HORTSCIENCE 44(6):1557–1561. 2009. Miaja et al., 2001), amplified fragment length polymorphism (AFLP) (Ferrari et al., 2004), and simple sequence repeat (SSR) (Boccacci Genetic Characterization of Hazelnut et al., 2006; 2008; Gokirmak et al., 2009). Palme and Vendramin (2002) used polymer- (Corylus avellana L.) Cultivars from ase chain reaction (PCR)-RFLP to look at chloroplast variation in wild hazelnut popu- Turkey Using Molecular Markers lations. The objective of this study was to evaluate RAPD, ISSR, and AFLP markers for Salih Kafkas1 and Yıldız Dog˘an identifying 18 hazelnut cultivars that are Department of Horticulture, Faculty of Agriculture, University of Cxukurova, economically important in Turkey. Adana, 01330, Turkey Materials and Methods Ali Sabır Department of Horticulture, Faculty of Agriculture, University of Selcxuk, Plant materials and DNA isolation. Eigh- teen hazelnut cultivars, grown primarily in Konya, 42070, Turkey the Giresun, Ordu, Trabzon, Duzce, and Ali Turan and Hasbi Seker Izmit provinces, were used (Table 1). Leaf samples were collected from the germplasm Hazelnut Research Institute, Giresun, 28200, Turkey collection of the Hazelnut Research Institute Additional index words. RAPD, ISSR, AFLP, genetic similarity, PCoA in Giresun, Turkey. DNA from young leaves was extracted according to the CTAB-based Abstract. Genetic relationships among 18 Turkish hazelnut (Corylus avellana L.) cultivars method (Doyle and Doyle, 1987) with minor were investigated using randomly amplified polymorphic DNA (RAPD), intersimple modifications (Kafkas et al., 2006). The con- sequence repeat (ISSR), and amplified fragment length polymorphism (AFLP) markers. centration of DNA solution was estimated by Twenty-five RAPD primers, 25 ISSR primers, and eight AFLP primer pairs generated comparing band intensity with l DNA of a total of 434 polymorphic marker loci. The three marker systems were able to known concentrations, after 0.8% agarose gel differentiate the cultivars. Genetic similarity index values ranged from a high of 0.96 electrophoresis and ethidium bromide stain- for ‘Kan’ and ‘UzunMusa’ to a low of 0.73 for ‘Yassi Badem’ and ‘Kalinkara’. The ing. DNA was diluted to 5 ngÁmL–1 for RAPD genetic relationships were presented as an unweighted pair group method with and ISSR analysis and to 50 ngÁmL–1 for arithmetic average (UPGMA) dendrogram and a three-dimensional principal coordinate AFLP reactions. analysis (PCoA) plot. The UPGMA dendrogram showed two main clusters, while PCoA Primer selection. Initially, 160 RAPD analysis showed three groups. Cultivar-specific markers were produced by all marker [100 from UBC set #4 (Michael Smith Labo- systems for 10 cultivars. This study demonstrates the usefulness of molecular markers for ratories, University of British Columbia, Van- identification of hazelnut cultivars. couver, BC, Canada) and 60 in Operon sets A, G, H, R, AA, and AC from Operon Technol- ogies, Alameda, CA], 100 ISSR primers (UBC The genus Corylus belongs to the Betula- province, and from there its cultivation set #9), and 24 AFLP primer pairs were ceae of the order Fagales. The number of spread to adjacent Ordu province. Hazelnuts screened using seven Turkish cultivars: ‘Aci’, species in the genus has varied depending are now grown along the Black Sea coast, ‘Cakildak’, ‘Incekara’, ‘Kalinkara’, ‘Palaz’, on the authorities due to recognition of where ecological conditions are favorable, ‘Sivri’, and ‘Yuvarlak Badem’. Based on the some species as a distinct species or a sub- and plantings today cover about 400,000 ha numbers of reproducible and polymorphic species, or within a certain species (Rehder, (Ayfer et al., 1986). Turkey is the world’s fragments, 25 RAPD and 25 ISSR primers, as 1947; Kasapligil, 1972; Mehlenbacher, 1991; leading producer and exporter of hazelnuts well as eight AFLP primer combinations were Thompson et al., 1996). The commercially (Yavuz and Ercisli, 2006), accounting for used to characterize the 18 hazelnut cultivars. important European hazelnut (Corylus avel- about 70% of total production. Other impor- RAPD, ISSR, and AFLP analysis. RAPD lana L.) is native to most of Europe, Turkey, tant hazelnut-producing countries are Italy, and ISSR analysis followed the protocol and the Caucasus mountains, and wild pop- the United States, Azerbaijan, Iran, Spain, and described by Williams et al. (1990) and ulations can be found in these areas today. Georgia (Food and Agricultural Organization Zietkiewicz et al. (1994), respectively, with Ayfer et al. (1986) stated that Turkish cultivars of the United Nations, 2007). minor modifications (Kafkas et al., 2006). In are usually spontaneous hybrids of C. avellana Hazelnuts from Turkey are classified the AFLP assay, adaptor and primer sequ- · C. maxima, although the latter was sug- based on the shape of the nut and quality of ences, PCR conditions for preselective and gested as a distinct species (Mehlenbacher, the kernel. The best-quality hazelnuts have selective amplifications, and selective primer 1991; Thompson et al., 1996). Centuries ago, a round shape, a high oil content, and