BIHAREAN BIOLOGIST 12 (2): 61-64 ©Biharean Biologist, Oradea, Romania, 2018 Article No.: e181101 http://biozoojournals.ro/bihbiol/index.html

Determination of genetic diversity of Sonchus erzincanicus Matthews (Asteraceae), a Critically Endangered plant endemic to Turkey using RAPD markers

Nalan Yıldırım DOĞAN, Pınar KURT* and Etem OSMA

Department of Biology, Faculty of Arts and Science, Erzincan University, Erzincan, Turkey. * Corresponding author, P. Kurt, E-mail: [email protected]

Received: 20. August 2017 / Accepted: 26. March 2018 / Available online: 28. March 2018 / Printed: December 2018

Abstract. This study was carried out to determine the genetic structure of three Sonchus erzincanicus (one of the endemic species in Turkey that is on the edge of extinction) populations from Ekşisu, Beşsaray and Saztepe districts in Erzincan province, using Random Amplified Polymorphic DNA (RAPD) marker. In this study, 15 samples from each population were used and sixteen RAPD (Random Amplified Polymorphic DNA) produced 125 bands at a size of 250-3000 bp. Beşsaray has the maximum polymorphism rate (84.80%) among the populations, whereas Ekşisu has the minimum rate (58.40%). Beşsaray and Ekşisu were the closest populations according to Nei's genetic distance values (D=0.9671), and Ekşisu and Saztepe were the farthest populations (D=0.0639). The gene flow between populations (Nm) was calculated as 1.71, while genetic differentiation (Gst) was calculated as 0.127.

Key words: endemic, genetic structure, RAPD, Sonchus erzincanicus.

Introduction sity of populations of genus Sonchus erzincanus, which is at the Critically Endangered (CR) level according to the IUCN Turkey has a very rich flora due to its geographical location, threat category in three different locations in Erzincan, Tur- as well as its climatic and physical characteristics. The ratio key. of endemic plant species in the country is approximately 33.5%. The Asteraceae is the family which has the most en- demic species with a rate of 38% endemism (Kaya & Aksakal Material and Methods

2005). Plant materials: In the study, three populations of genus Sonchus erz- The genus Sonchus L. belonging to Asteraceae family has incanicus, which are in Erzincan province (Ekşisu, Saztepe, Beşsaray a wide distribution in the world with the exception of Cen- regions), were sampled by selecting 15 individuals for each popula- tral America and South America. The genus Sonchus has 14 tion using random sampling method. Healthy and fresh leaf samples taxas in the European flora (Leventer 2012). were collected from their natural habitat and stored at -80 C until Sonchus erzincanicus is a perennial that is an herbaceous DNAs were isolated. For each population, DNA samples were ex- with average 130 cm in length, also is a seed breeding with a tracted separately. capitula of 1.5-2 cm wideness. Each face of its achene has 1-2 DNA extraction: DNA isolation protocol is based on the study of Cheng Lin et al. (2001), and several items of that protocol have been veins and the lower leaves are rosette, rectangular, and ellip- modified. tical shaped that are feathered and thorny. Leaf stalk is RAPD Amplification: In this study, 45 RAPD primers using OP- bright yellow; its achene is 3-4 mm long and slightly out- ERON Technologies (Alameda, Calif.) were tested and it was studied ward convoluted. In addition, the flowering period is be- on 16 primers, giving amplification. Tm values of the primers, used tween June and September (Kandemir et al. 2006). in the study are given in Table 1. Sonchus erzincanicus is an endemic species and only Table 1. Collected localities of S. erzincanicus. spreads around a limited area all over the world in Erzincan, Turkey. The species grows in humid soils where the Locality Coordinates Altitude groundwater level is not too high; also in environments with Ekşisu N 39° 42’58.21’’E 39° 34’11.28’’ 1153 light salt, strong alkaline, dense clay, lime and rich in terms Beşsaray N 39° 42’26.41’’E 39° 25’43.11’’ 1158 of organic matter. It can also grow in soil containing slightly Saztepe N 39° 42’19.14’’E 39° 37’31.15’’ 1150 basic and low organic matter at times (Kandemir et al. 2006). RAPD, emerged at first as a PCR-based method in which Protocol of RAPD-PCR: The protocol RAPD-PCR: it was pre- randomly selected primers were used, in 1990 (Öz Aydın pared in a way that total 20 μl, including 10x 2μlPCR buffer, 1 μl100 2004). The RAPD method amplifies with PCR, by randomly ng/lDNA, 0.5 μl10 MmdNTP, 1.25 μl25 MmMgCl2, 1 μl5 lPrimer, binding of a selected 9-10 bp oligonucleotide primer to ge- 1 μlTaqDNApolimeraz (5 Unit) and 13.25 μl pure water. The prepared samples were placed in a PCR automated thermo- nomic DNA at low binding temperature. The obtained am- cycle instrument (Eppendorf Mastercycler Gradient Authorized plified product by this method is carried out on standard gel Thermal Cycle) and subjected to the following cycles. The PCR in- electrophoresis and the amplified products are observed on strument was automatically held for 5 min at 94 C, and held for 1 the gel in bands, and the presence or absence of the bands on min 30 sec 94 C, 1 min 30 sec 37 C, 3 min 72 C respectively, in 4 the gel are watched when evaluating (Williams et al. 1990). cycles. Then, it was held in 41 cycles for 1 minute at 94 C, 1 minute The RAPD method is a reliable method used in individual at 36 C, 1 minute at 42C, 3 minutes at 72 C, respectively. Finally, cases, fingerprinting studies, forensic-medical cases, clinical the process was completed by holding at 72 C for 7 minutes. Sam- trials, population genetics, a diversity of genetic resources, ples removed from the PCR instrument were stored at 4 C. genetic mapping and ecological studies (Rafalski et al. 1994). Electrophoresis: The PCR products (20 μl) were loaded onto an agarose (1.5 % w/v) gel electrophoresis in 1 XTBE (Tris-Borate- In this study, it is aimed to investigate the genetic diver- EDTA) buffer at 70 V (volts) for 150 min. The gel was stained in 62 N.Y. Doğan et al.

