Genet Resour Crop Evol (2012) 59:1727–1741 DOI 10.1007/s10722-012-9794-x RESEARCH ARTICLE Castanea spp. biodiversity conservation: collection and characterization of the genetic diversity of an endangered species M. G. Mellano • G. L. Beccaro • D. Donno • D. Torello Marinoni • P. Boccacci • S. Canterino A. K. Cerutti • G. Bounous • Received: 24 October 2011 / Accepted: 9 January 2012 / Published online: 24 March 2012 Ó Springer Science+Business Media B.V. 2012 Abstract Centuries of co-evolution between Castanea spp. biodiversity and human populations has resulted in the spread of rich and varied chestnut genetic diversity throughout most of the world, especially in mountainous and forested regions. Its plasticity and adaptability to different pedoclimates and the wide genetic variability of the species determined the spread of many different ecotypes and varieties in the wild. Throughout the centuries, man has used, selected and preserved these different genotypes, vegetatively propagating them by grafting, for many applications: fresh consumption, production of flour, animal nutrition, timber production, thereby actively contributing to the maintenance of the natural biodiversity of the species, and providing an excellent example of conservation horticulture. Nonetheless, currently the genetic variability of the species is critically endangered and hundreds of ecotypes and varieties are at risk of being lost due to a number of phytosanitary problems (canker blight, Chryphonectria parasitica; ink disease, Phytophthora spp.; gall wasp, Dryocosmus kuriphilus), and because of the many years of decline and abandonment of chestnut cultivation, which resulted in the loss of the binomial M. G. Mellano
G. L. Beccaro (&)
D. Donno
D. T. Marinoni
P. Boccacci
S. Canterino
A. K. Cerutti
G. Bounous Department of Arboriculture, University of Turin, Via Leonardo da Vinci, 44, 10095 Grugliasco, TO, Italy e-mail: gabriele.beccaro@unito.it male chestnut. Recently, several research and experimentation programmes have attempted to develop strategies for the conservation of chestnut biodiversity. The purpose of this paper is to give an overview of the status of biodiversity conservation of the species and to present the results of a 7 year project aimed at the individuation and study of genetic diversity and conservation of Castanea spp. germplasm. Keywords Castanea
Chestnut
Conservation and horticulture
Ex situ conservation
Germplasm identification
Piedmont Region Introduction Fagaceae (Cupuliferae) includes eight genera (Castanea, Castanopsis, Fagus, Lithocarpus, Nothofagus, Quercus, Trigonobalanis, Chrysolepis) and about a 600–800 species. The genus Castanea is widespread in the Boreal Hemisphere (Fig. 1) and includes 12 or 13 species according to classification (Table 1). The natural distribution of the European chestnut (Castanea sativa) includes Europe and all of the Mediterranean countries. In Asia (China, Korea, Japan, Vietnam) C. crenata, C. mollissima, C. seguinii, C. henryi occur. In North America, C. dentata is found between Ontario and Maine, along the Appalachian Mountain Range into Georgia and Alabama (Camus 1929) and C. pumila is found in the southeastern states. 123 1728 Genet Resour Crop Evol (2012) 59:1727–1741 Fig. 1 Main areas of chestnut (Castanea spp.) distribution in the world a and in Europe b Table 1 Origin and distribution of Castanea spp. Origin Section Species Common name Orchards Prevalent use Europe Castanea C. sativa Mill. European or sweet chestnut Europe, Asia Minor, North Africa Nut, timber Asia Castanea C. crenata Sieb. et Zucc. Japanese chestnut Japan, Korea Nut C. mollissima Blume Chinese chestnut China Nut China Firewood China Firewood China Timber North America Timber C. seguinii Dode C. davidii Dode Hypocastanon America Castanea Balanocastanon C. henryi (Skan) Rehd. et E. H. Wils C. dentata (Marsh.) Borkh. Willow leaf or pearl chestnut American chestnut C. pumila Mill. var. pumila Allegheny chinkapin Southeast USA Nut C. pumila Mill. var. ozarkensis (Ashe) Tucker Ozark chinkapin USA (AR, MI, OK) Timber C. floridana (Sarg.) Ashe Florida chinkapin Southeast USA Ornamental C. ashei (Sudw.) Ashe Ashe chinkapin Southeast USA Ornamental C. alnifolia Nutt Creeping chinkapin Southern USA (AL–FL) – Southern USA (TX–LA) – C. paucispina Ashe All species are diploids (x = 12; 2n = 24) (Jaynes 1962). The genus is taxonomically divided into 3 sections: Castanea, Balanocastanon, and Hypocastanon, 123 