TAXONOMIC VARIATION AMONG SCHINUS MOLLE L. PLANTS ASSOCIATED WITH A SLIGHT CHANGE IN ELEVATION

This study examined the degree of variations in DNA fingerprints associated with slight altitudinal change of Schinus molle grown in Abha region, Saudi Arabia. Seven populations from Schinus molle plants located at 2193.0, 2246.0, 2197.7, 2441.0, 2372.0, 2250.6 and 2175.0 meters had been investigated. The degree of genetic variability was evaluated using random amplified polymorphic DNA (RAPD), mixed RAPD and intersimple sequence repeat markers (ISSR). The genetic similarity coefficients from RAPD analysis revealed the maximum similarity value (89.9%) was between population at 2250.6 m and population at 2175.0 m. The genetic similarity coefficients from mixed RAPD primers displayed the highest similarity value (87.6%) between population at 2246.0 m and population at 2197.7 m. Similarity coefficients from ISSR analysis revealed the highest similarity value (86.2%) among populations at 2193.0 m, 2246.0 m, 2441.0 m and at 2250.6 m. Super tree analysis (RAPD + mixed RAPD + ISSR) showed the highest similarity value (85.5%) between population at 2441.0 m and population at 2250.6 m. In conclusion, marker systems including RAPD, mixed RAPD and ISSR, alone or combined can be effectively used in determining the genetic relationship among Schinus molle plants even at very close populations. Introduction Abha region has a specialized environmental condition among all other areas in the Kingdom of Saudi Arabia which have an indirect effect on the weed plants growth. S. molle plants (family, Anacardiaceae) are among the most common weed in Saudi Arabia especially in Tharawat Mountains. The tree of S. molle plant is an evergreen, dioecious, grows up to 20 meters in height. Flowers are small, with yellowish white petals and all plant parts especially fruits having strong aroma (Lim, 2012). S. molle is common weed in South America and recently into many tropical and subtropical countries (Olafsson et al., 1997). In Abha region, S. molle tree has been planted in many areas as in valleys, public gardens and for house decorations. After that, the plant became a common weed in many areas of Abha city growing beside road and next the wall of houses as it is reproduced by seeds. S. molle plant showed to be resistant to the harsh environmental condition such as high temperature, cold and increasing soil salinity (Lim, 2012). In addition, S. molle plants usually used for the restoration of degraded areas and showed tolerance to heavy metals (Doganlar et al., 2012; Pereira et al., 2016). Toward this approach examine the genetic diversity of S. molle plant is highly needed as no reports available. In recent years, a number of randomly amplified polymorphic DNA–polymerase chain reaction (RAPD-PCR) and inter-simple sequence repeat–polymerase chain reaction (ISSR-PCR) markers had been used to study genetic diversity among plant species. For example, RAPD Corresponding author. Email: mfmostfa@kku.edu.sa Research Center for Advanced Materials Science (RCAMS), King Khalid University, Abha, Saudi Arabia. Department of Botany, Faculty of Science, South Valley University, Qena, Egypt.


Introduction
Abha region has a specialized environmental condition among all other areas in the Kingdom of Saudi Arabia which have an indirect effect on the weed plants growth.S. molle plants (family, Anacardiaceae) are among the most common weed in Saudi Arabia especially in Tharawat Mountains.The tree of S. molle plant is an evergreen, dioecious, grows up to 20 meters in height.Flowers are small, with yellowish white petals and all plant parts especially fruits having strong aroma (Lim, 2012).S. molle is common weed in South America and recently into many tropical and subtropical countries (Olafsson et al., 1997).In Abha region, S. molle tree has been planted in many areas as in valleys, public gardens and for house decorations.After that, the plant became a common weed in many areas of Abha city growing beside road and next the wall of houses as it is reproduced by seeds.S. molle plant showed to be resistant to the harsh environmental condition such as high temperature, cold and increasing soil salinity (Lim, 2012).In addition, S. molle plants usually used for the restoration of degraded areas and showed tolerance to heavy metals (Doganlar et al., 2012;Pereira et al., 2016).Toward this approach examine the genetic diversity of S. molle plant is highly needed as no reports available.
In recent years, a number of randomly amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) and inter-simple sequence repeat-polymerase chain reaction (ISSR-PCR) markers had been used to study genetic diversity among plant species.For example, RAPD technique was successfully applied genetically to distinguish among Ocimum spp.(Vieria et al., 2003), to study the genetic diversity in Monodora myristica (Uyoh et al., 2014) and various population of Ziziphus spina-christi L. (Moustafa et al., 2016).ISSR technique was used to study genetic diversity of the Lens spp.(Fikiru et al., 2007), and genetic relationships of Chukrasia spp.(Wu et al., 2014).
Therefore, the aim of this research is to study genetic diversity of S. molle plants growing at close locations in Abha region, KSA.To the best of our knowledge, there are few reports indicating the use of mix primer to estimate the genetic diversity among plant /or to study plant DNA fingerprint.Therefore, this study also aimed to check the status of DAN fingerprints using mixed primers.

