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Determination of genetic diversity of natural sage populations in Muğla region of Turkey

  • D. Altindal
Open Access
Original Paper
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Abstract

Turkey, which is a very rich country in terms of medicinal and aromatic plants including sage, is one of the world’s leading gene sources. Thyme and sage that is the medicinal and aromatic plant are naturally grown in the Aegean region. In the present study, 16 ISSR molecular markers were used in the determination of genetic correlations among 8 sages picked up from forests and mountains in Muğla province in the Aegean region. In this study, the average polymorphism rate was determined as 69.84%; in addition, 44 polymorphic bands, totally 63 bands were obtained. As a result of unweighted pair group method with arithmetical average (UPGMA) analysis, the most distant sages to each other were the ones belonging to the Bodrum and the Yerkesik locations, and the ones nearest to each other were obtained from Bodrum and Marmaris locations. On the other hand, in terms of the genetic variation, Dalyan, Köyceğiz, Marmaris and Bodrum locations constituted the first group, and Göcek, Dalaman and Fethiye locations constituted the second group, whereas the sages belonging to the Yerkesik location formed a group by itself. This study revealed that there was a significant genetic variation among the populations of sages grown naturally in Muğla region.

Keywords

Sage Biodiversity Molecular marker ISSR Population 

Introduction

Turkey has rich flora with 12.000 plant taxon due to the differences in geographical factors or cultivation environments of various plants and contains a large number of medicinal and aromatic plants. Therefore, too many plant species form plant populations and do not exhibit the same characteristics in various regions, as a result of the climate and soil characteristics, the local differences emerge and endemic medicinal and aromatic plants grow.

The Western Mediterranean flora, which is included in Muğla, is in the Mediterranean climate zone and is warm and rainy in the winter and hot and dry in the summer. One of the endemic species in Turkey is the sage that grows naturally in the mountains from İzmir to the shores of Muğla.

Sage, considered as a medicinal plant throughout history, is a fragrant plant belonging to the genus Salvia officinalis of Labiatae family. Its motherland is the Mediterranean. It grows well especially in sandy and loamy soils rich with nutrient. Sage is a bush, grown as single-year or perennial, generally 40 to 100 cm in length, and has purple-blue colored cluster-like flowers, and its leaves are gray-green, feathered and pointed. The plants contain both male and female organs or may have also a single female organ. It has two male organs. The varieties of essential oils in plant leaves are at the highest rates before flowering. Therefore, sage leaves are picked up before or at the beginning of flowering in May–June. The leaves collected during the noon hours when active substances reach the peak are left to dry in a shady and narrow area.

The most important ingredient of sage leaves is essential oil (Oleum Salviae) that varies in the rate of 1–2.5% in leaves. Essential oils of sage contain thujone, cineol, linalool, borneol, salven, pinene and camphor, tannins, tritierpotides, flavones being important in pharmacological terms, estrogen-like substances and resins Triterpenes and flavones are present in the essential oils’ composition. In addition, the plant contains water, protein, carbohydrates, calcium, iron, magnesium, phosphorus, potassium, sodium and zinc elements as well as vitamins A and C and niacin (vitamin B3).

In Turkey, Salvia L. has 97 species, 4 subspecies and a total of 109 taxa belonging to 8 varieties. 51 of these species are endemic. 58 of the 97 species grown in Turkey (59.7%) are in Iran-Turan region, 27 (27.8%) in the Mediterranean, 5 (5.0%) in European Siberian phytogeographic region, while 7 (7.0%) are spread in more than one Phytogeographic region. Anatolia Sage (Salvia fruticosa Mill.) populations which shows the natural spread in Muğla province, have been widely used in public medicine for many years (Karık 2015). Salvia L. species, which is used as gas extractor, sedative, carminative, diuretic, stomach healer, sweat preventer, external wound healer and antiseptic in public medicine, have a wide range of biological effects such as antibacterial, antifungal, antiviral, antiseptic, analgesic, antioxidant, astringent, antispasmodic, hallucinogenic, central nervous system depressant, antisudorific, antidiabetic, anticancer, tuberculostatic, cardiovascular therapeutic and insecticide (Lu and Yeap 2002).

Despite many studies on Salvia officinalis, there is very little information about the endemic sage of Turkey. Salvia verbenaca (2n = 14–64) is the species grown almost everywhere in Europe and in Turkey. S. hasankeyfensis, a new species native to Turkey (Dirmenci, Celep and Ö Güner), has recently been added to the Verbenaca group as a species grown at a height of 650–700 m in the hills and surrounding of Hasankeyf (Batman, southeastern Turkey) (Mahdjoub et al. 2018).