but further revisions are expected (Johnson 1988) as a result of new genetic studies, which contest the validity of this classification (Santamour et al. 1986). Genet Resour Crop Evol (2012) 59:1727–1741 Castanea species biodiversity is very wide, reflecting the adaptation of the genus to different environmental conditions. It shows variability for morphological and ecological traits, vegetative and reproductive habits, nut size, wood characteristics, adaptability, and resistance to biotic and abiotic stresses, and the burden between natural biodiversity and human selection is very weak and sometimes unclear (Bounous and Beccaro 2002). Species in the type section Castanea, which includes the most economically important species, display high genetic diversity. Different species are found in very different pedoclimates, but they prefer deep, soft, acidic soils (pH ranging from 4.0 to 6.5), temperate climates, and rainfall ranging from 700 to 1,500 mm/year. The latitudinal distribution is related to altitude. At low latitudes chestnut trees are found above 1,500 m a.s.l., as on the slopes of Mount Etna in Italy (Polacco 1938), on the Sierra Nevada in Spain, and in Caucasus, where the species thrives at an elevation of 1,800 m (Fenaroli 1945). Tree shape and form are variable. Castanea dentata and C. sativa are upright, tall and slender trees, but some species have smaller size, round foliage and branches that start from the base. Other species are dwarf shrubs. Co-evolution between Castanea spp. biodiversity and human populations has resulted in the spread of rich and varied chestnut genetic diversity throughout most of the world, especially in mountainous regions. Yet, currently, 100s of ecotypes and varieties are at risk of being lost due to a number of phytosanitary problems (canker blight, Chryphonectria parasitica; ink disease, Phytophthora spp; gall wasp, Dryocosmus kuriphilus) and because of the many years of decline and abandonment of chestnut cultivation (Sartor et al. 2009). The purpose of this paper is to give an overview of the biodiversity of the species, and to present the results of a 7-year project aimed at the individuation, description and conservation of Castanea spp. germplasm. History and taxonomy UPGMA analysis of isozyme-based genetic distance estimates (Dane et al. 2003) and phylogenetic analysis based on cpDNA sequence data (Lang et al. 2006) suggest that Castanea species are geographically structured. This is inconsistent with the current 1729 phylogeny based on cupule characteristics. The section Castanea appears to be paraphyletic with differentiation among species being best explained by their current geographical ranges. C. crenata appears to be the most basal taxa and sister to the remainder of the genus. The three Chinese species [C. mollissima, C. seguinii (Castanea) and C. henryi (Hypocastanon)] are supported as a single monophyletic clade and sister to a group containing the North American and European species. There appears to be weak but consistent support for a sister-group relationship between the North American species and European species. The biogeographical history of the genus has been inferred from cpDNA data and molecular clock theory (Lang et al. 2007). A unique westward expansion of extant Castanea species has been hypothesized with Castanea originating in eastern Asia, followed by intercontinental dispersion and divergence between the Chinese and European/North American species during the middle Eocene, followed by subsequent divergence between the European and North American species during the late Eocene. European species Castanea sativa Mill. (European chestnut or sweet chestnut) The genus Castanea appeared at the end of Miocene (15 million years ago) (Giordano 1993; Bounous et al. 2001) and its indicators (Cupuliferae dissemination) include oak and beech. Leaves and one fossil chestnut resembling European chestnut dating back to 8.5 million years ago were found in Coiron Massif, France (Breisch 1995). During the quaternary era glaciations, chestnut trees receded southwards (at the end of Würmian glaciation). In Europe, there were two taxa of chestnut: C. sativa and C. latifolia Sord. (Paganelli 1997). At the end of the last glaciation (Würmian), as pollen charts demonstrated, only C. sativa survived. C. sativa is now the only native species in Mediterranean and Central European regions. In Europe, C. sativa is found from Turkey to Portugal and Spain. The