevispica [56], and genetic variation in natural populations of A. visco [57]. Taylor and Dhileepan [58] Plants 2020, 9, 243 4 of 18 showed the effect of the change in phylogenetic relationships of Acacia species on the biological control of Vachellia nilotica ssp. Also, Monks et al. [59] studied the recovery of some threatened plant species and their habitats in the biodiversity hotspot of the Southwest Australian Floristic Region.
The present study investigated the genetic description and phylogeny of some Egyptian Acacia species and subspecies by calculating the morphological, quantitative, and qualitative variations among the species, detecting the biochemical markers based on peroxidase activity and proline content, and estimating the level of polymorphism using RAPD-PCR markers.

Morphological Variations of Acacia Species
The morphological variations among five Acacia species (A. farnesiana, A. tortilis ssp. raddiana, A. saligna, A. sclerosperma, and A. stenophylla) were determined, and the results are illustrated in Table 1. Some morphological differentiations were also recorded in Figures 1 and 2 Regarding these parameters, ANOVA revealed very significant differences among the Acacia species, confirming the hypothesis of the possibility of identification from such vegetative characteristics. Concerning spine length (mm), data in Table 1 clearly indicate highly significant variation among the different species. We recorded the longest spines in A. tortilis ssp. raddiana (collected from Siwa Oasis), followed by A. tortilis ssp. raddiana (Borg Al-Arab city), with means of 28.7 and 19.3 mm, respectively. The shortest spines were found in A. farnesiana, i.e., 6.5 mm. No spines were present in the other three species; A. saligna, A. sclerosperma, and A. stenophylla had no spines. Regarding these parameters, ANOVA revealed very significant differences among the Acacia species, confirming the hypothesis of the possibility of identification from such vegetative characteristics. Concerning spine length (mm), data in Table 1 clearly indicate highly significant variation among the different species. We recorded the longest spines in A. tortilis ssp. raddiana (collected from Siwa Oasis), followed by A. tortilis ssp. raddiana (Borg Al-Arab city), with means of 28.7 and 19.3 mm, respectively. The shortest spines were found in A. farnesiana, i.e., 6.5 mm. No spines were present in the other three species; A. saligna, A. sclerosperma, and A. stenophylla had no spines. The results in Table 1 demonstrate an inverse relationship between the spine and pinna length, especially in the desert localities, where these species grow under very unfavorable conditions of drought and high salinity. A. tortilis ssp. raddiana collected from Siwa and Borg Al-Arab had the shortest pinna length (0.68 and 0.98 cm, respectively) and A. farnesiana had the longest pinna length (4.3 cm). As per the phyllode length (cm) data in Table 1, A. sclerosperma and A. saligna had the longest mean leaf length values of 19.3 cm and 26.1 cm, respectively. The mean shortest leaf lengths recorded were 2.85 cm for A. tortilis ssp. raddiana collected from (Siwa), 3.2 cm for A. tortilis ssp. raddiana collected from Borg Al-Arab, and finally 3.80 cm for A. farnesiana. The means ranged from 2.0 to 2.5 mm for A. tortilis ssp. raddiana (collected from Siwa Oasis = 2 mm), followed by A. tortilis ssp. raddiana (collected from Borg Al-Arab = 2.50) and A. farnesiana (2.25 mm).
Based on the morphological characterization (via qualitative description) we found some similarities and differences that are taxonomically important for comparing taxonomical grouping with morphological data for the genetic description of Acacia species. Based on the qualitative results, there were high similarities between A. farnesiana and A. tortilis ssp. raddiana, as both species possessed pinnately compound leaves. On the other hand, A. saligna, A. sclerosperma, and A. stenophylla had simple leaves ( Table 2). The same trend was observed for the growth type, with shrub/small trees being compared to other species that were shrubs or trees. The highest number of stems was recorded for A. farnesiana (2-5 stems), followed by both A. tortilis ssp. (1-4 stems) and A. stenophylla (typically 1 stem). Concerning spine shape, the spines were small in A. farnesiana but were long, white, and straight in both A. tortilis ssp., as shown in Figures 1 and 2. Morphological studies were conducted on Acacia species in different Arabian countries, such as Morocco, by El Ayadi et al. [60]. In their study, the authors assessed the variability in eight pod traits of 300 genotypes (mother-tree) of A. tortilis ssp. raddiana (Savi) 'Brenan', collected from the southern regions of Morocco. The ANOVA results showed that A. raddiana exhibited significant differences in traits due to genotypes within provenances, i.e., in the pod length, seed weight per pod, seed number per pod, infected seed number per pod, and 100-seed weight.
In general, the phenotypic coefficient of variation was higher than the genotypic coefficient of variation, indicating the predominant role of the environment. Their results also demonstrated great heritability and genotypic gain for the 100-seed weight (92.75% and 17.20%, respectively), empty pod weight (53.68% and 21.18%, respectively), and pod weight (46.45% and 16.13%, respectively), indicating additive gene action.
Our results are consistent with those of Quentin et al. [61], who reported that A. saligna grows as a small, dense, sprawling tree with a short trunk and a weeping habit. It grows up to 8 m in height. Like many Acacia species, it has phyllodes rather than true leaves and these phyllodes can reach a length of 25 cm.

