This study aimed at investigating genetic diversity and qualitative variation within Syrian accessions of Rosa damascena in order to determine the best oil bearing one for commercial production. The experiments were conducted at Suleyman Demirel University in Turkey and Damascus University in Syria. Microsatellite technique was used to analyze the genetic diversity of seven accessions of R. damascena collected across major and minor rose oil production areas in Syria and one accession Control collected from Isparta province in Turkey. The microsatellite DNA allele counting-peak ratios method (MAC-PR) was used. The accessions were clustered using the un-weighted pair group method for arithmetic averages (UPGMA) by the statistical program marked as Popgene 1.31. Gas chromatography/mass spectrometry (GC/MS) Analysis of Rose oil distilled from each accession was used to compare oil quality within genotypes. The analysis results were statistically analyzed by the program marked as SPSS. Six different genotypes have been obtained from Rosa damascena accessions collected from Syria. Two accesions, Almarah1 and Bab Alnayrab, were identical to the Turkish gynotype. GC/MS analysis identified the main components of oil such as: Geraniol (28-31%), Citronellol (26-30%), Nerol (12-14%), Germacrene-D (6-8%), Nonadecane (4-6%) and Linalool (1-3%). In addition, many trace compounds were detected such as: Eicosane, Eugenol, Citral, Hexadecane and Rose oxide. This study showed for the first time the existence of genetic diversity within Rosa damascena cultivated in Syria. Almarah1 and Bab Alnayrab accessions are recommended to be used to broaden the production of rose oil.
PDF Abstract XML References Citation
How to cite this article
Rosa damascena Mill. is the most important rose species for rose oil production (Moein et al., 2010). In recent years, antioxidant, antibacterial and anticancer activities of R. damascena essential oil have been demonstrated (Shahriari et al., 2007; Rakhshandeh et al., 2008; Gholamhoseinian et al., 2008). As Rosa damascena was originally introduced from the Middle East into Western Europe, it is thought that its origin and diversity can be found in this region (Babaei et al., 2008). In Syria, cultivation and consumption of Rosa damascena has a long history especially in Kalamoon Mountains where the so-called "village of Rosa damascena" Almarah is located (Alsemaan et al., 2011). Rosa damascena has been commercially cultivated in two regions in Syria which are Aleppo and Rural Damascus (Sulayman, 2010). In recent genetic diversity studies using RAPDs and SSRs with Bulgarian Damask roses (Rusanov et al., 2005) and AFLPs with Turkish Damask roses (Baydar et al., 2004), no variation was revealed among the Damask roses cultivated for oil in these two countries. Thus, all production material in Bulgaria and Turkey consists of only one oil bearing genotype (Babaei et al., 2007). Microsatellite analysis showed the existence of multiple genotypes within Rosa damascena in Iran (Babaei et al., 2007). During the last few decades, the use of molecular markers, revealing polymorphism at the DNA level, has been playing an increasing part in plant biotechnology and their genetics studies. There are different types of markers viz. morphological, biochemical and DNA based molecular markers. These DNA based markers are differentiates in two types first non PCR based (RFLP) and second is PCR based markers (Random Amplified Polymorphic DNA (RAPD), Amplified Fragment Length Polymorphic DNA (AFLP, Simple Sequence Repeats OR Microsatellites (SSR), Simple Nucleotide Polymorphism (SNP) etc.), amongst others, the microsatellite DNA marker has been the most widely used, due to its easy use by simple PCR, followed by a denaturing gel electrophoresis for allele size determination and to the high degree of information provided by its large number of alleles per locus (Kumar et al., 2009). The strengths of microsatellites include the codominance of alleles, their high genomic abundance in eukaryotes and their random distribution throughout the genome, with preferential association in low-copy regions (Morgante et al., 2002). Because the technique is PCR-based, only low quantities of template DNA (10-100 ng per reaction) are required. Due to the use of long PCR primers, the reproducibility of microsatellites is high and analyses do not require high quality DNA. Although microsatellite analysis is, in principle, a single-locus technique, multiple microsatellites may be multiplexed during PCR or gel electrophoresis if the size ranges of the alleles of different loci do not overlap (Ghislain et al., 2004). This decreases significantly the analytical costs. Rose essential oil should be extracted from fresh flowers picked before 8 am in the morning, by steam distillation (Gunes, 2005). Rose essential oil is also called otto of rose and attar of rose. The chemical composition of rose oil is complex. It contains more than 300 known compounds. The main chemical components of rose oil can be listed as -citronellol, phenyl ethanol, geraniol, nerol, with traces of other components (Yousefi et al., 2009). Baydar et al. (2004) indicated that the qualitative variation among Turkish accessions could be caused by ecological conditions which resulted in mutations. In contrary (Babaei et al., 2007) expected that qualitative variation might be genetically explained. Sulayman (2010) found that different accessions of Rosa damascena showed different yields and different rose oil qualities. According to the big climatic differences of the areas in which accessions of Rosa damascene cultivated in Syria, this investigation was conducted to examine genetic diversity and qualitative variation within them.
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).
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.
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.
- Babaei, A., S.R. Tabaei-Aghdaei, M. Khosh-Khui, R. Omidbaigi, M.R. Naghavi, G.D. Esselink and M.JM.D. Smulders, 2007. Microsatellite analysis of Damask rose (Rosa damascena Mill.) accessions from various regions reveals multiple genotypes. BMC Plant Biol., 10: 7-12.
- Babaei, A., S.R. Tabaei-Aghdaei, M.R. Naghavi, M. Khosh-Khui, R. Omidbaigi and M.H. Assareh, 2008. Rosa damascena (Rosaceae) characters and their heritability analysis in Iran. Iran. J. Bot., 14: 75-80.
- Baydar, N.G., H. Baydar and T. Debener, 2004. Analysis of genetic relationships among Rosa damascena plants grown in Turkey by using AFLP and microsatellite markers. J. Biotechnol., 111: 263-267.
- Ghislain, M., D.M. Spooner, F. Rodriguez, F. Villamon and C. Nunez et al., 2004. Selection of highly informative and user-friendly microsatellites (SSRs) for genotyping of cultivated potato. Theor. Applied Genet., 108: 881-890.
- Kiani, M., Z. Zamani, A. Khalighi, R. Fatahi and D.H. Byrne, 2009. Microsatellite analysis of Iranian Damask rose (Rosa damascena Mill.) germplasm. Plant Breed., 129: 551-557.
- Kumar, P., V.K. Gupta, A.K. Misra, D.R. Modi and B.K. Pandey, 2009. Potential of molecular markers in plant biotechnology. Plant Omics J., 2: 141-162.
- Moein, M., F. Karami, H. Tavallali and Y. Ghasemi, 2010. Composition of the essential oil of Rosa damascena Mill. Iranian J. Pharma. Sci., 6: 59-62.
- Rusanov, K., N. Kovacheva, B. Vosman, L. Zhang, S. Rajapakse, A. Atanassov and I. Atanassov, 2005. Microsatellite analysis of Rosa damascena Mill. Accessions reveals genetic similarity between genotypes used for rose oil production and old Damask rose varieties. Theor. Applied Genet., 111: 804-809.
- Loghmani-Khouzani, H., O. Sabzi Fini and J. Safari, 2007. Essential oil composition of Rosa damascena mill cultivated in central Iran. Scientia Iranica, 14: 316-319.
- Yousefi, B., S.R. Tabaei-Aghdaei, F. Darvish and M.H. Assareh, 2009. Flower yield performance and stability of various Rosa damascena Mill. Landraces under different ecological conditions. Sci. Hortic., 121: 333-339.