MATERIALS AND METHODS

Eight accessions of Rosa damascena were examined. Seven of them were collected from commercial production fields in seven sites located in two regions of Syria's. They were (Bab Alnayrab) in Aleppo, (Almarah1, Almarah2, Rankoos, Ernah, Issal Alward and Mesraba) in Rural Damascus. The eighth accession "Control" was collected from the experimental field of Suleyman Demirel University in the province of Isparta in Turkey. Table 1 specifies each area mentioned above (Fig. 1). All accessions have been grown in the experimental field of faculty of agriculture at Damascus University since 2008. Eight robust microsatellite markers were used as linkage groups on the genetic map of rose to differentiate genotypes (Babaei et al., 2007). Primer pairs were amplified using the Qiagen PCR multiplex kit (India) after the extraction of DNA from the young leaves (Baydar et al., 2004).

Table 1:	Geographical origins of Rosa damascena accessions in Syria and Turkey

Fig. 1:	Agro-ecological Zones in Syria

Fluorescent amplification products were detected using an ABI Prism 3700 DNA Analyzer (Applied Biosystems) and all samples were genotyped in accordance with reference alleles for each locus as described by Babaei et al. (2007) using Genotyper 3.5 NT (Applied Biosystems). The microsatellite DNA allele counting-peak ratios method (MAC-PR) provided by Genotyper software which calculates ratios between the peak areas for each two alleles in which they occurred together was used. The accessions were clustered using the unweighted pair group method for arithmetic averages (UPGMA) by the statistical program marked as Popgene 1.31. Rose oil from each accession was obtained through Clevenger. Then, 20 μL of essential oil was analyzed using gas chromatography/mass spectrometry GC/MS in three replications following these parameters: injection temperature: 240°C, Flow: 10 psi, Ionization Mode: EI (70 eV), Oven Program: 60°C up to 240°C. Gas: Helium, Column: Cp W AX 52 CB m* 10.32 mm, 1.2 μm and Library: Wiley, Nist, Tutor. The analysis results were analyzed statistically using LSD (Lowest Significant Difference) by the statistical program marked as SPSS. Duncan test was also used to compare mean averages. GC/MS software was used to draw the chromatograms for each studied accession (Fig. 3).

RESULTS AND DISCUSSION

Genetic diversity: In this investigation, six different genotypes have been obtained from Rosa damascena accessions collected from Syria by using microsatellite marker analysis. Two of them were identical to the gynotype collected from Turkey (province of Isparta), which supports. All markers detected polymorphisms among the accessions. For all studied accessions, the MAC-PR method showed the allelic configurations at six loci (RhE2b, RhD221, RhAB73, RhB303, RhAB40, RhP50) (Table 2). The dendrogram of Cluster analysis shows six different genotypes. The biggest group consists of the three accessions of (Bab Alnayrab, Almarah1 and Isparta) confirming that they are identical. The Genotypes used for rose oil production are genetically related and perhaps the same genotype is used in several countries such as Turkey, Bulgaria and Iran. That supports (Baydar et al., 2004; Rusanov et al., 2005; Babaei et al., 2007; Kianiet al., 2009). This genotype is being used for attar production in Syria (Rural Damascus and Aleppo) (Alsemaan et al., 2011). Despite All other genotypes identified in dendrogram represent different genotypes, a close genetic relation within them was found.

Fig. 2:	Genetic relation tree using UPGMA (Popgene 1.31)

Table 2:	Allelic configurations of MAC-PR analyses

Table 3:	Percentage of essential oil components for each investigated accession
- : No significant difference. Numbers followed with different letters are significantly different at 99% confidence

Figure 2 shows that there is a close genetic relation (90%) between Issal Alward genotype and the accessions mentioned before. While Mesraba genotype showsed the furthest genetic relation to them (70%). That supports (Sulayman, 2010).

Qualitative variation: Table 3 shows that six components, representing 76-89% of the oil, were characterized. Geraniol (28-31%), Citronellol (26-30%), Nerol (12-14%), Germacrene-D (6-8%), Nonadecane (4-6%) and Linalool (1-3%), were found to be major constituents, which supports (Baydar et al., 2004; Loghmani-Khouzani et al., 2007) It also shows the qualitative variation within the accessions investgated (Fig. 3). No significant differences in their content of Heneicosane, Methyl Eugeno, PEA, 9-Nonadecene, Phenyl Ethyl Acetate, Geranyl acetat, Linalyl, Propionate, Germacrene D, Gamma-Muurolene, Rose oxide, beta-Caryophyllene, alpha-humulene, β-selinene and Hexadecane were found. In contrary, Almarah1, Bab Alnayrab and Isparta accessions were significantly superior to others in their content of Linalool, Nonadecane, Geraniol, Nerol and Eicosane, While Erna accession was significantly superior in its content of Citral and Citronellyl Acetate to all other accessions. The genetic relations found might explain the qualitative variation noticed within the accessions, which supports (Sulayman, 2010). But, the qualitative variation between Bab Alnayrab accession on one hand and Almarah1 and Isparta accessions on the other hand could not be explained genetically. Otherwise, agro-ecological variation should be considered. The high percentages of main components of Rose oil are considered as a positive quality parameter. Contradictory, another positive quality parameter is the low content of Eugenol and Citronellol which was noticed in Almarah1 and Isparta accessions. The high content of Eugenol in rose oil may make it inedible. Besides, as Citronellol percentage is higher, as the rose oil decomposition is faster (Yousefi et al., 2009).

Fig. 3:	Chromatograms of GC/MS analysis for all the accessions

It was remarkable that the content of Rose oxide was high in all the accessions, which could be attributed to either insufficient distillation machines or the delayed flower harvest (Moein, 2010).

CONCLUSION

This study showed for the first time the existence of genetic diversity within Rosa damascena cultivated in Syria. GC/MS analysis indicated that different genotypes may have qualitative differences in composition of essential oil. Almarah1 and Bab Alnayrab accessions are recommended to be used to broaden the production of rose oil.

ACKNOWLEDGMENTS

The authors would like to thank Prof Souheil HADDD for his collaboration. Also, we acknowledge Prof Adel SAFAR the minister of agriculture in Syria for his support, General Commission of Biotechnology in Syria for its laboratories, Institute of Rose and Aromatic Plants in Isparta (Turkey) for providing leaf material of damask roses. This work was financed by Damascus University, General Commission of Biotechnology in Syria, Suleyman Demirel University and the higher commission for scientific research in Syria.