Isozyme Assay
Peroxidase isozymes exhibited a wide range of variability among the different species at different localities (that ranged in Siwa from deep depression that reaches blew sea level, to about -19 matters, to hot semi-arid climate with moderate temperatures in Alexandria, to hot desert climate in Marsa Matruh). One cathodal (Pex.c1) band was observed as a common band for all samples. The results revealed five anodal (Pex.a1; Pex.a2; Pex.a3, Pex.a4, and Pex.a5) bands recorded for all species. Pex.a1 was recorded in A. farnesiana specie, Pex.a2 was recorded in A. tortilis ssp., and Pex.a3 was recorded in both A. stenophylla, and A. sclerosperma, and Pex.a5 was observed in A. sclerosperma (Figure 3).

Isozyme Assay
Peroxidase isozymes exhibited a wide range of variability among the different species at different localities (that ranged in Siwa from deep depression that reaches blew sea level, to about -19 matters, to hot semi-arid climate with moderate temperatures in Alexandria, to hot desert climate in Marsa Matruh). One cathodal (Pex.c1) band was observed as a common band for all samples. The results revealed five anodal (Pex.a1; Pex.a2; Pex.a3, Pex.a4, and Pex.a5) bands recorded for all species. Pex.a1 was recorded in A. farnesiana specie, Pex.a2 was recorded in A. tortilis ssp., and Pex.a3 was recorded in both A. stenophylla, and A. sclerosperma, and Pex.a5 was observed in A. sclerosperma ( Figure  3). The data indicate that the peroxidase patterns in the leaves of wild A. raddiana (Siwa) and the five domesticated Acacia species showed two kinds of banding profiles. First, it was evident that all plants expressed Px.c1, and the five domesticated plants exhibited the same banding profile containing this one locus. This indicated that one common locus was consistently monomorphically expressed. Second, the A. raddiana (Siwa) wild types shared one common locus (Px.a1). The banding pattern activity of Acacia displayed a unique marker band at the Px.1a and Px.5a loci, indicating that the Px.a2, Px.a3, and Px.a4 loci are specifically polymorphic. The similarity and genetic distance of Acacia spp. are presented in Figure 4. The data indicate that the peroxidase patterns in the leaves of wild A. raddiana (Siwa) and the five domesticated Acacia species showed two kinds of banding profiles. First, it was evident that all plants expressed Px.c1, and the five domesticated plants exhibited the same banding profile containing this one locus. This indicated that one common locus was consistently monomorphically expressed. Second, the A. raddiana (Siwa) wild types shared one common locus (Px.a1). The banding pattern activity of Acacia displayed a unique marker band at the Px.1a and Px.5a loci, indicating that the Px.a2, Px.a3, and Px.a4 loci are specifically polymorphic. The similarity and genetic distance of Acacia spp. are presented in Figure 4. Bands common among all examined species were estimated using average values and were standardized prior to cluster analysis. Data shown in Figure 4 reveal strong similarities among the Acacia species located in the first cluster (69%), between A. raddiana (Siwa and Borg Al-Arab) and A. saligna, followed by all Acacia species (50%) in the last cluster (except for A. farnesiana), and the similarity between all Acacia species was 33%. Salt stress was found to increase the intensity of the peroxidase bands. A. tortilis ssp. raddiana exhibited greater band intensity compared to the other species.