Azores archipelago (25°–31°W) is the most Occidental point for C. sativa and the Canary Islands is the most Southern point (27°–29°N). Towards the north, chestnut fruit production reaches 52°N latitude 123 1730 to the south of the United Kingdom, northern Germany, Poland and Ukraine. It is found at sea level in some littoral areas of the northern Iberian peninsula, Calabria (39°N, 16°W) in Italy and Thessalia (Middle Eastern Greece, 39°N, 22°100 W) and at up to 1,100 m in the highest mountains of Trás-os-Montes (Northeast Portugal; 41°N, 07°W). However at lower latitudes the chestnut grows at up to 1,500 m in Sierra Nevada (Granada, Southern Spain; 37°N, 03°W) and on the slopes of Etna in Sicily (37°N, 14°W) or even at up to 1,800 m in the Caucasus Mountains (42°N, 42°W) (Bounous 2002a; Pereira-Lorenzo et al. 2001; Gomes-Laranjo et al. 2007; Fernández De Ana Magán et al. 1997). In South America, C. sativa was introduced into Chile by European settlers at the beginning of the nineteenth century. It is mainly to be found along the Andes mountain range (34°S, 69°W–41°S, 72°W) (Bounous 2002a). Castanea sativa is a tall tree of majestic appearance; it is vigorous and can exceed 30 m height and 400 years of age. Some century-old trees measure 6–7 m in girth. The nut (10–30 g) has a white–cream pulp and it can have pellicle intrusions into the kernel. In Europe, the germplasm is very broad and the risk of genetic erosion is high, mostly in marginal or abandoned zones (Bounous et al. 2001; Pisani 1992). Conservation of the most interesting genetic materials, selected over centuries, is necessary to maintain valuable biodiversity. There are hundreds of cultivar names for chestnuts, many of which are synonyms or homonyms (Botta et al. 2001). Asian species Castanea crenata Sieb. et Zucc. (Japanese chestnut) Castanea crenata can be dated by fossil findings to the middle of Jomon Civilization (1,000–4,000 BCE). From its zone of origin it spread from Japan to Korea and to Northeast China, and it was naturalized in South Korea and in Taiwan (Tanaka et al. 2005). The species has been cultivated in Central and South Japan for 2,000 years. It can be found between paddy fields and conifer forests, on fertile, recent volcanic soils. The species is distributed throughout the island (41°N, 141°W–31°N, 131°W), but it is primarily cultivated at around 37°N latitude and it is not found in the Okinawa region. 123 Genet Resour Crop Evol (2012) 59:1727–1741 It prefers a mild summer climate, not too cold in winter, with high rainfall (1,200–1,400 mm/year) in summer. On the southern Japanese Islands, where there is abundant summer rain and mild winters, C. crenata grows to 1,300 m a.s.l. It is not as cold resistant as American and Chinese species (Rutter et al. 1991) and early flowering makes it sensitive to late spring frosts (Breisch 1995). The tree does not normally exceed 8–10 m in height but can reach 15 m, and 60 cm in diameter. The adaxial side of leaves is dark-green and the abaxial side is light green. Leaves are acute with strongly marked edges, and leaf margins are crenate. Young leaves have scattered, disk-shaped trichomes and have long, protective, whitish pubescence on major veins (Camus 1929). The nuts of C. crenata vary greatly from tree to tree; some are the largest in the genus and can weigh more than 30 g. The hilum scar is very wide and reaches the middle of the chestnut. They are not usually sweet, are sometimes astringent, and have an adherent pellicle, which is difficult to separate from the kernel (Tanaka and Kotobuki 1992). In France, C. crenata germplasm has been widely used in breeding programs to obtain Phytophthoraresistant trees (Salesses et al. 1993). Castanea mollissima Blume (Chinese Chestnut) This species owes its name to the thick pubescence on buds and on the abaxial side of the leaves. This is the most widespread native species in China. C. mollissima grows in sub-tropical, temperate-continental, and temperate-maritime regions with mild winters and hot summers where rainfall is about 1,000 mm/year (mostly in the summer). Chinese chestnut has been recently introduced into many countries for its plasticity and adaptability to different pedoclimates. Castanea mollissima thrives from 41°290 N latitude in Jilin Province, close to Korea, to 18°310 N latitude North of Hainan Island. It grows in Hebei and Shandong, in the Yangtze Valley, from west to east and in Sichuan, Hubei, Anhui, Jiangsu and, in the