Plant material
Seven locations at various elevations in Abha region, KSA,include 2193.0,2246.0,2197.7,2441.0,2372.0,2250.6 and 2175.0 meters have been selected (Fig. 1).At each site, random samples of young fresh leaves from S. molle trees having a height 1500 cm had been collected.Extraction the genomic DNA from leaves of S. molle plants Genomic DNA was extracted from fresh young leaves of S. molle plants by using DNeasy plant mini kit.DNA concentration was estimated by a Thermo Scientific™ BioMate 3S UV-Visible at 260 nm.

PCR amplification
Eight RAPD, nine ISSR and eight mixed RAPD markers were used in this study (Table 1).PCR reaction consists from 1 X GoTaq Green Master Mix, 4 µl from each primer, 20 ng of genomic DNA and nuclease-free water to get a final 25 µl volume.PTC 200 Peltier Thermal Cycler (MJ Research -USA) adjusted as follows: Initial degree at 94°C for 5 minutes followed by forty nine cycles at 92°C for 1 minute, primer annealing temperature at 29°C for 1 minute, extension at 72°C for 2 minutes and final process for primer extension at 72°C for 7 minutes.An equal amount of each amplified product of 20 ul was separated by electrophoresis using 1.3 % agarose gels in 0.5X TBE buffer.Stained gel with ethidium bromide was photographed by gel documentation system using UV transilluminator at 365 nm (Hashemi et al., 2009).Each experiment was repeated three times and molecular weight of RAPD-PCR, mixed RAPD-PCR and ISSR-PCR fragments were estimated using marker 1 kb DNA ladder between 250 to 10,000 bp.

Data analysis
All scored fragments gained from RAPD-PCR, mixed RAPD-PCR and ISSR-PCR were manually recorded as present (1) or absent (0).Matrix of similarity based on binary-double zeros-S3, and squared Euclidean distance was used to calculate the distances and to generate dendrogram (Sneath and Sokal, 1973).Polymorphism percentage was estimated by calculating polymorphic bands/total number of bands.

RAPD analysis
RAPD primers produced a total of 109 scorable bands from genotypes of S. molle, out of which 23.0 (21.1%) were found to be polymorphic, 1.00 (0.91%) to be monomorphic bands and 85.0 (77.9%) to be unique bands.Primer Oligo 345, yielded the maximum number of bands (20.0 bands) while the lowest number of bands (3.00 bands) obtained from Primer Oligo 214.The percentage of polymorphism ranged from 0.00% (Primer Oligo 33 and Primer Oligo 214) to 57.1% (Primer Oligo 342).The maximum number of unique bands (17.0 bands) was recorded from Primer Oligo 33, while the lowest number of unique bands (3.00 bands) from the Primer Oligo 342 and Primer Oligo 214 (Table 2 and Fig. 2 Panel A).
The genetic similarity coefficients (Table 3) revealed that the maximum similarity value (89.9%) was between population at 2250.6 m and population at 2175.0 m, while the least similarity value (72.5%) between population at 2246.0 m and population at 2372.0 m.
Dendrogram analysis (Fig. 3 Panel A) showed that population at 2193.0, 2197.7 and 2441.0 m found to be forming one cluster whereas population at 2246.0 m separated from them in a single cluster while population at 2372.0, 2250.6 and 2175.0 m found to be forming another one cluster.

Mixed RAPD analysis
Mixed RAPD primers generated a total of 100 reproducible bands of which (19.0%) were polymorphic bands, (81.0%) unique bands, and no any monomorphic bands (Table 4 and Fig. 2 Panel B).Primer OPK-8 produced the highest number of bands (21.0) while primer Oligo 42 gave the minimum number of bands (3.00).Primer Oligo 33 showed the highest percentage value of polymorphism of 50.0% and the zero polymorphism rate gained from the primer Oligo 42 and primer Oligo 214.The maximum number of unique bands were (18.0 bands) gained from primer OPK-8, while the minimum numbers were (3.00) gained from primer Oligo 42.The genetic similarity coefficients displayed the highest similarity value (87.6%) between population at 2246.0 m and population at 2197.7 m, while the least similarity value (72.6%) was recorded between population at 2372.0 m and population at 2175.0 m (Table 5).
Resulted dendrogram showed that populations at 2193.0, 2246.0 and 2197.7 m found to be forming one cluster whereas population at 2441.0 m and population at 2372.0 m clustered together as well as population at 2250.6 m and population at 2175.0 m (Fig. 3 Panel B).