Proline Content
The highest content according the Table 3, A. tortilis ssp. raddiana (Siwa Oasis) had the highest proline content (43.4 μmol/g fresh weight), whereas A. sclerosperma had the lowest value (7.6 μmol/g fresh weight) ( Table 3). There was highly significant variation among all species in relation to the proline content, and this variation was associated with environmental effects and conditions. Acacia tortilis ssp. raddiana (Borg Al-Arab) had the highest proline content (23.1 μmol/g fresh weight). The results supported the conclusion that proline accumulates in greater concentrations in plants growing in salty, dry soil and may be useful as a salt injury indicator in plants. This variation in proline could be useful in the selection for salt tolerance and as a marker of salt-tolerant plants. Genotypic variations in proline accumulation have been observed [62], and attempts have been made to correlate its accumulation with stress tolerance in plants. This apparent correlation between proline accumulation and environmental stress suggests that proline might exhibit a protective function [63]. Bands common among all examined species were estimated using average values and were standardized prior to cluster analysis. Data shown in Figure 4 reveal strong similarities among the Acacia species located in the first cluster (69%), between A. raddiana (Siwa and Borg Al-Arab) and A. saligna, followed by all Acacia species (50%) in the last cluster (except for A. farnesiana), and the similarity between all Acacia species was 33%. Salt stress was found to increase the intensity of the peroxidase bands. A. tortilis ssp. raddiana exhibited greater band intensity compared to the other species.

Proline Content
The highest content according the Table 3, A. tortilis ssp. raddiana (Siwa Oasis) had the highest proline content (43.4 µmol/g fresh weight), whereas A. sclerosperma had the lowest value (7.6 µmol/g fresh weight) ( Table 3). There was highly significant variation among all species in relation to the proline content, and this variation was associated with environmental effects and conditions. Acacia tortilis ssp. raddiana (Borg Al-Arab) had the highest proline content (23.1 µmol/g fresh weight). The results supported the conclusion that proline accumulates in greater concentrations in plants growing in salty, dry soil and may be useful as a salt injury indicator in plants. This variation in proline could be useful in the selection for salt tolerance and as a marker of salt-tolerant plants. Genotypic variations in proline accumulation have been observed [62], and attempts have been made to correlate its accumulation with stress tolerance in plants. This apparent correlation between proline accumulation and environmental stress suggests that proline might exhibit a protective function [63].

Random Amplified Polymorphic DNA (RAPD) Analysis
In the present study, the genetic variability and relationships of different Acacia species were studied based on RAPD analysis. The initial screening of 52 primers with six samples resulted in 43 primers that produced informative and polymorphic products resolvable by agarose gel electrophoresis (Table 4). Profile examples of Acacia species on agarose gel, amplified with RAPD primers, are found in ( Figure 5).  Out of 563 amplification fragments, 190 fragments were monomorphic, and 373 fragments were polymorphic ( Table 4). The maximum number of amplification fragments recorded was 21 for primer OPB-20, compared with the lowest, i.e., seven for primer OPA-20. The average number of amplification fragments was 13.09 for all 43 primers (Table 4).
Concerning the monomorphic fragments, eight was the highest number of fragments recorded with primers OPB-03 and OPN-04, and one was the lowest number of fragments recorded for primer OPA-20. The general mean of the monomorphic fragments was 4.42 for all primers. Regarding polymorphic fragments, 14 was the highest value, recorded for primers OPA-16, OPB-20 and OPQ- Out of 563 amplification fragments, 190 fragments were monomorphic, and 373 fragments were polymorphic ( Table 4). The maximum number of amplification fragments recorded was 21 for primer OPB-20, compared with the lowest, i.e., seven for primer OPA-20. The average number of amplification fragments was 13.09 for all 43 primers (Table 4).
Concerning the monomorphic fragments, eight was the highest number of fragments recorded with primers OPB-03 and OPN-04, and one was the lowest number of fragments recorded for primer OPA-20. The general mean of the monomorphic fragments was 4.42 for all primers. Regarding polymorphic fragments, 14 was the highest value, recorded for primers OPA-16, OPB-20 and OPQ-14, while four fragments was the lowest detected value (OPA-09). The average number of polymorphic amplification fragments was 8.67 (Table 4). The percent polymorphic loci (PIC%) values ranged from 85.71% for primer OPA-20 to 42.85% for primer OPN-04. The PIC% between the six Acacia species using 43 RAPD primers was 66.46% (Table 4 and Figure 6). 14, while four fragments was the lowest detected value (OPA-09). The average number of polymorphic amplification fragments was 8.67 (Table 4).