As in all over the world, the natural flora plants have been used in the food, insecticide and cosmetics industry through the human history and has become a traditional activity. However, with the modernization, the richness of the plant could not be sufficiently and regularly benefited. Therefore, endemic medicinal-aromatic plant species have been lost. Twenty-five percent of the endemic plant species in Turkey is about to be depleted due to unconscious use. This genetic diversity of Turkey which is among the important countries in terms of biological diversity in the world should be preserved sufficiently and transferred to the future generations. The construction of buildings in Muğla flora, where these species are spreading, is irregular, and intensive animal grazing reduces the population of these species, causing the continuity of species to be compromised over time. Fragmentation or disappearance of natural populations can lead to gene flow among populations with certain genetic characteristics (Ouborg et al. 2006), causing to the lack of genes in populations due to genetic drift, thus increasing the genetic differentiation between populations and genetic structures (Hatmaker et al. 2018).

There is much talk about the disappearance of plant species as a result of erosion in natural plantations where genetic variability is wide, but the number of visible measures is still limited. The decrease in genetic diversity, identification and evaluation of the genetic erosion is the first priority. By improving scientific and technical possibilities, genetic resources of plants can increase. For this reason, the results of genetic erosion must be determined quickly, and genetic resources must be protected. To determine endemic and rare medicinal aromatic plant species, scientific research should be carried out and studies to determine antioxidant properties of medicinal aromatic plants and to learn phytochemical structures should be given priority.

The genetic variability among 6 Salvia species gathered from different parts of the Iran Alborz mountain region was investigated through Inter-Simple Sequence Repeats (ISSR) molecular markers (Safaei et al. 2016).

To compare genetic and metabolic diversity in S. officinalis, the genetic difference among 7 different sage population grown in Greece was also investigated. The significant genetic differences among 7 sage populations grouped in three main clusters according to the UPGMA ISSR data-based dendrogram and Principle Coordinate Analysis were revealed by using Inter Simple Sequence Repeats (ISSR) Analysis (Sarrou et al. 2017).

The ISSR and start codon targeted (ScoT) markers were used to determine the genetic variation of the 9 sage species collected from different geographical regions of Iran. The study illustrated the high genetic diversity, in addition, it was shown that there was a similar polymorphism distribution between ISSR and SCot techniques and that SCot markers could be used as reliable and informative techniques for the evaluation of genetic diversity and the relationships among Salvia species (Etminan et al. 2018).

In this study, 8 naturally grown sages in Muğla were picked up and genetic diversity among the sage populations was investigated by ISSR molecular marker technique.

Materials and methods

In the study, genetic variability was determined by gathering the sage that grows in natural conditions in the coastal areas and nearby provinces of Muğla city. In the study, 8 sages were used as material (Table 1). To perform ISSR-PCR analysis on these species, 20 ng DNA, 50 mM 10 × buffer, 25 mM MgCl2, 1.25 mM dNTP, 20 μM primer and 5 U/μl Taq DNA polymerase were used as stocks for a total of 25 μl of the optimal PCR mix in each tube. In addition, 14 of 16 primers (Altındal 2014; Table 2) which produce polymorphic bands, belonging to LOL, PHV and UBC sets, were used through the ISSR-PCR method. The Bio-rad Cycler thermal PCR device was used for PCR, and the program carried out as 1 cycle at 3 min at 94 °C, 30 s at 94 °C, 45 s at 38–59 °C (Table 2), then 35 cycles at 1 min at 72 °C, followed by 10 min at 72 °C, and ended at 4 °C. After the PCR process, the replicated DNA was run in 2% agarose gel (containing ethidium bromide) in electrophoresis using 1 × TBE buffer at 120 V for 120 min. Then the amplified bands were displayed under UV light and photographed. The ISSR-PCR reactions were repeated three times. The bands that are visible and easily counted on the gel are recorded as existent or not existent (1/0). The generated data matrix was examined using the Numerical Taxonomy and Multivariate Analysis System (NTSYS-pc ver. 2.2) (Rohlf 1991) software. In addition, principal components analysis (PCA) was performed to investigate the genetic variation among sage populations.
Table 1

Locations where the sage populations were picked up

Toplandığı yer

Rakım (m)

Ort yağış (mm)

Ort sıcaklık (°C)

Fethiye

319

983

17.7

Göcek

345

998

17.9

Dalaman

5

1018

18.1

Köyceğiz

50

1032

18.3

Yerkesik

654

1088

15.3

Dalyan

5

1023

18.3

Marmaris

109

928

18.8

Bodrum

8

765

18.9

Table 2

Nucleotide sequences and Tm values of the ISSR primers

No.