southwest, in Yunnan Province, close to the Vietnamese border. C. mollissima, along with C. crenata, is also found in Korea: the former in northern Korea (40°N, 126°W) while the latter is more frequently Genet Resour Crop Evol (2012) 59:1727–1741 found in the southern Korean Peninsula (36°N, 127°W), which is the main production area (Kim et al. 2005). It grows from 50 to 2,800 m a.s.l. in a wide range of climatic conditions. Many varieties and local ecotypes have been described, of which about 50 are cultivated. They are divided into six groups with different morphological, physiological, horticultural, and geographical features. Interesting germplasm includes plants with burs that turn red in early autumn, plants with hanging branches, and some very precocious dwarf types (Liu 1993). Castanea mollissima is a medium-sized tree: 12 m tall and with trunk diameters of up to 75–80 cm. Leaf serrations are large, irregular, not well pronounced, and have a hairy, mucronate point. The adaxial leaf surface is bright green, the abaxial surface is whitishgrey or velvet due to pubescence. The nuts are round or elliptical and show a long torch (the tip of the nut, formed by the remains of the styles) covered by a thick, white-cream pubescence; the pulp is very sweet, but not as sweet as the American chestnut, and it is richer in proteins than the Japanese and European species. The hilum scar is wide but less developed than in C. crenata. Chestnuts show thin, easy-to-peel pellicles (not invading the kernel); kernels are sweet and ripen early. In the Northern regions, chestnuts are small (\15 g), show bright colour, and have a good, sweet taste. In subtropical regions, the nuts of most cultivars are large (15–20 g) with high starch content. Castanea seguinii Dode This small tree or shrub is scattered in subtropical regions and in southwestern China. The very small nuts (2–4 g) are harvested for nourishment by rural people. Trees are periodically coppiced to produce firewood. They have early flowering and continue to flower throughout the bearing season until frost (Bounous et al. 2001). Other genotypes, coming from Jiangsu province, show shoots with 10–20 burs. The reflowering feature appears to be regulated by two recessive genes and early flowering depends on one dominant gene (Jaynes 1962). Genetic diversity has been studied through isoenzymes (Huang and Norton 1992) with the aim of finding compatible genotypes to produce dwarf rootstocks. 1731 Castanea davidii Dode Some authors consider C. davidii a variety of C. seguinii based on the many affinities. Castanea henryi (Skan) Rehd. et E. H. Wils Known as the willow leaved chestnut, or pearl chestnut, the species is native to the warm temperate subtropical climates of China. It grows along the Yangtze River Valley and in southern regions. It is cultivated for timber in Fujian and Zhejiang provinces. Castanea henryi is a forest species that grows rapidly with upright (slender) trunk, over 30 m tall. The chestnuts (1 per bur) are small (3–6 g) and marketed to some extent. North American species Castanea dentata (Marsh.) Borkh. (American chestnut) Castanea dentata grew in Long Island 30,000– 50,000 years ago, as evidenced by pollen dating back to the last inter-glacial periods. It is native to the eastern United States and Canada and it spread from Ontario and Maine (on the Appalachian Range, 47°N, 66°W–32°N, 87°W) to Georgia and Alabama, where it was long a dominant species. Its natural range once covered more than 200 million acres from the Canadian border to the Gulf of Mexico (Rosengarten 1984). It grows rapidly, with an upright, slender trunk that can exceed 30 m in height and has a diameter of 3 m or more. The destruction of C. dentata by canker blight, Cryphonectria parasitica, was the greatest disaster in the history of forest pathology (Roane et al. 1986; Anagnostakis 1987). The canker, first identified in New York in 1904 at the Bronx Zoological Park, led to the complete removal of the species from the forest canopy. West of the native range it is possible to find adult trees that escaped the blight. Castanea dentata is the most cold resistant species of the genus. Northern zone genotypes can survive to -35°C (Ashworth 1964). Stems are small, sharp, brown and hairless. Leaves are similar in shape and dimension to C. sativa, and are generally hairless, sometimes having just a few hairs 123 1732 on the mid-vein, and thin. Nuts are sweet, not astringent, and very