primer. While the number of polymorphic bands of OPK-19 and OPBA-03 primers was the highest, the number of poly- morphic bands of primers OPD-20 and A-8 was the lowest (Table 2).

Table 3. Genetic diversity with in S. erzincanicus populations detected by RAPD markers.

Population Na Ne h I P %P Saztepe 1.6560 1.2645 0.1715 0.2737 82 65.60 Beşsaray 1.8480 1.2454 0.1725 0.2909 106 84.80 Ekşisu 1.5840 1.2637 0.1676 0.2631 73 58.40 Average 1.9920 1.2750 0.1946 0.3307 124 99.20

Na: Observed number of alleles, ne: Effective number of alleles, H: Nei’s (1973) gene diversity, I: Shannon's information index,

Figure 1. Amplification products generated from 40 individuals of P: Number of polymorphic loci, %P: %Percentage of polymorphic loci.

Beşsaray populations of S. erzincanicus using primer A-1 (250-10000

bp). Among the populations, the rate of polymorphism of

Beşsaray was the highest (84.80%), and Ekşisu polymor- ethidium bromide solution for 40 min and visualized under ultravio- phism rate was the lowest (58.40%). In addition, the highest let (UV) light in BioDoc Image Analysis System with Uvisoft analysis observed number of alleles was in the Beşsaray with 1.8480 package (Cambridge, UK). per location; and Ekşisu population allocated at the lowest Data analysis: The reproducible and well-defined bands were point with 1.5840. Meanwhile, with an average of 1.2750, the only scored. The matrix was then used for the following analyses: number of effective alleles per location was at the highest the percentage of polymorphicband (PPB), observed number of al- leles (na), effective number of alleles (ne), Shannon’s information in- value with 1.2645 in Saztepe population, while in the Beş- dex (I) and Nei’s gene diversity (H) (Lewontin 1972, Nei 1973). These saray population was at the lowest value with 1.2454. Shan- measures were obtained at both species level and population level non information index (I) and Nei's gene diversity (h) were using the software package POPGENE (Yeh et al. 1997). A dendro- found at the highest level in Beşsaray population with 0.2909 gram was also constructed based on Nei’s genetic distance (D) using and 0.1725 and at the lowest level in Ekşisu population with an unweighted group method of cluster analysis employing arithme- 0.2631 and 0.1676, while their average was calculated as tic averages (UPGMA) using NTSYS-pc version 2.02c (Rohlf 2007). (0.3307 and 0.1946). The average number of alleles observed