Genetic Similarity and Phylogenetic Relationships Among Acacia Species
Genetic similarities and phylogenetic relationships among the six tested samples of Acacia species were examined using RAPD-PCR analysis, and the obtained data were subjected to cluster analysis with a Dice equation using SPSS (ver. 15) to calculate the proximity matrix and design dendrogram. Genetic similarity values generated from the RAPD markers varied between 0.60 and 0.78, with an average of 0.69. The dendrogram was based on similarity values (Table 5) from RAPD and was constructed using SPSS (ver. 15) to reveal similarities between the five different Acacia species. The dendrogram ( Figure 6) demonstrated that the six Acacia spp. fell into three main groups. The first group included A. tortilis ssp. raddiana (Siwa Oasis), the second group included A. tortilis ssp. raddiana (Borg Al-Arab), and the third group included the other Acacia species.

Genetic Similarity and Phylogenetic Relationships Among Acacia Species
Genetic similarities and phylogenetic relationships among the six tested samples of Acacia species were examined using RAPD-PCR analysis, and the obtained data were subjected to cluster analysis with a Dice equation using SPSS (ver. 15) to calculate the proximity matrix and design dendrogram. Genetic similarity values generated from the RAPD markers varied between 0.60 and 0.78, with an average of 0.69. The dendrogram was based on similarity values (Table 5) from RAPD and was constructed using SPSS (ver. 15) to reveal similarities between the five different Acacia species. The dendrogram ( Figure 6) demonstrated that the six Acacia spp. fell into three main groups. The first group included A. tortilis ssp. raddiana (Siwa Oasis), the second group included A. tortilis ssp. raddiana (Borg Al-Arab), and the third group included the other Acacia species. RAPD-PCR markers are used routinely to identify genetic variations [64]. RAPD markers have also been used successfully in various taxonomic and phylogenetic studies by Kazan et al. [65] and Wilkie et al. [66]. As a molecular marker system, RAPD has also been successfully applied in cultivar identification. RAPD analysis is normally easy to perform, but it has a major disadvantage in that reproducibility is difficult to achieve between different laboratories, and often even between different people in the same laboratory. The previous results are consistent with Fagg and Allison [67], who reported variation in the chemical composition as well as molecular and morphological characteristics between Ugandan and Sudanese populations of A. senegal. Our results are in line with those of Shrestha et al. [68], who investigated A. raddiana populations and reported a great degree of polymorphism contrary to the conventional expectation of small, isolated populations. The maintenance of genetic variation is important because future evolutionary adaptation depends on the existence of genetic variation.

Leaf Samples
Entire leaves of the following species were randomly collected from 20 individuals in natural Acacia tree habitats: A. tortilis ssp. raddiana collected from (Siwa Oasis) is an urban oasis in Egypt between the Qattara Depression and the Great Sand Sea in the Western Desert, nearly 50 km east of Libyan border and 650 km from Cairo, 29 •

Morphological Analysis
Four samples were collected for each parameter. The following morphological parameters were measured in the vegetative parts of the tree: pinna length (cm), leaf length (cm), leaflet length (mm), and spine length (mm) (if present) were measured with a millimetric ruler. Some qualitative characters were also recorded, such as crown shape (round, flat, spread, or undefined), growth habit, and number of stems counted from the ground level (1, 2-5, or > 5). The spine shape observed in individual plants was determined to be either straight or mixed, i.e., straight and curved.