small with a thin pellicle which is easily removed from the kernel. Castanea pumila (L.) Mill. This polymorphic species is divided into two botanical varieties: C. pumila var. pumila (Allegheny chinkapin) and C. pumila var. ozarkensis (Ashe) Tucker (Ozark chinkapin) (Johnson 1987). Other authors include C. floridana and C. alnifolia into C. pumila. It is native in the United States from the east and southeast to the Ozark mountains of Arkansas and to Missouri and Oklahoma (Camus 1929). Chinkapin tree shapes can be bushy (pumila), creeping (with some reported to be stoloniferous) or 20 m tall (ozarkensis) (Pardo 1978; Johnson 1988). A high variability of leaf form, size and colour has been observed in the same plant. Burs are small (1–5 cm in diameter) with soft thorns. They remain on the branches and contain a single nut, sometimes remaining all winter long. These sweet and tasty chestnuts are very small (1 g). Genet Resour Crop Evol (2012) 59:1727–1741 Materials and methods Within the framework of different research projects begun at the beginning of the twenty-first Century, the individuation, study of genetic diversity and conservation of Castanea spp. germplasm is still in progress at the Department of Arboriculture of the University of Turin (Italy). The aim of the research is to collect, identify, describe and preserve ex situ the germplasm of Castanea spp. in order to provide additional strategies to complement current efforts to protect the species. Through collaboration with many international research Institutions, to date more than 300 ancient trees, representative of chestnut genetic variability, have been individuated in different areas of Europe, Asia and USA. Each tree was numbered and localized by GPS in order to obtain a detailed cartography and genetic material (scions) was collected from each tree in order to realize a collection field in North West Italy. Morphological and phenological observations Castanea floridana Ashe (Sarg.) This is a decorative bushy plant native to the southeastern US from FL to TX, where it is known as Florida chinkapin. It can be 6–7 m high. The nuts (1 per bur) are very small, and the plants flower much later in the season than C. pumila. Castanea ashei (Sudw.) Ashe Ashe chinkapin is a 6–7 m tall tree scattered throughout NC, GA and FL. Castanea alnifolia Nutt Shrub or creeping chinkapin is a creeping shrub (30–60 cm) originating in southern US, from AL to FL. Castanea paucispina Ashe The distribution area of this creeping shrub (30–60 cm) includes TX and LA. 123 Leaves, flowers and nuts of analyzed cultivars were sampled in situ and ex situ in the collection field. Through direct observation, or published information, when available, phenological (bud break, times of male and female flowering, harvesting date) and morphological traits (shape of the leaf; morphology of the flower; nut size; nut stripes; nut colour through colorimetric analysis; percentage of double fruits; kernel quality; pellicle adhesion) were used to characterize the genotypes (Breviglieri 1955; Bounous 2002a; Beccaro et al. 2005, 2009). Nut descriptors are very useful for understanding the presumed traditional uses of a variety, but phenological and morphological observations are not considered effective enough for genotype identification, being subject to environmental and developmental factors, and they may not be enough to individuate homonymous and synonymous among the different genotypes. Therefore the accessions are characterized both by phenological and morphological analysis conducted in situ and ex situ and by microsatellite markers (SSR). Genet Resour Crop Evol (2012) 59:1727–1741 Molecular analysis Samples of young fresh leaves are collected in spring for molecular analysis; DNA analysis is successfully performed by multiplex analysis of seven microsatellite loci isolated in C. sativa (Marinoni et al. 2003; Botta et al. 2001) and Quercus petraea (Steinkellner et al. 1997; Casasoli et al. 2006; Goulao et al. 2001; Marinoni et al. 2003; Buck et al. 2003; Yamamoto et al. 2003): CsCAT-1, -8, -14, -15, -16, -17, and -41. Loci were chosen based on the ease of allele scoring, multiplexing ability, and their linkage group assignment (Barreneche et al. 2004). PCR is performed in 20 ll reaction volumes containing 50 ng of DNA, 0.5 Units of AmpliTaq DNA polymerase (Applied Biosystems, Foster City, CA, USA), 2 ll of GeneAmp 109 PCR buffer (100 mM Tris–HCl pH 8.3, 500 mM KCl), 1 ll of 10% Bovine