Open Access

Microsatellite analysis of Damask rose (Rosa damascena Mill.) accessions from various regions in Iran reveals multiple genotypes

  • Alireza Babaei1,
  • Seyed Reza Tabaei-Aghdaei2,
  • Morteza Khosh-Khui3,
  • Reza Omidbaigi1,
  • Mohammad Reza Naghavi4,
  • Gerhard D Esselink5 and
  • Marinus JM Smulders5Email author
BMC Plant Biology20077:12

DOI: 10.1186/1471-2229-7-12

Received: 24 November 2006

Accepted: 08 March 2007

Published: 08 March 2007

Abstract

Background

Damask roses (Rosa damascena Mill.) are mainly used for essential oil production. Previous studies have indicated that all production material in Bulgaria and Turkey consists of only one genotype. Nine polymorphic microsatellite markers were used to analyze the genetic diversity of 40 accessions of R. damascena collected across major and minor rose oil production areas in Iran.

Results

All microsatellite markers showed a high level of polymorphism (5–15 alleles per microsatellite marker, with an average of 9.11 alleles per locus). Cluster analysis of genetic similarities revealed that these microsatellites identified a total of nine different genotypes. The genotype from Isfahan province, which is the major production area, was by far the most common genotype (27/40 accessions). It was identical to the Bulgarian genotype. Other genotypes (each represented by 1–4 accessions) were collected from minor production areas in several provinces, notably in the mountainous Northwest of Iran.

Conclusion

This is the first study that uncovered genetic diversity within Damask rose. Our results will guide new collection activities to establish larger collections and manage the Iranian Damask rose genetic resources. The genotypes identified here may be directly useful for breeding.

Background

There are almost 200 species and more than 18000 cultivars in the genus Rosa [1]. They are mostly shrubs, distributed in the temperate zones of the Northern hemisphere [2]. One of the important Rosa species is Rosa damascena Mill., which is commercially used for essential oil production and cultivated as garden rose [3]. In recent years, antioxidant, antibacterial and antimicrobial activities of R. damascena essential oil have been demonstrated [47]. Three recent studies on molecular analyses of genetic diversity of Rosa damascena Mill. with RAPD, AFLP and SSR markers did not show any polymorphism among R. damascena plants from various plantations in Turkey[8, 9] and Bulgaria[3], indicating that commercial production of essential oil is in fact done by large scale propagation of only one or very few genotypes.

R. damascena can now be found in the wild in Morocco, Andalusia, the Middle East, and the Caucasus. As Damask roses were originally introduced from the Middle East into Western Europe, it is thought that the origin and centre of diversity of Damask roses can be found in this region. In Iran, cultivation and consumption of Damask roses has a long history. Crude distillation of roses was probably developed in Persia in the late 7th century A.D. [3, 1012].

In order to study genetic diversity of R. damascena in Iran, all relevant geographical regions of Iran were sampled. Some samples were taken from large production fields in the main rose oil production area in the centre of the country, but most of the samples were collected from smaller production fields and abandoned fields in remote and mountainous areas. In this way 40 Damask rose accessions were collected from 28 provinces of Iran. Results on morphology and oil content variation suggest that this collection may include multiple genotypes [13].

In this investigation, a microsatellite marker analysis of the Iranian collection of R. damascena is reported. We show that we have obtained as much as nine different genotypes, of which some have been used for regional production of Damask rose oil.

Results

Microsatellite analysis

In this study 40 accessions of Rosa damascena (Table 1) that showed a high level of phenotypic and oil content variation were analyzed with nine microsatellite markers. All markers detected polymorphisms among the samples. The number of alleles ranged from 5 to 15 with an average of 9.11 (Table 2). Using the MAC-PR method, we determined the allelic configurations at six loci (RhP519, RhB303, RhEO506, RhD221, RhP50, RhE2b) for all investigated accessions (Table 3).
Table 1

Geographical origins of Iranian Damask rose accessions

Origin site no.

