Running Title : REDISCOVERY of HISTORICAL Vitis vinifera VARIETIES FROM SOUTH ANATOLIA REGION Title : REDISCOVERY of HISTORICAL Vitis vinifera VARIETIES FROM SOUTH ANATOLIA REGION BY USING AMPLIFIED FRAGMENT LENGHT POLYMORPHISM and SIMPLE SEQUENCE REPEAT DNA FINGERPRINTING METHODS Kaan Yilancioglu1, Selim Cetiner1* 1Faculty of Engineering and Natural Sciences, Sabanci University, Orhanlı, Istanbul, 34956 Turkey. *Corresponding Author: 1Faculty of Engineering and Natural Sciences, Sabanci University, Orhanlı, Istanbul, 34956 Turkey, Telephone Number : (+90) 216 483 9000 (9545), Fax Number: (+90) 216 483 95 50, E‐mail : [email protected] ABSTRACT Anatolia is thought to have played an important role in the diversification and spread of economically important crop plants including Vitis vinifera. Although, various biodiversity studies have been already conducted with local grape varieties in different regions of Anatolia, our aim is to contribute to gain better knowledge on the biodiversity of historical grape varieties in North, Adana region of South Anatolia. In the present study, microsatellites were used for pre‐selection of 27 collection varieties among 72 grape accessions obtained from National Germplasm Vineyards, for further determination of genetic relatedness among studied grape accessions amplified fragment length polymorphims (AFLPs) were utilized. The unweighted pair group method with arithmatic mean (UPMGA) cluster and principal component analyses revealed that Saimbeyli accessions form a distict group which are distantly related to collection varieties from all over Turkey. The results of this study revealed the genetic similarities of some isolated historical Anatolian grape varieties cultivated for decades in local vineyards in Saimbeyli, Adana. To our knowledge, this is the first study conducted with these varieties. Further preservation and use of these varieties will be helpful to avoid genetic erosion and diversity loss in this part of Anatolia. Keywords ; Vitis vinifera, AFLP Markers, SSR Makers, Genetic Characterization, Cultivar Identification INTRODUCTION The domestication history of the commercially important plant species, Vitis vinifera, has been hotly debated for years. Some authors accept the view that the first domestications of Vitis vinifera were in the Caucasian area. Because of the strategic location of Anatolia between Asia and Europe, it has been widely accepted that most crop plants originated and spread from this region (Ergul et al. 2006; Ergul 2011; McGowern 2003). Under the light of the previous studies, the history of the domestication of this important crop plant stretches back to ca. 8000 BP in Anatolia and the neighbouring Transcaucasia regions (This et al. 2006). The unique location of Anatolia, the Asian part of Turkey between the Caucasus, southwest Asia, the Mediterranean and Europe is thought to have given it a crucial role for diversification of current grape varieties around the world (Ergul et al. 2006). Therefore, the Anatolia region is becoming one of the most important areas for diversity and characterization studies of grape varieties however very few studies have been conducted in this area to date (Ergul et al. 2006; Ergul 2011; Karatas and Agaoglu 2010). As well as a long history of cultivation of this important crop plant, the very rich variety of biotic and abiotic features of the Anatolian region give another significant opportunity to identify diverse wild and hybrid grape varieties which are currently locally cultivated in private vineyards. Therefore, the oppotunity to obtain new hybrids with many desired traits via utilization of modern molecular breeding techniques among these varieties has arisen. Another important issue is that the rich germplasm of local varieties and genetic resources are being pressurized by climatic changes and cultivation of mostly industrial varieties. For this reason, the need for characterization and preservation of Anatolian local varieties is becoming necessary to stop loss of variability and genetic resources. As characterization studies of grape varieties were mostly based on ampelographic techniques, the germplasm repositories have the same problem that most of the varieties deposited are more or less synonyms or homonyms. This prevents the precise and reliable determination of different genotypes between varieties, and the standardization of breeding efforts. After molecular fingerprinting techniques began to be utilized in pyhlogenetic studies anaylzing diversity between species, bottlenecks in reliable characterizations resulting from dependence on developmental stage and environmental factors of ampelographic markers were excluded. The most popular techniques used in previous molecular phylogenetic studies