Primer

Nükleotid sekansı (5′–3′)

Tm dereceleri (°C)

1

LOL-3

(CA)6AC

41.0

2

LOL-4

(CA)6GT

41.0

3

LOL-5

(GA)6GG

44.0

4

LOL-8

(GT)6CC

44.0

5

LOL-9

(CAC)3GC

38.0

6

LOL-10

(GAG)3GC

38.0

7

PHV-4

GGC(GT)8

59.0

8

PHV-5

ACG(CA)8

57.0

9

PHV-6

CCA(CT)8

57.0

10

UBC-807

(AG)8T

50.0

11

UBC-810

(GA)8T

50.0

12

UBC-811

(GA)8C

52.0

13

UBC-812

(GA)8A

50.0

14

UBC-814

(CT)8A

54.0

15

UBC-826

(AC)8C

52.0

16

UBC-854

(CT)8RG

55.0

Fethiye district where the sage is gathered is located on the west of the Mediterranean region and east of Muğla province. In Fethiye where the characteristic Mediterranean climate is seen, the summers are hot and dry, the winters are warm and rainy. Sage was picked up from the 319-m altitude locations of Fethiye Ölüdeniz region. The altitude of Göcek, which is one of the other locations, is 345, located to the west of the Mediterranean region and to the east of Muğla province, and is surrounded by the Fethiye on the east, the Mediterranean on the west, Dalaman Province on the northwest, and Denizli Province on the north. The characteristic Mediterranean climate is seen in Gocek, its summers are hot and dry, and the winters are warm and rainy. Another location is Dalaman District that is located between Muğla Province-Köyceğiz, Ortaca and Fethiye district and Denizli Province Çameli district. The average annual temperature of Dalaman district is 18.1 °C, annual average rainfall amount is 1018 mm and the altitude is 5 m. Koycegiz district is located where the Mediterranean and Aegean regions meet and has 50 m altitude. The Mediterranean and continental climate are seen in the coastal and mountainous regions of Köycegiz, respectively. The annual average temperature of the district is 18.3 °C, annual average rainfall is 1032 mm. The distance of Yerkesik district is approximately 10 km to the city center of Muğla with altitude 654 m. A warm and temperate climate exist in the region. In winter, there is much more rainfall than in the summer months. The average annual temperature is 15.3 °C, and the average annual rainfall is 1088 mm. Marmaris and Bodrum districts and Dalyan, which is the town of Ortaca district, have a warm and mild climate, with more rainfall in the winter than in the summer months. The altitude of these three locations is 109, 8 and 5 m, respectively. The annual rainfall in Marmaris and Bodrum (928 and 765 mm respectively) is less than other districts (Table 1) (Anonymous 2018).

The Maximum band-producing primers are LOL-4 and UBC-812 primer (Fig. 1), with 4 and 5 polymorphic bands, respectively. UBC-854 primer gave the least with 3 bands (Table 3).
Fig. 1

Gel image obtained by UBC-812 primer

Table 3

Band characteristics of the ISSR primers

Primer

Toplam bant sayısı

Polimorfik bant sayısı

Polimorfizm (%)

LOL-3

8

2

25.00

LOL-4

16

4

25.00

LOL-5

5

1

20.00

LOL-9

13

4

30.77

LOL-10

5

1

20.00

PHV-5

11

3

27.27

PHV-6

15

3

20.00

UBC-807

10

2

20.00

UBC-810

10

4

40.00

UBC-811

8

2

25.00

UBC-812

16

5

31.25

UBC-814

4

3

75.00

UBC-826

4

2

25.00

UBC-854

3

1

33.33

Toplam

128

41

28.91

Results and discussion

Fourteen of the primers used in the study gave reproducible and reliable polymorphic ISSR-PCR products. The band characteristics of these primers showing amplification are given in Table 3. According to Table 3, a total of 128 bands were obtained from 14 primers showing amplification in the sage populations. Forty-one of these amplified bands displayed polymorphic bands. Total polymorphism rate was 28.91%.