Province(s) included

Accession name

Climatea

Genotypeb

Os1

Isfahan

Isf01

Cool temperate – semi arid

G_I

  

Isf02

 

G_I

  

Isf03

 

G_I

  

Isf04

 

G_I

  

Isf05

 

G_I

  

Isf06

 

G_I

  

Isf07

 

G_I

  

Isf08

 

G_I

  

Isf09

 

G_I

  

Isf10

 

G_I

Os2

East & West Azarbayjan, Ardabil

EastAzar

Cool temperate – semi arid

G_II

  

WestAzar

 

G_V

  

Ardabil

 

G_V

Os3

Kermanshah, Eilam

Kermanshah

Temperate – semi humid

G_VII

  

Eilam

 

G_I

Os4

Tehran, Markazi

Tehran

Cool temperate – semi arid

G_VI

  

Arak

 

G_I

Os5

Chaharmahall, Kohkilooie, Lorestan

Chaharmahall

Temperate – semi arid

G_I

  

Kohkilooie

 

G_I

  

Lorestan

 

G_I

Os6

Razavi Khorasan, South Khorasan

Khor01

Temperate – arid

G_I

  

Khor02

 

G_I

Os7

Khoozestan, Hormozgan, Baloochestan

Khooz

Warm – arid

G_I

  

Hormoz

 

G_I

  

Baloochestan

 

G_I

Os8

Zanjan, Qazvin

Zanjan

Cool temperate – semi arid

G_II

  

Qazvin

 

G_II

Os9

Semnan, Qom

Semnan01

Warm temperate – arid

G_I

  

Semnan02

 

G_I

  

Qom

 

G_III

Os10

Fars, Kerman

Fars01

Temperate – semi arid

G_IX

  

Fars02

 

G_I

  

Kerman

 

G_I

Os11

Kurdistan, Hamedan

Kurdistan

Cool – semi arid

G_I

  

Hamedan

 

G_II

Os12

Guilan, Mazandaran, Golestan

Guilan

Temperate – humid

G_VIII

  

Mazan

 

G_IV

  

Golestan

 

G_IV

Os13

Yazd

Yazd01

Warm temperate – arid

G_I

  

Yazd02

 

G_I

a Yearly mean temperature in warm, temperate and cool climates are 15–25°C, 10–15°C and 0–5°C, respectively. Yearly mean rainfalls in semi humid, semi arid and arid climates are 600–1400 mm, 300–600 mm and 100–300 mm, respectively.

bGenotypes as identified in this study

Table 2

Characteristics of the microsatellite markers used.

Locus

Label

Linkage groupa

Number of alleles

RhP519

6FAM

n.d.b

6

RhB303

HEX

n.d.b

11

RhO517

NED

1

5

RhEO506

6FAM

2

13

RhD221

HEX

4

7

RhAB73

NED

7

9

RhP50

6FAM

3

15

RhAB40

HEX

4

8

RhE2b

NED

6

8

Average

  

9.11

a from Debener et al. [14] and Yan et al. [15]

b n.d.= not determined

Table 3

Allele configuration of the nine different R. damascena genotypes based on MAC-PR analyses

Genotype

Number of accessions

Marker

  