relied on randomly amplified polymorphic DNAs, RAPDs (Karatas and Agaoglu 2008; Siles et al. 2000), simple sequence repeats, SSRs (Adam‐Blondon et al. 2004; Cipriani et al. 2010; Faria et al. 2000) and amplified fragment length polymorphisms, AFLPs (Ergul et al. 2006; Fanizza et al. 2003; Vorwerk and Forneck 2007). In this study two of these powerful techniques based on SSRs and AFLPs were successfully utilized for determination of genetic relatedness of locally obtained Saimbeyli and collection cultivars obtained from all over Turkey. Saimbeyli is a town located 157 km north of Adana city in the Mediterranean region of Turkey. The region has an ancient history demonstrated by the remains of Hittite civilization found in the area, and more importantly reported to have exported wine to France when the town was still populated by Armenians in early 1900’s. One of the most important commercial products in Saimbeyli is grape which is approximately one fourth of the total grape production of the Adana region. As most of the grape varieties produced in small, local private vineyards are facing extinction, the varieties which may have ancient history and important genetic resources are about to be lost. In this study, genetic relatedness and diversity among locally obtained Saimbeyli grape varieties and collection cultivars obtained from the national repository were studied. Our results clearly showed that the grape varieties collected from local vineyards in Saimbeyli, Adana form a distinct group among 72 selected collection varieties obtained from the National Germplasm Repository, showing lower genetic relatedness to collection cultivars. In addition, some of the synonym and homonym varieties were found among studied varieties in both Saimbeyli and repository cultivars. MATERIALS AND METHODS Plant Material The leaves of the grape accessions studied in this research were obtained from the National Grapevine Germplasm Vineyard at the Institute of Viticulture in Tekirdag, Turkey and were locally obtained from local vineyards in Saimbeyli, Adana. Accessions were shown in Table 1. DNA Isolation DNA isolations were carried out according to Bowers et. al. (Bowers et al. 1993). DNA quantifications and purity analysis were carried out using a NanoDrop® ND‐1000 spectrophotometer at 260 nm and electrophoresis on %1 w/v agarose gels. SSR Analysis In order to preselect collection varieties showing higher genetic relatedness according to the similarity scores (>0.500) to Saimbeyli local cultivars, SSR markers were used. By this approach sample size was minimized, by excluding more distantly related collection varieties before AFLP analysis was done. 4 microsatellite core loci were selected within Genres #081 European Project (VVMD27, VVMD7, VrZAG62, VrZAG79) (This et al. 2004). PCR reactions were carried out in a final volume of 25ul, containing 50ng of genomic DNA, 1.5 units of Taq polymerase (Fermentas), 1X Taq polymerase buffer (Fermentas), 1.5mM MgCl2 (Fermentas), 0.25mM of each dNTP (Fermentas) and 200uM of each forward and reverse primer. Amplification reactions were carried out on thermal cycler using the following cycling profile: 94 °C for 2 min followed by 40 cycles at 94 °C for 30 s, 52–56 °C for 1 min, and 72 °C for 2 min, and a final extension step at 72 °C for 15 min. Aliquots of the amplification products were checked by 1% agarose gel electrophoresis and visualized with ethidium bromide. The remaining PCR products were separated on 6% (w/v) 20cm vertical polyacrylamide gels, visualized by ethidium bromide staining and documented with UV gel illuminator/photography system. AFLP Analysis AFLP analysis was conducted using the LI‐COR® template preparation and 2‐dye selective amplification kits (LI‐COR Biosciences, USA), and visualized with Li‐COR 4300 DNA analyzer (LI‐COR, USA). Briefly, Genomic DNA (250ng) was digested with 1uL of mixture of EcoRI / MseI (1.25units/uL) at 37 °C for 2 hours and then ligated to EcoRI / MseI adapters with 1.5uL (1unit/uL) of T4 DNA ligase at 25 °C for 2 hours. For the pre‐ amplification reaction 1:10 diluted ligation reaction mixture was used. The pre‐amplification mixture was prepared according to the manufacturer’s instructions and the PCR reaction was performed as follows: 30 cycles at 94 °C for 15s, 56 °C for 30s and 72 °C for 1 min, then 72°C for 3 min. For the selective amplification, infrared fluorescence dye (IRD‐700nm and IRD‐800nm) labeled EcoRI primers and non‐ labeled MseI primers were utilized as described in Table 4. Selective amplification mixture was prepared according to the manufacturer’s instructions and then PCR reaction was performed by a touchdown program as follows: 13 cycles at 94 °C for 15s, 65 °C for 30s – 0.7 °C / cycle, and 72 °C for 1 min, then 30 cycles at