in all populations was 1.9920, the number of polymorphic Results loci was 124, and the rate of polymorphic loci was 99.20% (Table 3). According to these results, the closest populations Results of our RAPD analysis are summarized in Table 2 and to each other according to genetic distance values were Beş- 3. Forty-five RAPD primers were tested to determine the saray and Ekşisu (D=0.9671), and the farthest populations to proportion of polymorphisms in three different populations each other were Ekşisu and Saztepe (D = 0.0639). Genetic di- of S. erzincanicus, but only 16 of them gave reproducible and versity in the total set of the population (Ht) and average polymorphic bands (Table 2). These bands are 250-3000 bp in gene diversity within the population (Hs) calculated as 0.195 size. A total of 125 bands were observed and 113 polymor- and 0.170, respectively (Table 4). The gene flow (Nm) be- phic bands were detected. The rate of total polymorphism tween the populations was calculated as 1.71 and the genetic was 90.4%. The maximum band number was observed in the differentiation value (Gst) as 0.127. OPA-13 (11) and the minimum band number in the OPD-20 The genetic distance between 3 populations of SJonchus

Table 2. List od the selected primes code and the degree of polimorphism obtained among the three populations.

Primer Sequence 5’→3’ Tm Length of amplified bands No. of bands No. of polymorphic bands P(%) OPK19 CACAGGCGGA 34oC 900-2700 10 10 100 OPBB- 3 TCACGTGGCT 32oC 750-2500 8 8 100 OPA– 4 AATCGGGCTG 32oC 750-2500 9 9 100 OPA-13 CAGCACCCAC 34oC 900-3000 11 9 81.8 OPH- 17 CACTCTCCTC 32oC 750-2900 10 9 90 OPK-4 CCGCCCAAAC 34oC 600-2800 9 8 88.8 OPY-6 AAGGCTCACC 32oC 750-2500 9 9 100 OPC-11 AAAGCTGCGG 32oC 600-1800 7 6 85.7 OPW– 17 GTCCTGGGTT 32oC 750-2200 9 9 100 OPBA-03 GTGCGAGAAC 32oC 750-2600 10 10 100 OPL-09 TGCGAGAGTC 32oC 850-2000 6 6 100 OPD-20 ACCCGGTCAC 34oC 750-1100 4 3 75 OPN-16 AAGCGACCTG 32oC 900-2000 5 4 80 A-1 AGTCAGCCAC 32oC 250-2000 6 4 66.6 A-3 GGGTAACGC 30oC 250-2000 6 6 100 A-8 GTGACGTAGG 32oC 250-2000 6 3 50 Total 250-3000 125 113 90.4

Determination of genetic diversity of Sonchus erzincanicus Matthews using RAPD markers 63

Table 4. Nei’ genetic distance of three S. erzincanicus populations. important factor in determining genetic diversity in plant

Populations Saztepe Beşsaray Ekşisu species (Hamrick 1982). While genetic diversity is less in Saztepe **** 0.9601 0.9381 Ht 0.195 self-fertilized taxa, it is higher in externally fertilized or hy- Beşsaray 0.0407 **** 0.9671 Hs 0.170 brid species (Nybom & Bartish 2000). S. erzincanicus species Ekşisu 0.0639 0.0334 **** Gst 0.127 was grown through seeds. However, That the seed could not

Ht- Gene diversity in total set of populations; Hs- average gene diversity germinate due to environmental conditions caused the spe- within population; Gst- Nei’s genetic differentiation index among popula- cies to head to vegetative reproduction. tions. The Gst value could be between 0 and 1. It was empha-

sized that Gst value is higher than 0.5 in self-fertilized spe- cies, and is between 0.1 and 0.2 in species fertilized with seed (Hamrick & Godt 1989). In the study results, the Gst value was found to In the conservation genetic studies of endemic plants, this value was found to be as 0.3849, 0.3577 and 0.3670 for Limonium sinense using RAPD, ISSR and AFLP markers (Ding et al. 2013), and for Mentha cervina as 0.532 (Rodrigues et al. 2013), while for endemic Plantago species was calculated to be 0.1873 (Ferreria et al. 2013). Further- more, in a study conducted with RAPD markers in Centaurea wiedemanniana, an endemic species growing in Turkey, high genetic differentiation (GST = 0.223) was detected among the populations. In this study, it was aimed to evaluate genetic structure of Sonchus erzincanicus Matthews (Asteraceae) populations

that are naturally distributed and endemic around Erzincan, Figure 2. UPGMA clustering for three S. erzincanicus populations Turkey using RAPD markers. The genome of the species we based on RAPD markers. studied was not available, random amplified Polymorphic

