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Species Using RAPD Markers

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Species Using RAPD Markers İ. ERÖZ POYRAZ, E. SÖZEN, E. ATAŞLAR, İ. POYRAZ Turk J Biol 36 (2012) 293-302 © TÜBİTAK doi:10.3906/biy-1012-177 Determination of genetic relationships among Velezia L. (Caryophyllaceae) species using RAPD markers İlham ERÖZ POYRAZ1, Emel SÖZEN2, Ebru ATAŞLAR3, İsmail POYRAZ4 1Department of Pharmaceutical Botany, Faculty of Pharmacy, Anadolu University, 26470 Eskişehir - TURKEY 2Department of Biology, Faculty of Sciences, Anadolu University, 26470 Eskişehir - TURKEY 3Department of Biology, Faculty of Science and Literature, Eskişehir Osmangazi University, 26480 Eskişehir - TURKEY 4Department of Molecular Biology and Genetics, Faculty of Science and Letters, Bilecik University, 11210 Gülümbe, Bilecik - TURKEY Received: 24.12.2010 Abstract: Random amplifi ed polymorphic DNA (RAPD) markers were used to determine genetic relationships among Velezia L. species from Turkey. A total of 432 amplifi ed bands were obtained using 14 RAPD primers. Th e polymorphism in RAPD markers was high (98.61%) and was suffi cient to distinguish each species. Th e degree of band-sharing was used for evaluating the genetic similarity between species and for constructing a dendrogram by the unweighted pair group method with arithmetic mean (UPGMA). Genetic relationships among the species were found to be fully consistent with those obtained by the use of morphological characters. Data obtained from our study demonstrated that the RAPD technique could be successfully used for the determination of genetic relationships among Velezia species and for species identifi cation. Furthermore, it can be effi ciently employed in future studies to provide preliminary data for conservation of endangered Velezia species. Key words: Genetic relationships, RAPD-PCR, Velezia L., Turkey RAPD markörleri kullanılarak Velezia L. (Caryophyllaceae) türlerinin genetik akrabalıklarının belirlenmesi Özet: Bu çalışmada, RAPD (Random Amplifi ed Polymorphic DNA) belirteçleri Türkiye’de yetişen Velezia L. türlerinin genetik akrabalık seviyelerini belirlemede kullanılmıştır. 14 RAPD primerinden, toplam 432 bant elde edilmiştir. RAPD belirteçlerindeki polimorfi zm oranı yüksek (% 98,6) ve her türü ayırt etmek için yeterlidir. Paylaşılan ortak bant seviyesi türler arasındaki genetik benzerliği değerlendirmek ve UPGMA (unweighted pair group method with arithmetic mean) tabanlı bir dendrogram oluşturmak için kullanılmıştır. Türler arasındaki genetik akrabalıkların, morfolojik karakterlerin kullanımıyla elde edilen verilerle tamamen uyumlu olduğu bulunmuştur. Elde edilen sonuçlar RAPD tekniğinin Velezia türleri arasındaki genetik akrabalıkların belirlenmesinde olduğu kadar türlerin tanımlanmasında da başarıyla kullanılabileceğini göstermiştir. Ayrıca bu teknik gelecekteki çalışmalarda, özellikle tehlike altındaki Velezia türlerinin korunması için ön verileri sağlamada etkin olarak kullanılabilir. Anahtar sözcükler: Genetik akrabalık, RAPD-PCR, Velezia L., Türkiye 293 Determination of genetic relationships among Velezia L. (Caryophyllaceae) species using RAPD markers Introduction for revealing genetic relationships following the In Turkey, the genus Velezia L. (Caryophyllaceae) optimization of polymerase chain reaction (PCR) comprises 6 species: V. hispida Boiss. & Balansa, V. conditions (3,10-17). quadridentata Sibth. & Sm., V. fasciculata Boiss., V. Th e taxonomy of Velezia is restricted to the rigida L., V. pseudorigida Hub.