Albumin Serum (BSA), 1.5 mM MgCl2, 0.2 mM dNTPs, 0.5 lM labelled forward primer and 0.5 lM reverse primer. The forward primers are labelled with a fluorochrome (6-FAM, HEX or NED). Samples are analysed on an ABI 3130 capillary sequencer (Applied Biosystems, Foster City, CA, USA). Data is processed by the GeneMapper Software 4.0 (Applied Biosystems) and alleles defined by their size (in bp), compared with the standard, GeneScan500 LIZ. Genetic relationships among the accessions were investigated by UPGMA cluster analysis. Ex situ conservation The realization of a germplasm collection field of chestnut genetic diversity is in progress. Vegetal material is collected from each mother-tree and grafted into a collection field (3 plants for each accession) located at the Chestnut Regional Repository in Chiusa Pesio, Cuneo Province (North Western Italy), on the border with France (44°190 N; 7°400 E; 575 m a.s.l.). The area has a typical temperate climate and is largely given over to chestnut culture. It is in the phytoclimatic transition zone of ‘‘cold Castanetum’’ and ‘‘hot Fagetum’’, according to Mayr–Pavari’s classification (De Philippis 1937) which identifies five phytoclimatic zones by means of the dominant tree species. Soils are made of river deposits, with a high concentration of sand and the actual soil depth (between 30 and 60 cm) is limited because of coarse 1733 gravel (Tani et al. 2007). Currently 5 hectares are given over to the Castanea arboretum. The collection field was created with an 8 9 11.50 m planting layout, in order to use the arboretum both for scientific research and for landscape and tourist activities. Results and discussion Morphological and phenological observations The main passport data for 105 ecotypes and varieties are shown in Table 2. Very high variability within the same genotype and among different genotypes is observed. Ecotypes and varieties are classified in longistaminate (e.g. Solenga), mesostaminate, brachistaminate (e.g. Pelosa) and astaminate (e.g. Neirana, Marrone and Gioviasca), depending on the catkin flower morphology. Morphological observations of leaves (Fig. 2) and flowers (Fig. 3) as well as phenological observations do not yield relevant differences that were able to separate one genotype from the others. Nut descriptors are also not particularly effective in discriminating among the accessions (Table 3; Fig. 4). However, very wide biodiversity is observed among the different genotypes (Fig. 5). One of the most important nut descriptors is the mono-embryonic versus polyembryonic. Mono-embryonic nuts were named ‘‘marroni’’ type in Italy in the Middle Ages. Genotypes producing poly-embryonic nuts were named chestnut type. In Europe, most genotypes produce mono-embryonic nuts and some of them are ‘‘marroni’’ type (Breisch 1995; Bounous et al. 2001; Pereira-Lorenzo et al. 2006). In the past, many botanists tried to describe chestnut genotypes through the definition of morphological descriptors. However, because of the plasticity of the species, a morphological descriptor able to separate each different variety from another has not been individuated. The botanist Pier Antonio Micheli (1679–1737) [Enumeratio rariorum plantarum (Manoscr.)], (cited by Breviglieri 1955) was the first to describe chestnut varieties based on their bur, fruit, leaf and flower characteristics. Later, about 300 varieties were classified by region and harvesting time in Italy Piccioli (1922), and Remondino (1926) referenced nearly 1,000 denominations, many of them synonymous. Breviglieri (1955) established the base descriptors for chestnuts that are used today. Solignat and Chapa 123 1734 Genet Resour Crop Evol (2012) 59:1727–1741 Table 2 Castanea accessions in collection in the chestnut regional repository of Chiusa Pesio (Italy) Species Country Region Allegheny chinkapin C. pumila var. pumila USA Eastern North America Arizinca C. sativa France Corse Bastarda Rossa Belle Epine C. sativa C. sativa Italy France Tuscany Dordogne / Bouche de Bétizac C. sativa 9 C. crenata France Bouche Rouge C. sativa France Ardèche Bracalla C. sativa Italy Piedmont Brunette C. sativa Italy Piedmont C. dentata C. dentata USA Eastern North America Capannaccia C. sativa Italy Tuscany Carigliettara C. sativa Italy Calabria Castagna dei 100 Cavalli C. sativa Italy Sicilia Castagna della Madonna C. sativa Italy Piedmont Castagna di Canepina C. sativa Italy Tuscany Cecio C. sativa Italy Tuscany Cesarucca C. sativa