RhP519

RhB303

RhEO506

RhD221

RhP50

RhE2b

G_I

27

232 232 232 232

119 125 127 128

210 222 228 260

209 217 223 226

349 371 404 420

168 170 177 180

G_II

4

219 232 232 241

127 128 128 130

207 213 219 260

209 217 217 223

326 363 371 396

168 168 177 182

G_III

1

219 232 232 241

127 128 128 130

207 213 219 263

209 217 217 223

326 363 371 396

168 168 177 182

G_IV

2

219 232 232 238

122 127 127 129

213 213 228 240

200 209 220 223

343 371 433 433

168 180 180 189

G_V

2

232 232 232 241

127 128 128 130

210 213 213 228

211 217 217 223

349 363 371 396

168 168 180 189

G_VI

1

219 232 232 232

119 125 127 127

210 213 222 228

209 211 223 223

343 371 396 404

168 177 180 189

G_VII

1

219 219 232 238

122 125 145 146

222 225 228 228

217 223 226 226

374 374 404 404

168 168 174 189

G_VIII

1

219 219 232 232

119 125 127 129

213 213 228 228

211 211 223 223

326 354 396 396

180 180 189 189

G_IX

1

219 219 232 232 247 235

117 119 125 128 129 133

195 201 213 234 246 260

211 217 220 220 223 232

340 354 380 396 399 411

168 168 180 182 189 199

Genotype identification

Cluster analysis resulted in grouping of the 40 accessions into nine distinct genotypes (Fig. 1). The main group consisted of 27 landraces that showed the same microsatellite profile. This group included all accessions from the main rose oil production sites of Damask rose in Iran. The pattern of this group was identical to that of an accession from Bulgarian production areas. Rusanov et al. showed that all Bulgarian Damask roses are this genotype [3].
https://static-content.springer.com/image/art%3A10.1186%2F1471-2229-7-12/MediaObjects/12870_2006_Article_153_Fig1_HTML.jpg
Figure 1

1a UPGMA clustering of Dice genetic similarities based on dominant scores of microsatellite alleles, among all accessions of Damask rose included in this study. Note that 1 (similarity) = genetically identical. 1b UPGMA clustering of genetic distances based on pairwise Fst among the nine unique genotypes, derived from codominant scores of six microsatellite loci. Note that 0 (distance) = genetically identical.

The other genotypes that we identified in the cluster analyses were present in much smaller numbers. Some genotypes were unique (accessions from Tehran, Guilan, Kermanshah, Qom provinces and one accession from Fars province); others were present as two or four accessions (Fig. 1a and Table 1). The unique accessions were from mountainous and remote areas in the Northwest of Iran where roses are cultivated on small scale. In addition, the accessions from the humid area near the Caspian Sea were different from all other accessions as well.

The accessions from Fars province formed two distinct clusters in the dendrogram. They are from an environmentally very distinct region, far to the South of Iran. One of these samples was hexaploid, while all other samples were tetraploid, as expected for R. damascena.

As expected, the absolute magnitude of genetic distances based on codominant scoring is much smaller than that of dominant scores, as more alleles are shared, but the topologies of the trees (Figure 1a and Figure 1b) are largely comparable for those samples that were not too genetically distant.

Discussion

It seems that for commercial rose production only one and the same genotype is used in several countries. This makes it likely that also in Turkey this genotype is being used for large-scale production, but this remains to be confirmed as samples from Turkey were not included in the study of Rusanov et al. [3] nor in the present study.

Except one plant, all genotypes identified here were tetraploid, consistent with the general literature. One plant was hexaploid. At this moment, we do not know whether this is the first of more hexaploid R. damascena plants. It may be misclassified, but cuttings from all plants have been evaluated by several experienced taxonomists after cultivation for 2–3 years in a common garden.

The genetic distances among accessions were not correlated with geographical distances among their places of origins (not shown). Clearly, a larger sample of genotypes will be necessary to determine whether there is some relationship with geographical distance, whether there is isolation of populations due to barriers in gene flow, or whether different climatic conditions lead to differentiation within the species.

In MAC-PR analysis we determined the allelic configuration based on six loci, because in the other three loci, not all alleles were present in plants in completely heterozygous configurations, which is necessary to be able to accurately determine the relative amplification of each allele [16]. Genotype G_II and G_III differ by only one allele at locus RhEO506. This is surprising as genotypes in roses are usually identical (due to vegetative propagation) or very different (due to segregation of alleles from the heterozygous parents) [17]. Remarkably, this small difference is confirmed in the MAC-PR analysis, as no differences were found in allele frequencies at the other five loci. Although this does not completely rule out that the two plants are close relatives, a mutation leading to an allele that is one repeat longer is a more likely possibility. Genotype G_III was from Qom, which borders the three provinces in which genotype G_II was found.