-Mor., and V. tunicoides morphological observations made by Davis (1), P.H.Davis. Among these, V. rigida is a cosmopolite and, to date, no study has been undertaken for the species that is widespread in Turkey and throughout identifi cation and evaluation of genetic relationships the world; V. quadridentata is mainly distributed over of Velezia based on molecular methods. In Flora of the northwestern parts of the Marmara and Aegean Turkey, the dichotomous diagnostic key for Velezia regions; V. tunicoides, a narrow, local endemic species, L. was built based on the diff erences in indumentum, is distributed only in the Antalya-Göynük region calyx, petal, and infl orescence. It should be mentioned (southern Turkey); V. hispida, an endemic species, that identifi cation of Velezia species solely on the is distributed only in the middle and southeastern basis of the morphology of reproductive structures is parts of the Aegean region; and V. pseudorigida is very tedious, especially when accurate identifi cation also an endemic species that is widespread from of 2 morphologically similar species, such as V. the mid-Aegean region to the southwestern parts pseudorigida and V. rigida, is undertaken. of Turkey (1). During the fi eld study, no specimens Th us, the aim of the present study was to evaluate for V. fasciculata could be found in the A5 Amasya the usefulness of the RAPD technique in generating and Çorum regions of northern central Turkey. It is DNA markers for identifying morphologically possible that the distribution sites and population similar Velezia species from Turkey and determining size of this species decreased with time due to the genetic relationships among them. habitat destruction (2). Recently, V. tunicoides and V. pseudorigida have been categorized as critically endangered and V. hispida has been categorized as Materials and methods near threatened (2). Plant materials Accurate identifi cation of the species and a precise Whole plant samples from each species of Velezia diagnostic key are very important for fl oristic studies; were collected at diff erent locations in Turkey (Figure precise identifi cation also aids in conservation eff orts 1, Table 1) between June 2003 and July 2007. Since and the rational use of genetic resources. For this V. rigida is a widespread species, 15 populations reason, diff erent types of molecular markers were from diff erent regions were included in the study. developed for facilitating taxonomic, evolutionary, V. hispida was sampled from 2 localities (B3 Afyon and phylogenetic studies based solely on morphology and C3 Isparta), and Isparta was a new record for V. (3-5). As DNA markers are unlimited in number, hispida. For the other Velezia species, plant samples produce high polymorphism, and are independent were collected from single localities. Samples were of environmental interactions, they are considered identifi ed based on morphology (Table 1), and valuable tools for determining genetic relationships voucher specimens were deposited in the herbarium and diversity. Random amplifi ed polymorphic DNA of Eskişehir Osmangazi University (OUFE). (RAPD) data are known to be generated faster and more easily than other methods such as restriction DNA extraction fragment length polymorphism (RFLP) and For DNA analysis, 5 plant samples collected from microsatellites (6,7). However, the