Italy Tuscany Châtaigne de Laguepie C. sativa France Sud Est France Colossal C. sativa 9 C. crenata USA Eastern North America Contessa Dorée de Lyon C. sativa C. sativa Italy France Piedmont Central-west France Ederra C. sativa 9 C. crenata Spain/France Pais Vasco Epinerere C. sativa Italy Aosta Valley Gabiana C. sativa Italy Piedmont Garrone nero C. sativa Italy Piedmont Garrone Rosso C. sativa Italy Piedmont Gentile C. sativa Italy Piedmont Gioviasca C. sativa Italy Piedmont Grossulee C. sativa Italy Aosta Valley Gynyose C. crenata Japan Japan, Korea, Northeast China, Taiwan Hakury C. crenata Japan Japan, Korea, Northeast China, Taiwan Herria C. sativa Spain Pais Vasco Idae C. mollissima 9 C. crenata Korea / Injerta C. sativa Spain / Ipharra Ishyzuki early C. sativa 9 C. crenata C. crenata Spain/France Japan Pais Vasco Japan, Korea, Northeast China, Taiwan Ishyzuki late C. crenata Japan Japan, Korea, Northeast China, Taiwan Janfau C. sativa Portugal/Spain Padrela Judia C. sativa Portugal/Spain Padrela Longal C. sativa Portugal/Spain Terra fria Lusenta C. sativa Italy Piedmont Mansa C. sativa Spain Canary Islands Maraval C. sativa 9 C. crenata France / Maridonne C. sativa 9 C. crenata France / Marigoule C. sativa 9 C. crenata France / 123 Genet Resour Crop Evol (2012) 59:1727–1741 1735 Table 2 continued Species Country Region Marissard C. sativa 9 C. crenata France / Marky C. sativa 9 C. crenata France / Marron Comballe Marron de Goujounac C. sativa C. sativa France France Ardèche Ardèche Marron de Redon C. sativa France Ardèche Marron d’Olargue C. sativa France Hérault Marron Sauvage C. sativa France Ardèche Marrone Caprese Michelangelo C. sativa Italy Tuscany Marrone della Garfagnana C. sativa Italy Tuscany Marrone dell’Etna C. sativa Italy Sicily Marrone di Castel del Rio C. sativa Italy Emilia Romagna Marrone di Castione C. sativa Italy Trentino Alto Adige Marrone di Gemonio C. sativa Italy Lombardia Marrone di Marradi IGP C. sativa Italy Tuscany Marrone di Roccamonfina C. sativa Italy Campania Marrone di San Mauro Saline C. sativa Italy Veneto Marrone di Segni C. sativa Italy Latium Marrone di Val Susa C. sativa Italy Piedmont Marrone di Viterbo Marrone di Zocca C. sativa C. sativa Italy Italy Latium Emilia Romagna Marrone Ossola C. sativa Italy Piedmont Marrone Pellice C. sativa Italy Piedmont Marrubia C. sativa Italy Piedmont Marrubia Susa C. sativa Italy Piedmont / Marsol C. sativa 9 C. crenata France Montagne C. sativa France Dordogne Monte Arso ecot. 3 C. sativa Italy Sicily Monte Scarello ecot. 2 C. sativa Italy Sicily Negral C. sativa Portugal Padrela Neirana C. sativa Italy Piedmont Neirana Susa C. sativa Italy Piedmont Nouzillard C. sativa Spain Pais Vasco Pelosa C. sativa Spain Canary Islands Pelosora Pontecosa Garfagnana C. sativa C. sativa Italy Italy Tuscany Tuscany Precoce de Vans C. sativa France Dordogne, Lot, Cantal Precoce di Brignola C. sativa Italy Piedmont Precoce Migoule C. sativa 9 C. crenata France / Primato C. sativa Italy Piedmont Primitiva di Roccamonfina C. sativa Italy Campania Riggiola C. sativa Italy Calabria Rossane C. sativa Italy Aosta Valley Rossolino C. sativa Italy Tuscany Rouffinette C. sativa Italy Aosta Valley 123 1736 Genet Resour Crop Evol (2012) 59:1727–1741 Table 2 continued Species Country Region Rousse de Nay C. sativa France Dordogne, Lot, Cantal Rubiera C. sativa Italy Piedmont Ruiana Sardonne C. sativa C. sativa Italy France Piedmont Dordogne, Lot, Cantal Savoye C. sativa France Dordogne, Lot, Cantal Selvaschina C. sativa Italy Piedmont Selvaschina ecotype 2 C. sativa Italy Piedmont Servaj ‘d l’oca C. sativa Italy Piedmont Solenga C. sativa Italy Piedmont Tarvisò C. sativa Italy Piedmont Tempuriva C. sativa Italy Piedmont Torcione Nero C. sativa Switzerland Ticino Toumive C. sativa France Dordogne, Lot, Cantal Tsukuba C. crenata Japan Japan, Korea, Northeast China, Taiwan Verdale C. sativa France Dordogne, Lot, Cantal Vignols C. crenata 9 C. sativa France Dordogne, Lot, Cantal Yeuillaz C. sativa Italy Aosta Valley Fig. 2 Leaf morphology in a C. sativa, Marrone di Chiusa Pesio, b C. sativa, Monte Arso ecotype 2, c C.mollissima 9 C. crenata, Idae, d C. dentata (1975) described French cultivars, and classified them into main or local interest. Those descriptions were updated by Bergougnoux et al. (1978) but focused on clonal selection of the main cultivars, and included 123 hybrids used in the new plantations mixed with European chestnut, as in