ISSR analysis
A total of 231 counted bands were generated by using the nine ISSR primers from S. molle genetic materials (Table 6 and Fig. 2 Panel C).Sixty-two polymorphic bands (26.8%), 1.00 (0.43%) monomorphic bands, 168 (72.7%) unique bands with polymorphism rate 23.2% were recorded.Primer UBC 826 generated the maximum number of bands (63.0), while primer UBC 824 showed the minimum number of bands (6.00).Primer (1) showed the highest rate of polymorphism (51.4%) and Primer (3) showed the least rate numbers (4.54%).The highest number of unique bands (46.0) was recorded from primer UBC 826, while the least number of unique bands (5.00) resulted from primer UBC 824.
Resulted genetic similarity coefficients exhibited the highest similarity value among populations at 2193.0 m, 2246.0 m, 2441.0 m and population at 2250.6 m recording 86.2%, while the least similarity value between population at 2246.0 m and population at 2175.0 m with value of 69.1% (Table 7)

Discussion
This research article reports the use of the RAPD, mixed RAPD and ISSR makers to the S. molle plant and revealed its efficiency to determinate the DNA fingerprints.Also it revealed that RAPD, mixed RAPD and ISSR markers could be used alone or in combination to estimate the genetic diversifications of S. molle plants.Polymorphism rate obtained either from RAPD, mixed RAPD, ISSR or from combined analysis all showed that there was high genetic variability among S. molle at a very close distance populations.The percentage of polymorphism almost same to that detected in other examined plants that they have a wide genetic variability.For example, Adawy et al. (2004) and Hussein et al. (2005) found that RAPD polymorphism rate in various Egyptian date palm cultivars (Phoniex dactylifera L.) is in the range of 25.2% and for ISSR technique in the range of 28.6%.Among the studied Pistacia vera (L.) various cultivars polymorphism rate based on ISSR markers was 46.4% and 100% among Mangifera indica (L.) based on ISSR markers (Noroozi et al., 2009;Souza et al., 2011).RADP, mixed RAPD and ISSR showed various degrees in their ability to detect the diversifications among populations of S. molle plants.This variation may be due to that the genome S. molle plants having a considerable number of alleles per locus/or loci that vary in their distribution.Izzatullayeva et al. (2014) reported that such difference between RAPD and ISSR markers due to the fact of abundant nature of microsatellites that results from slippage in DNA replication.This explains why this plant can be found in various habitats vary from salinity soil to alkalinity soil and in different temperature condition ranging from very low to very high (Lim, 2012).In this study, total number of unique bands obtained from ISSR-PCR of S. molle plant more than that of RAPD-PCR and mixed RAPD-PCR.The results also showed that ISSR fingerprinting had a high number of scored bands and high polymorphic percentage rate.This in agreement with earlier studies showed that ISSR fingerprinting was more efficient than the RAPD assay in assessing genetic variation in Arthrocnemum macrostachyum (Saleh, 2011).Again this variation among RAPD, mixed RAPD and ISSR probably due to that amplified profiles of PCR of RAPD, ISSR, or mixed RAPD originated from different variable numbers of repetitive and non-repetitive sequence on the genomes of S. molle plant (Thormann et al., 1994).The presence of monomorphic bands from RAPD-PCR or from ISSR-PCR indication to the sharing characters based on the DNA fragment in genomic S. molle plants.Cluster analysis based on RAPD, mixed RAPD and ISSR markers individually or combined showed that the three markers differ from each other in the manner of distributing S. molle populations.
In our study, the amount of genetic similarity among various populations of S. molle plants based on RAPD markers were in range between 72.5% to 89.9% and for mixed RAPD between 72.6% to 87.6% and for ISSR 69.1% to 86.2% and for the sum of all data between 71.9% to 85.5%.These values to some extent are in accordance with the basis proofed by Weier et al. (1982) that operational taxonomic units between 85 to 100% among the same plant species and more than 65% between the same plant genus.In conclusion, our study confirms that there were a wide genetic diversity among S. molle plants that can be evaluated by using RAPD, mixed RAPD and ISSR markers.
. A dendrogram pattern revealed that population at 2193.0 m and population at 2246.0 m formed one cluster whereas the populations at 2197.7 m, 2441.0 m, 2250.6 m and 2372.0 m found to be in another cluster and population at 2175.0 m formed out-group from the in-group including populations at 2193.0 m, 2246.0 m, 2197.7 m, 2441.0 m, 2250.6 m and 2372.0 m (Fig. 3 Panel C).