Conclusion

Our analysis showed for the first time the existence of multiple genotypes within Rosa damascena. We are currently performing an analysis of oil production across several years, in order to determine whether different genotypes also have a qualitative difference in production and/or composition of essential oil. If so, these genotypes may be used to broaden the production of rose oil, and they can also be used as the basis of a breeding program. As these nine genotypes were found after sampling only 40 large and small production fields, we expect that a more intensive sampling will be valuable in order to find more genetic diversity. For this, we will focus on the areas where we have found the unique genotypes, i.e., the Western and Northern provinces.

Methods

Plant material

A total of 40 Damask rose accessions were collected from 28 provinces of Iran (Table 1), in order to obtain a good geographical coverage of the country and a good coverage of the 13 different climatic regions that have been identified [13]. Samples were taken from commercial production fields and from small (< 5 ha) or abandoned production fields. All accessions were grown from 2000 onwards in experimental field of the Research Institute of Forests and Rangelands (RIFR), Tehran, Iran. DNA was extracted from fresh young leaves using the Qiagen DNeasy Plant Mini Kit (Westburg, The Netherlands).

Microsatellite analysis

A set of nine robust microsatellite markers were selected from Esselink et al. [17] and Yan et al. [15] representing different linkage groups on the genetic map of rose (Table 2). These markers are highly polymorphic in hybrid tea rose [17] and in other Rosa species [1820], and hence have a high discriminative power to differentiate genotypes. Fluorescently labelled (6FAM, HEX or NED) primer pairs were amplified in three multiplexes using the Qiagen PCR multiplex kit (Westburg, The Netherlands). The PCR program for amplification were as follows: 94°C for 15 min; 30 cycles of 94°C for 30 s, ramp to 50°C (1°C/s), 50°C for 30 s, ramp to 72°C (1°C/s), 72°C for 2 min; and a final elongation step at 72°C for 10 min. 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 Vosman et al. [21], using Genotyper 3.5 NT (Applied Biosystems).

MAC-PR and statistical analysis

The microsatellite DNA allele counting – peak ratios method (MAC-PR), which was developed for the tetraploid hybrid tea rose (Rosa × hybrida L.) varieties by Esselink et al. [16], assigns precise allelic configurations (the actual genotype) based on quantitative values for peak areas provided by the Genotyper software. For each locus, all alleles were analyzed in pairwise combinations in order to determine their copy number in the individual samples. This was accomplished by calculating ratios between the peak areas for two alleles in all samples in which these two alleles occurred together.

Genetic distances were calculated either as Dice similarities on the basis of dominant scoring of individual alleles in NTSYS 2.1 (Applied Biostatistics) or as pairwise Fst of the MAC-PR genotypes using SPAGeDi 1.2 [22]. The use of Dice (Nei & Li) coefficient is more suitable for codominant markers such as SSRs when they are scored dominantly [23, 24]. The accessions were clustered using the unweighted pair group method using arithmetic averages (UPGMA) module of NTSYS.

Declarations

Acknowledgements

The authors would like to thank Yolanda Noordijk for her kind assistance in laboratory procedures. Also, we acknowledge Ivan Atanassov, AgroBioInstitute, Sofia, Bulgaria and Natasha Kovacheva, Institute of Rose and Aromatic Plants, Kazanlak, Bulgaria for providing leaf material of Bulgarian damask roses. Ben Vosman and Paul Arens are greatly acknowledged for their critical comments. This work is partly financed by the Ministry of Science, Research and Technology of Iran (MSRTI) through a travel grant for A. Babaei.

Authors’ Affiliations

(1)
Department of Horticultural Science, Faculty of Agriculture, Tarbiat Modares University
(2)
Biotechnology Research Department of Natural Resources, Research Institute of Forests and Rangelands
(3)
Department of Horticultural Science, Faculty of Agriculture, Shiraz University
(4)
Department of Plant Breeding, Faculty of Agriculture, University of Tehran
(5)
Plant Research International, Wageningen UR

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Copyright

© Babaei et al; licensee BioMed Central Ltd. 2007

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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