DNA (RAPD) method was preferred in our study. erzincanicus being calculated according to Nei (1972) was plotted according to the UPGMA method (Fig. 2) and the populations were divided into 2 groups. Beşsaray and Ek- şisu were in the same group, while Saztepe was in a separate References group. Cheng, L.R., Ding, Z.S., Liliang, B.L., Kuang, T.Y. (2001): A rapid and efficient DANN mini preparation suitable for screening transgenic plants. Plant Molecular Biology Reporter 19: 379a-379e. Discussion Ding, G., Zhang, D., Yu, Y., Zhao, L., Zhang, B. (2013): Analysis of genetic variability and population structure of the endemic medicinal Limonium sinense using molecular markers. Gene 520: 189-193. Considering the results of the study in terms of the number Ferreira, V., Matos, M., Correia, S., Martins, N., Gonçalves, S., Romano, A., of alleles, it was found that the number of alleles that are ef- Pinto-Carnide O. (2013): Genetic diversity of two endemic and endangered Plantago species. Biochemical Systematicand Ecology 51: 37-44. fective for all populations is lower than it is expected. The Hamrick, J.L., Godt, M.J.W. (1989): Allozyme diversity in plant species. pp. 43- number of observed alleles will be equal to the number of ef- 63. In: Brown, A.H., Clegg, M.T., Kahle, A.L., Weir, B.S. (eds), Plant fective alleles only when the whole alleles are the same fre- Population Genetics, Breeding, and Genetic Resources. Sinauer Associates Inc., Sunderland, Massachusetts, USA. quency (Kimura & Crow 1978). Hamrick, J.L, (1982): Plant population genetics and evolution. American Journal Genetic variation and gene flow are important indicators of Botany 69: 1685–1693. for evaluating a species’ genetic makeup (Song et al. 2010). Kandemir, A., Makbul, S., Türkmen, Z., Yılmaz, M. (2006): Morphological, Anatomical and Palynological Investigation on Sonchus erzincanicus Hamrick & Godt (1989) reported the mean Nm value is 0.265 Matthews (Asteraceae). Turkish Journal of Botany 30: 405-411. for the species that are self-fertilized, spread their seeds and Kaya, Y., Aksakal, Ö. (2005): Endemik Bitkilerin Dünya ve Türkiye’deki pollens to very short (2-3 m diameter) distances, and is 4,750 Dağılımı. Erzincan Eğitim Fakültesi Dergisi 7: 85. for the species that are fertilized externally and are able to Kimura, M., Crow, J.F. (1978): The number of alleles that can be maintained in a finite population. Genetics 49: 725–738. spread their seeds and pollens to remote distances by vari- Leventer, S. (2012): The Morphological, Anatomical and Palynological ous carriers. On the other hand, the critical Nm for popula- Investigations on Sonchus L. (Asteraceae) in European Part of Turkey. tions is 0.50, and values above this value indicate that the Master Thesis, Trakya University Institute of Naturel Sciences, 1-2. 90pages, Turkey. gene flow is at amount which can prevent genetic drift. In Lewontin, R.C. (1972): Testing the theory of natural selection. Nature 236: 181- addition, the Nm value of less than 1 indicates that differen- 182. tiation has begun due to genetic drift in populations (Wright Nei, M. (1973): Genetic Distance Between Populations. The American Naturalist 106: 283-292. 1969). Nybom, H., Bartish, I.V. (2000): Effects of life history traits and sampling The protective gene of Tuberaria major (Cistaceae), one of strategies on genetic diversity estimates obtained with RAPD markers in the endemic species, was studied with ISSR markers and the plants. Perspectives in Plant Ecology, Evolution and Systematics 3: 93–114. Öz Aydın, S. (2004): RAPD (Rastgele arttırılmış polimofik DNA) belirleyicileri Nm value was found to be 2.199 (Trindade et al. 2012). For ve bitki sistematiği. DPÜ Fen Bilimleri Enstitüsü Dergisi 6: 113-130. Limonium sinense, Nm value at the RAPD, ISSR and AFLP Rafalskı, A., Tingey, S.V., Williams, J.G.K. (1994): (RAPD) markers. Plant markers were calculated as 0.581, 0.618, 0.612, respectively Molecular Biology Mannual 114: 1-8. (Ding et al. 2013). Reproductive systems are thought to be an 64 N.Y. Doğan et al.

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