In a similar study, ISSR and RAPD molecular techniques were used to evaluate the genetic correlations among 21 ecotypes of 8 Salvia species. As a result, they determined that genomic DNA amplification produced 280 bands, 91% of which was polymorphic (Yousefiazarkhanian et al. 2016). Safaei et al. (2016) reported that the highest polymorphism rate (57.14%) was found in S. limbata sage and the lowest polymorphism rate (28.5%) was found in S. reuterana. In another study conducted with ISSR method, the polymorphic rate of 9 sage species was determined to be 0.38% (Etminan et al. 2018).

The similarity ratios of PCR products obtained by polymorphic primers are given in Table 4. The highest similarity rate (60%) was obtained between Marmaris and Bodrum sages, while the lowest (10%) was found between Yerkesik and Bodrum sages. According to the obtained data; the most distant sages to each other were the Yerkesik and Bodrum sages, the nearest ones were the Marmaris and Bodrum sages. We examined the location information where sage populations are picked up (Table 1), and because the Yerkesik location has the highest altitude, the average annual precipitation is higher, the average annual temperature is lower and the geographical location is different, it appears that the sage belonging to this location is not similar to the sage populations growing at other locations. In addition, although the altitude difference between Marmaris and Bodrum is very high, the sage collected from these locations is similar because the average annual rainfall and temperature are the same in both districts. This can be explained by the fact that sage populations may be linked to climate and ecological characteristics of the places where they are grown, or may emerge independently of these characteristics.
Table 4

Similarity matrix values of sage populations

 

Fethiye

Göcek

Dalaman

Köyceğiz

Yerkesik

Dalyan

Marmaris

Bodrum

Fethiye

1

       

Göcek

0.51

1

      

Dalaman

0.41

0.51

1

     

Köyceğiz

0.33

0.48

0.43

1

    

Yerkesik

0.26

0.20

0.23

0.31

1

   

Dalyan

0.47

0.25

0.39

0.40

0.34

1

  

Marmaris

0.32

0.17

0.30

0.42

0.32

0.46

1

 

Bodrum

0.29

0.27

0.41

0.54

0.10

0.30

0.60

1

Bold values indicate similarity ratio

In the present study, 2 main clusters were formed in the dendrogram (Fig. 2) which was created according to the similarity index of 8 sage. In the first main set, while the Yerkesik sage is located alone, the other main set is divided into two subsets. Bodrum sage were similar to Köycegiz sage in the rate of 60% and to Marmaris sage in the rate of 42%. The other subset is also divided into two subsets. The similarity between Göcek and Dalaman sages is 51% and between Fethiye and Dalyan sages is 47%.
Fig. 2

Dendrogram showing the genetic relationship among sage populations

In this study, principal component analysis (PCA) was performed to determine the genetic variation among sage populations (Fig. 3). According to the results, in terms of genetic variation, the sages belonging to Marmaris, Bodrum, Dalyan and Köyceğiz locations were in the same group. Sages, which is provided from Fethiye, Dalaman, and Göcek, is included in the same group and Yerkesik sage is classified in the separate group.
Fig. 3

Principal components analysis for sage populations

The sages took place in 3 groups (except Dalyan) similarly to the dendrogram obtained from the genetic similarity equation. Although Dalyan, Göcek, Fethiye and Dalaman are placed in the same group in the dendrogram, the sage belonging to the Dalyan location was included in the different group by the principal component analysis (PCA). This can be explained by the fact that the location where the sage grows naturally has different ecological conditions or different genetic characteristics.

Conclusion

In the research area, the climate characteristics are rainy and temperate and at the same time, the ecological structure has suitable conditions, which causes different plant vegetation to develop and thus plant diversity is increasing. A detailed inventory of the biological diversity and Genetic Resources of Muğla province, morphological examination of its genetic resources, molecular characterization, identification and conservation should be realized. In addition, some legal and technical measures should be taken, gene banks should be established, the benefits of biological wealth should be taught with educations, and public awareness should be made on this issue.

Notes

Acknowledgments

The authors wish to thank all who assisted in conducting this work.

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OpenAccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

Authors and Affiliations

  1. 1.Department of Crop and Animal Production, Organic Farming Programme, Fethiye Ali Sıtkı Mefharet Koçman Vocational SchoolMuğla Sıtkı Koçman UniversityMuğlaTurkey

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