Iso-Enzyme Electrophoresis
Agar starch polyvinyl pyrolidine (PVP) gel electrophoresis was conducted according to the protocol described by Andrews [13]. The extracts were made by grinding young leaf tissue in a mortar with 10 µL of electrode buffer and centrifuging for 15 s; a 10 µL sample of the homogenate was then absorbed onto a small rectangle (approximately 4 × 2 mm) of filter paper that was placed on the origin line of gel plates, which was removed after storage at 4 • C for 30 min. The buffer was prepared by dissolving 92.75 g of 0.3 M boric acid and 12 g sodium hydroxide in 5 L of distilled water; then, the solution was adjusted to pH 8.3 according to Ahmed [74]. The gel buffer used was 0.07 M Tris 0.007 M citric acid, pH 8.3. One liter of the gel buffer was prepared by dissolving 9.21 g Tris and l.05 g citric acid in distilled water and stored in a refrigerator until use. Ten grams of PVP gel were dissolved.
The mixture was vigorously shaken and cooked in a boiling water bath until the solution was transparent. The hot liquid gel was poured over glass plates (20 × 30 cm) to produce a smooth surface layer with a thickness of 0.8-0.9 mm and stored at 4 • C until use [75]. The electrophoresis experiment was conducted in an incubated refrigerator set to 4 • C using a 250 V AC electrical current with constant voltage during the 90 min running period. A phosphate buffer (0.l M, pH = 7.0) was used as a staining buffer by adding 39 mL of a 0.l M solution of monobasic sodium phosphate to 61 mL of a 0.1 M solution of sodium dibasic phosphate and increasing the final volume to 200 mL using distilled water. Each gel was incubated in l00 mL phosphate buffer, pH 7.0, containing 20 mg α-naphthyl acetate (α-NA) and 20 mg β-naphthyl acetate (NA), dissolved in l mL acetone, and brought to 5 mL using distilled water. Fast blue RR salt (C 15 H 14 ClN 3 O 3 · 1 / 2 ZnCl 2 ) (50 mg dissolved in 5 mL distilled water) was added 3 min after the addition of αand β-NA. This implies that an additional incubation was carried out for 30 min at 27-30 • C under complete darkness. Plates were then washed with distilled water [76].

Proline Content
Proline was determined according to the method presented by Bates et al. [77]. The mixture was warmed by agitation until dissolved, then kept cool at 4 • C until use. A 0.5 g sample of leaf material was homogenized in 10 mL extraction buffer. The homogenate was filtered through Whatman filter paper No. 2, and 2 mL of filtrate was reacted with 2 mL acid ninhydrin and 2 mL glacial acetic acid in a test tube for 1 h at 100 • C. The reaction was terminated using an ice bath. The reaction mixture was extracted with 4 mL of toluene mixed vigorously in a test tube with a stirrer for 15-20 s. The chromosphere containing toluene was aspirated from the aqueous phase, and the absorbance was determined using a spectrophotometer at 520 nm using toluene as a blank. The proline content was expressed as fresh weight on a standard curve, using standard L-proline according to the previous method developed by Hasan et al. [78]. Briefly, as follows: µmol proline g of fresh plant material = [(µg proline/mL − × mL toluene)/115.5 µg/µmol/(g sample/5)] [63].

Random Amplified Polymorphic DNA (RAPD) Analysis
Fifty-two random amplified polymorphic DNA (RAPD) primers (10-mer primers) were initially screened using six Acacia species to determine the suitability of each primer for this study. Primers were selected for further analysis based on their ability to produce distinct, clearly resolved, and polymorphic amplified products between Acacia species. To ensure reproducibility, primers generating no, weak, or complex patterns were discarded [79]. DNA was extracted from 50 mg samples of Acacia leaves using the DNeasy ® Plant System (Operon Technologies Inc., Alameda, CA). The DNA extraction was modified, i.e., 700 µL of warm (up to 65 • C) buffer AP1 for lyses was required during homogenization. Samples were centrifuged for 30 s at low speed (4000× g), and 7 µL of RNase A stock solution (100 mg·mL −1 ) was mixed into each tube until no tissue clumps were visible.
The mixture was incubated for 20 min at 65 • C, and tubes were inverted twice during the incubation period. The addition of 228 µL buffer AP2 to the lysate and incubation on ice for 5 min precipitated the detergent, proteins, and polysaccharides. The column-tube assembly was centrifuged at maximum speed for 2 min in a microcentrifuge. At least 500 µL of clear filtrate was transferred to a 1.5 mL microfuge tube. Buffer AP3 and 100% ethanol (1:2 v/v) were added to the lysate, and the solution was gently mixed by pipetting. Up to 650 µL of the sample mixture (including any precipitate that formed) was applied to the DNeasy (Operon Technologies Inc., Alameda, CA) spin column, which was placed in a 2 mL collection tube. The assembly was centrifuged for 1 min at 12,000× g, causing the DNA to be bound to the column membrane, and the filtrate was discarded. The remaining sample mixture was applied to the same column, and the procedure was repeated. The collection tube was replaced by a clean 2 mL tube, and the column was washed two to three times by adding 500 µL buffer AW (containing ethanol) to the DNeasy column, centrifuging for 1 min at 12,000× g, and discarding the filtrate.
Following the final wash, the column-tube assembly was centrifuged for 2 min at 12,000× g to dry the column membrane, and the collection tube was discarded. The DNeasy column was transferred to a clean 1.5 mL microcentrifuge tube, and DNA was eluted from the membrane by pipetting 100 µL of preheated (65 • C) buffer AE directly onto the DNeasy column membrane. The sample was then incubated for 5 min and centrifuged for 1 min at 12,000× g or higher. The elution step was repeated using the same microcentrifuge tube and yielded a final volume of 50 µL DNA solution