superior designation were according to Vos et al. native wild hazelnut shrubs were growing in aroma and taste. In spite of the crop’s eco- (1995) with minor modifications (Kafkas close proximity to the mountains near the nomic importance, no extensive studies have et al., 2008). Selective amplification was with Black Sea in northern Turkey. It is believed been conducted on genetic diversity in Turk- eight primer pairs involving five MseI (M) that some Turkish cultivars originated over ish hazelnut germplasm. It appears that there and two EcoRI (E) primers: EAGA/MCAC, many centuries from selection within these are dozens of clones of each Turkish hazelnut EAGA/MCCC,EAGA/MCCG,EAGA/MCTC, local wild populations (Ayfer et al., 1986). cultivar in production, with a common name EAGT/MCAC,EAGT/MCAG,EAGT/MCCG, and Ozkurt (1950) hypothesized that hazelnuts used for each group. In the past, cultivar iden- EAGT/MCTC. Fragments were resolved using were first cultivated in Turkey in Giresun tification studies have been based on morpho- capillary electrophoresis on an ABI 3130xl logical characters (Ayfer et al., 1986; Caliskan, Genetic Analyzer [Applied Biosystems Inc. 1995; Bostan et al., 1997; Erdogan and (ABI), Foster City, CA] with version 3.0 of Mehlenbacher, 1997; Yao and Mehlenbacher, ABI data collection software. AFLP frag- Received for publication 1 June 2009. Accepted for 2000; Koksal, 2002). In recent years, re- ment analysis was performed with GeneScan publication 14 Aug. 2009. We express our gratitude to the Scientific Re- searchers around the globe have investi- Analysis Software 4.0 (ABI). search Projects Unit (Projects No. ZF2007BAP8) gated genetic relationships among hazelnut Data analysis. RAPD, ISSR, and AFLP of Cxukurova University for financial support. cultivars using several types of molecular bands were recorded as present (1) or absent 1To whom reprint requests should be addressed; markers, including randomly amplified poly- (0). Only the most reproducible bands were e-mail [email protected]. morphic DNA (RAPD) (Galderisi et al., 1999; scored and used for analysis. Scores for all HORTSCIENCE VOL. 44(6) OCTOBER 2009 1557 Table 1. Nut shape groups and main cultivation areas of 18 hazelnut cultivars from Turkey. per primer ranged from four to nine, with an No. Cultivars Main cultivation areaz Nut shape groupz average of 6.8. The highest number of total 1 Aci Ordu Globular bands was obtained from UBC primers 808, 2 Allahverdi Giresun Globular 823, and 824, while 836 yielded the fewest 3 Cakildak Ordu Globular bands. The number of polymorphic fragments 4 Cavcava Trabzon Globular per primer ranged from two to eight. The 5 Fosa Trabzon Globular highest level of polymorphism was for primer 6 Incekara Giresun Conical UBC857 (100%), the lowest was for primer 7 Kalinkara Giresun Globular UBC834 (28.57%), and the mean was 58.5%. 8 Kan Giresun Globular 9 Karafindik Du¨zce Globular PIC ranged from a high of 0.985 for primer 10 Kargalak Trabzon Globular UBC829 to a low of 0.335 for primer 11 Kus Giresun Conical UBC849, with a mean of 0.622. The average 12 Mincane Trabzon Globular resolving power was 4.07 and total Rp was 13 Palaz Ordu Globular 101.75. UBC857 had the highest Rp (10.22), 14 Sivri Giresun Conical while UBC829 had the lowest (0.44) (data not 15 Tombul Giresun Globular shown). 16 UzunMusa Ordu Globular The ISSR primers produced slightly more 17 Yassi Badem Izmit Long reproducible bands than the RAPD primers, in 18 Yuvarlak Badem Izmit_ Long z agreement with previous studies in different From Ayfer et al., (1986), Caliskan (1995), Okay (1999), and Koksal (2002). plant species (Goulao et al., 2001; Mattioni et al., 2002; Kafkas et al., 2006). Amplifica- Table 2. Number of bands, percentage of polymorphic bands, resolving power, and polymorphism tion products that were polymorphic and information content of RAPD, ISSR, and AFLP markers used to fingerprint 18 Turkish hazelnut easily scored were obtained from primers that cultivars. included TC, AC, AG, and CT repeats. AT- RAPD ISSR AFLP repeat primers yielded no amplification prod- Primer number (selected/screened) 25/160 25/100 8/24 ucts, although (AT)n is thought to be the most Total number of bands 165 170 582 abundant repeat motif in the plant kingdom Mean total number of bands 6.60 6.80 72.75 (Morgante and Olivieri, 1993). Similar results No. of polymorphic bands 96 99 239 were also reported by Casasoli et al. (2001) Mean no. of polymorphic bands 3.84 3.96 29.88 in chestnut (Castanea sativa Mill.) and by Polymorphism rate (%) 59.3 58.5 42.9 Kafkas et al. (2006) in pistachio (Pistacia Total resolving power 87.33 101.75 220.01 vera L.). ISSR analysis is quick, reproducible, Average resolving power 3.49 4.07 27.50 and generates sufficient polymorphism in Polymorphism information content 0.668 0.661 0.704 hazelnut. In the AFLP analysis, eight AFLP primer combinations revealed a total of 582 frag- three types of markers, polymorphic and chosen because they exhibited the highest ments, of which 239 (42.9%) were polymor- monomorphic, were analyzed using the level of polymorphism in the initial screen- phic.