use of RAPD has each locality were used. Total genomic DNA was been criticized for revealing unreliable phylogenies extracted from herbarium materials by a modifi ed owing to the occasional lack of reproducibility and hexadecyltrimethyl ammonium bromide (CTAB) a possible lack of homology in comigrating bands method (18). Th e quantity and quality of DNA (8,9). Despite this limitation, the RAPD method has were determined by using a Nanodrop® ND-1000 been successfully employed in several plant genera spectrophotometer (Wilmington, DE, USA). 294 İ. ERÖZ POYRAZ, E. SÖZEN, E. ATAŞLAR, İ. POYRAZ Table 1. Th e names and locations of Velezia species used for RAPD analyses. Accessio Species Locality and habitat Altitude no.(OUFE) Velezia tunicoides 13461 C3 Antalya: Göynük, dry stream bed, 12.07.2007, 36°38ʹ849ʺN, 30°32ʹ962.2ʺE 2 m P.H.Davis B3 Afyon: 2 km to Karakaya village, north hillsides, 24.06.2005, 21.07.2005, Velezia hispida Boiss. 13440 1160 m 29.07.2006, 02.08.2007, 38°53ʹ19.7ʺN, 30°48ʹ47.8ʺE C3 Isparta: Gölcük Lake National Park, roadsides, 23.07.2006, 37°43ʹ60ʺN, Velezia hispida Boiss. 13453 1200 m 30°30ʹE Velezia quadridentata B1 İzmir: Emiralem to Menemen, stony places, 21.05.2005, 38°27ʹ48.7ʺN, 13427 6 m Sibth. & Sm. 27°12ʹ18.7ʺE C2 Muğla: Muğla to Yılanlı Forest Management Planning Department, crossroads Velezia pseudorigida 13449 of power-plant, southeast, stony-calcareous hillside, 21.07.2006, 37°13ʹ32.1ʺN, 1250-1260 m Hub.-Mor. 26°27ʹ33.0ʺE Velezia rigida L. 10456 A3 Bilecik: Bozüyük to Bilecik, roadsides, 21.06.2003, 39°54ʹN, 30°2ʹE 873 m A3 Bolu: Nallıhan to Mudurnu, 16 km to Mudurnu, roadsides and under Velezia rigida L. 13415 1000 m Juniperus sp., 29.07.2004, 40°11ʹ1ʺN, 31°21ʹ2ʺE A5 Amasya: Eğerli Mountain, north stony hillsides, 28.06.2005, 40°46ʹ41.4ʺN, Velezia rigida L. 13416 910 m 35°13ʹ11.3ʺE A5 Amasya: Amasya to Ziyaret, west and east hillsides, roadsides, under Quercus Velezia rigida L. 13419 720 m sp., 06.07.2007, 40°43ʹ30.8ʺN, 35°49ʹ0.20ʺE Velezia rigida L. 13420 A5 Çorum: Çorum to Amasya, roadsides, 06.07.2007, 40°35ʹ72.5ʺN, 35°08ʹ61.1ʺE 1026 m Velezia rigida L. 13464 B1 Çanakkale: 11 km to Ayvacık, 20.05.2005, 39°34ʹ17.7ʺN, 26°32ʹ38.9ʺE 553 m Velezia rigida L. 13430 B2 Kütahya: Tavşanlı, 28.06.2006, 39°33ʹ07.8ʺN, 29°29ʹ52.7ʺE 938 m B2 Kütahya: Tunçbilek, near Ömerler mine, 25.06.2005, 39°40ʹ45.8ʺN, Velezia rigida L. 13431 830 m 29°27ʹ10.4ʺE Velezia rigida L. 13436 B2 Manisa: Sipil Mountain, west hillsides, 21.05.2005, 38°35ʹ31.2ʺN, 27°26ʹ01.1ʺE 700 m Velezia rigida L. 13438 B3 Eskişehir: Türkmen Mountain, 28.06.2007, 39°32ʹN, 30°15ʹE 1200 m B3 Konya: Akşehir, Tekke village, Sultan Mountains, 13.06.2004, 38°20ʹN, Velezia rigida L. 13441 1100 m 31°23ʹE C2 Muğla: Fethiye, Babadağ, parasailing area, 23.06.2005, 36°32ʹ19.9ʺN, Velezia rigida L. 13448 1816 m 29°10ʹ12.3ʺE Velezia rigida L. 13459 C3 Antalya: Akseki to Sadıklar, road sides, 15.06.2004, 36°57.663ʹN, 31°46.266ʹE 903 m Velezia rigida L. 13463 C5 Mersin: Gülek-Akçatekir (Tekir), 26.06.2004, 36°55ʹN, 34°53ʹE 1250 m C5 Adana: Kozan to Feke 20th km, roadsides in rubble, 25.6.2004, 37°49ʹN, Velezia rigida L. 13462 620 m 35°54ʹE 295 Determination of genetic relationships among Velezia L. (Caryophyllaceae) species using RAPD markers 12345678910 42° A 40° B 38° C 01020 km 36° 26° 28° 30° 32° 34° 36° 38° 40° 42° 44° Figure 1. Distribution map of Velezia species in Turkey used for RAPD analyses.
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