JOBNAME: horts 43#3 2008 PAGE: 1 OUTPUT: April 23 15:48:36 2008 tsp/horts/163067/02607 HORTSCIENCE 43(3):667–672. 2008. Giovanni, Tonda di Giffoni, and Tonda Gen- tile Romana) were introduced because of their high commercial value and have shown Genetic Diversity of Hazelnut good adaptation to the conditions of the area. Modern agriculture requires high yield for (Corylus avellana L.) Germplasm profitability, and consequently the number of cultivars planted has declined in recent years. in Northeastern Spain To avoid loss of local germplasm, efforts have been made on a worldwide scale to Paolo Boccacci1 and Roberto Botta1 collect and preserve genetic diversity. In Dipartimento di Colture Arboree, Universita` degli Studi di Torino, Via Tarragona, the diverse local hazelnut germ- Leonardo da Vinci 44, 10095 Grugliasco (Torino), Italy plasm was investigated by Tasias Valls (1975), who assigned cultivars to three groups: Merce` Rovira I) one main cultivar distributed in all hazelnut Institut de Recerca i Tecnologia Agroalimenta`ries (IRTA), Arboricultura cultivation areas of the province and in re- ` gular plantations; II) cultivars that are com- Mediterrania, Centre Mas de Bover, Ctra. Reus-El Morell, km 3.8, 43120 mon in some areas of the province and in Constantı´(Tarragona), Spain regular plantations; III) minor cultivars that Additional index words. filbert, genetic relationships, microsatellite, simple sequence repeat, are not cultivated in regular plantations but are occasionally found scattered in orchards SSR markers (Table 1). This germplasm has been collected Abstract. In Spain, hazelnut is mainly cultivated in Catalonia, a region in the northeast. and preserved in collection fields (Koksal,¨ The province of Tarragona accounts for 88% of the total Spanish area planted to 2000) to preserve the genetic variability for hazelnut. Almost 80% of the production in Tarragona is of the local cultivar Negret, with future use (Table 2). others cultivated to a lesser extent. Minor cultivars are only sporadically present in older Traditional methods to characterize and orchards, farm yards and gardens, and have been collected for preservation. In this identify hazelnut cultivars are based on phe- work, 16 SSR markers were used to fingerprint 18 minor hazelnut cultivars from notypic observations (Thompson et al., 1978; northeastern Spain. Their microsatellite profiles were combined with those of 15 Spanish UPOV, 1979), but this approach is subject to cultivars characterized in a previous work, and used to study the genetic diversity in 33 environmental influences and thus requires genotypes including local Spanish germplasm. The SSR analysis allowed development of several years to correctly define the traits of a unique profiles of each of the 18 cultivars, and no new case of synonymy was detected. A plant. Isozyme polymorphism was proposed high level of genetic diversity (mean He = 0.7) was observed in 33 genotypes, although a in the 1980s and early 1990s as an alternative high number of them showed a close genetic relationship. The dendrogram generated by and more effective method for cultivar iden- UPGMA cluster analysis placed the 33 accessions into nine main groups, related to their tification and studies of genetic relationships putative pedigrees or geographical area of cultivation. All investigated Negret-type (Ahmad et al., 1987; Rovira, 1997; Solar cultivars were found to be distinct from Negret, and only a few cultivars within this et al., 1997). During the last decade, DNA germplasm appeared to be seedlings of Negret. The results will be useful in the markers have proven to be convenient for conservation of hazelnut germplasm and in the selection of parents for use in breeding. accurately identifying cultivars due to their high discriminating power at a relatively low cost. Among the available DNA markers, mi- The European hazelnut (Corylus avellana the United States third with 4.1%, and Spain crosatellite or simple sequence repeat (SSR) L.) is one of the world’s major nut crops. Its fourth with 2.8%. Azerbaijan, Iran, Georgia, markers appear to be best-suited to cultivar geographic distribution extends from the China, France, and Greece are other impor- fingerprinting. They are generally codomi- Mediterranean coast of North Africa north- tant producers (FAOSTAT, 2007). nant, highly polymorphic, highly reproduc- ward to the British Isles and the Scandinavian In Spain, hazelnut is mainly cultivated in ible, and permit exchange of results among Peninsula, and eastward to the Ural Moun- Catalonia, a region in the northeast. The different laboratories as well as construction tains of Russia, the Caucasus Mountains, province of Tarragona accounts for 88% of of an integrated database. Microsatellite mar- Iran, and Lebanon (Thompson et al., 1996). the total Spanish area planted to hazelnut kers were recently developed in C. avellana Total worldwide hazelnut production is fifth (Fig. 1). Minor hazelnut-growing areas in- and evaluated in seven other Corylus species after that of