Polymerase Chain Reaction (PCR) Conditions
RAPD analysis was conducted using 10 oligonucleotide primers that were selected from the Operon Kit (Operon Technologies Inc., Alameda, CA). The polymerase chain reaction (PCR) mixture (25 µL) consisted of 0.8 U of Taq DNA polymerase, 25 pmol dNTPs, 25 pmol of primer, and 50 ng of genomic DNA. PCR amplification was performed in a Biometra T1 gradient thermal cycler (Phoretix International, Newcastle upon Tyne, UK) for 40 cycles after initial denaturation for 3 min at 94 • C. Each cycle consisted of denaturation at 94 • C for 1 min, annealing at 36 • C for 1 min, extension at 72 • C for 2 min, and a final extension at 72 • C for 10 min [79]. Amplification products were separated on 1% agarose gels at 100 V for 1.30 h, using 1 × TBE buffer. To detect the ethidium bromide/DNA complex, agarose gels were examined using an ultraviolet transilluminator (302 nm wavelength); subsequently, the lengths of the different DNA fragments were determined. For each sample, the reproducible DNA bands from two runs were scored for their presence or absence. Fragment scoring and lane matching were performed automatically on digital images of the gels using Phoretix 1D Advanced version 4.00 (Phoretix International, Newcastle upon Tyne, UK).
All but the faintest bands were scored, and, where necessary, scores and matches were manually corrected.
Based on the matrix of genetic similarity values (peroxidase isozymes data) and the dendrogram was generated from PAleontological STatistics (PASTA) program that runs on standard Windows computers and is available free of charge. PAST integrates spreadsheet-type data entry with univariate and multivariate statistics, curve fitting, time series analysis, data plotting, and simple phylogenetic analysis. Many of the functions are specific to paleontology and ecology, and these functions are not found in standard, more extensive, statistical packages. PAST also includes fourteen case studies (data files and exercises), illustrating use of the program for paleontological problems, making it a complete educational package for courses in quantitative methods (http://palaeo-electronica.org).

Statistical Analysis
Data of the morphological variations of Acacia species were statistically analyzed with one-way analysis of variance (ANOVA) using the SAS system [80]. Comparisons among means were measured using LSD 0.05 .

Conclusions
In the present study, genetic differentiation of some Acacia species growing in Egypt based on morphological, biochemical, and molecular markers were measured using twenty replicates of Acacia tree, collected from four different localities in Egypt. The results clearly indicated highly significant differences between Acacia species for morphological characteristics. In addition, the qualitative characteristics were used to detect the similarities and differences which are important in comparing the taxonomical grouping of Acacia species. RAPD-PCR proved to be a powerful tool for assessing the genetic diversity of several Acacia species in Egypt. Morphological parameters revealed highly significant variations among the Acacia species, confirming the hypothesis of the possibility of identification from such vegetative characteristics. Study of the genetic differentiation of Acacia species growing in Egypt is considered a primary step to genetic improvement in and documentation of this plant genetic resource in Egypt, especially Acacia tortilis ssp. Raddiana, which was collected from the isolated area Siwa Oasis in Egypt.