cashew (Anacardium occidentale clude Castello´n, Asturias, Paı´s Vasco, Ara- by Bassil et al. (2005a, 2005b) and Boccacci L.), almond [Prunus dulcis (Miller) D.A. go´n, and Navarra. Orchards in Tarragona et al. (2005). On the basis of their high level Webb], walnut (Juglans regia L.), and chest- province have been classified into two topo- of polymorphism, the most interesting loci nut (Castanea spp.). Turkey has long been graphic groups. The first group is of orchards were used to fingerprint and to identify the leading producer and exporter of hazel- of the inland mountain ranges of the province mistakes in hazelnut accessions from several nuts, accounting for about 71% of world (‘‘Priorat-Prades’’), located on hilly slopes germplasm repositories (Botta et al., 2005; production. Italy is second with over 13%, and characterized by a low level of mecha- G¨okirmak et al., 2005). Moreover, results nization and low nut yield (500–800 kgÁha–1). were used to verify synonymies and parent- The second group is of orchards in flat areas age hypotheses and to investigate genetic rela- Received for publication 11 Oct. 2007. Accepted of the region called ‘‘Camp de Tarragona’’ tionships among cultivars grown in important for publication 24 Jan. 2008. located near the Mediterranean coast that use (Boccacci et al., 2006; Ghanbari et al., 2005) The authors are grateful to Prof. Giovanni Me modern mechanized techniques and show production areas. SSR loci were also placed (Dipartimento di Colture Arboree, Torino, Italy) high nut yield (2,000–2,500 kgÁha–1). Most in a genetic map, and some of them were for his generous collaboration and Prof. Shawn A. commercial production is from this second found linked to a dominant allele for resis- Mehlenbacher (Oregon State University, Corvallis, area (Tous, 2005). Almost 80% of Tarrago- tance to eastern filbert blight caused by OR) for reviewing the manuscript. The research na’s production is of the native cultivar Anisogramma anomala (Peck) E. Mu¨ller was funded by Regione Piemonte Administration Negret. ‘Negret’ and ‘Pauetet’ are sold as (Mehlenbacher et al., 2006). (Italy), INIA and MAPYA (Spain), and by the European Union project SAFENUT (AGRI GEN the commercial type ‘‘negreta’’ that receives In the present work, 18 minor hazelnut RES 068). the best prices on the national markets. Other cultivars from northeastern Spain were DNA- 1To whom reprint requests should be addressed; Spanish cultivars (Grifoll, Gironell, Morell, typed using microsatellite markers. Their e-mail [email protected]., paolo.boccacci@ Culpla`, Ribet, and Trenet) are cultivated to a SSR profiles were combined with those unito.it lesser extent. A few Italian cultivars (i.e., San of 15 Spanish cultivars characterized by HORTSCIENCE VOL. 43(3) JUNE 2008 667 JOBNAME: horts 43#3 2008 PAGE: 2 OUTPUT: April 23 15:48:37 2008 tsp/horts/163067/02607 Fig. 1. Regions of Tarragona province (northeastern Spain) with hazelnut cultivation areas shown in dark (from Tasias Valls, 1975, modified). Boccacci et al. (2006) and used to study the containing 50 ng of DNA, 0.5 U of Taq-DNA Genetic distances (1000 bootstraps) were genetic diversity in this local germplasm, polymerase (AmpliTaq Gold, Applied Bio- computed as D = [1 – (proportion of shared including cultivars of international interest systems, Inc., Foster City, CA), 2 mLof10· alleles)] using the program Microsat (Minch, in cultivation and breeding. PCR buffer (100 mM Tris-HCl, pH 8.3, 500 1997). Cluster analysis was performed using mM KCl), 2 mM MgCl2, 200 mM dNTPs, and the Neighbor software in the Phylip v.3.5c Materials and methods 0.5 mM of each primer. PCR conditions were package (Felsenstein, 1989), and a dendro- as follows: an initial denaturation step at gram was constructed using the TreeView Plant material and DNA extraction. 95 °C for 9 min followed by 26 cycles of program (Page, 1996). The software Identity Leaves were sampled from 18 accessions denaturation (30 s at 95 °C), annealing (45 s 1.0 (Wagner and Sefc, 1999) was used to (Table 3) conserved in the germplasm col- at 55 °C and 50 °C for CaT-B502), and calculate expected (He) and observed (Ho) lection field of the Institut de Recerca i extension (90 s at 72 °C). The final elongation heterozygosities. Tecnologia Agroalimenta`ries (IRTA) of Reus step was at 72 °C for 45 min. The forward (Tarragona, Spain). Genomic DNA was ex- primers were labeled with a fluorochrome (6- Results and Discussion tracted from 0.2 g of leaves in a Tris-EDTA- FAM, HEX, NED, or PET), and amplifica- NaCl buffer containing 0.25 M NaCl, 0.2 M tion products were analyzed using an ABI In a previous study, Boccacci et al. (2006) Tris, pH 7.6, 2.5% PVP 40,000, 0.05 M Prism 377 sequencer (Applied Biosystems, characterized and investigated the genetic Na2EDTA, and 0.1% b-mercaptoethanol, using Inc.). Results of the run were then processed relationships among 78 hazelnut cultivars, a modified protocol described by Thomas with Genescan software and allele sizes were including 15 from Spain, using the same 16 et al.