Cevennes. Though the cultivation of chestnuts in Switzerland has mostly been abandoned, some cultivars were Genet Resour Crop Evol (2012) 59:1727–1741 1737 Fig. 3 Male flower morphology a astaminate (Marroni ecotypes), b brachistaminate, c mesostaminate, d longistaminate Table 3 Morphological data of five important Piedmont chestnut cultivars Cultivar Medium weight (g) Colour of the nut L a b Length (mm) Width (mm) Thickness (mm) Ilo length (mm) Ilo width (mm) Neirana 9.66a 29.4d 9.8b 4.67c 28.86ab 30.96b 18.22bc 20.8bc 11.08ab Gioviasca 9.54a 31.03bc 12.6a 7.06b 28.03b 30.48b 19.77ab 19.52c 10.29bc Solenga 4.96c 32.9a 12.78a 9.5a 24.52c 25.62c 16.15c 20.74bc 9.91c Pelosa 6.84b 31.78ab 12.58a 8.88a 25.84c 27.48c 11d 22.68a 10.96ab 30cd 12.92a 6.96b 30.12a 36.56a 21.56a 22.04ab 11.24a Marrone 10.6a Different letters for each cultivar indicate the significant differences at P \ 0.05 length width thickness 40 a 35 30 b b ab a b c 25 20 bc c c c a ab c 15 d 10 5 0 Neirana Gioviasca Solenga Pelosa Marrone Fig. 4 Width, length, thickness of five important Piedmont chestnut cultivars (mm) localised and described by Conedera et al. (1993), Conedera et al. (2004) and Gobbin et al. (2007). In Spain, Elorrieta (1949) was the first to report on Spanish cultivars. In 1996, the first inventory for Galician cultivars in North-western Spain was published, presenting the variability and a description of the local varieties (Pereira-Lorenzo et al. 1996a, b, 2006). These studies were extended to other important areas for nut production such as Asturias, Castile and León, Extremadura, Andalucı́a and the Canary Islands (Pereira-Lorenzo et al. 2001; Ramos-Cabrer and Pereira-Lorenzo 2005). 123 1738 Genet Resour Crop Evol (2012) 59:1727–1741 Fig. 5 a The Châtaigne de Laguepie (France) present large, mono-embryonic nuts, once selected for fresh consumption, b the Italian ecotype Ruiana was once used for animal feed Molecular analysis The reading of the profiles is clear most of the time and the amplification of all loci is always well balanced, with peaks of approximately the same height. So far, the most polymorphic loci have been CsCAT1, CsCAT16 and QpZAG110. The number of alleles totaled 59 and ranged from 6 to 11 per locus, with an average of 8.4. The loci with high numbers of alleles were CsCAT1 (10 alleles) and CsCAT41 (11 alleles). Expected heterozygosity (He) averaged 0.77 and ranged from 0.65 (for CsCAT15) to 0.85 (for CsCAT17), while observed heterozygosity (Ho) averaged 0.89 and ranged from 0.73 (for CsCAT16) to 1.00 (for CsCAT17). The estimated frequency of null alleles (r) was a positive value for locus CsCAT 16 (0.019). The total probability of identity at all loci was 2.99 9 10-8, thus cultivars with identical genotypes were considered synonyms. The results obtained in our set of accessions showed that microsatellite loci detected considerable polymorphism and confirmed that these markers are suitable for fingerprinting chestnut cultivars. The Table 4 Characteristics of 7 SSR loci in analyzed genotypes Locus Allele size range (bp) Na He Ho r CsCAT-1 186–222 10 0.79 0.92 -0.077 CsCAT-8 178–227 8 0.77 0.97 -0.112 CsCAT-14 133–162 6 0.71 0.92 -0.123 CsCAT-15 121–159 7 0.65 0.84 -0.116 CsCAT-16 118–150 8 0.77 0.73 0.019 CsCAT-17 130–161 9 0.85 1.00 -0.081 CsCAT-41 198–235 11 0.83 0.87 -0.024 Na number of alleles, He expected heterozigosity, Ho observed heterozigosity, r frequency of null allele 123 polymorphism and discriminant power of each locus were evaluated on the basis of number of alleles, expected and observed heterozygosity (Table 4). The mean values of these parameters were comparable to obtained in chestnut by Marinoni et al. (2003) and Gobbin et al. (2007). All loci analysed are highly polymorphic and thus particularly suitable for DNA typing of chestnut cultivars. The combined profiles across the 7 SSR loci show the presence of a total of 105 different genotypes: many synonymous varieties were found, especially among the Italian ecotypes and varieties of Marroni (e.g. Marrone di Gemonio, Marrone di Roccamonfina, Marrone di Castel del Rio, Marrone Caprese Michelangelo, Marrone di San Mauro Saline, Marrone di Segni, Marrone Val Susa and Marrone di Zocca have the same genetic profile). This indicates the existence of substantial genetic uniformity within some cultivated varieties but also the possibility of a polyclonal origin for some of the cultivated varieties; a second option is that individual species showing a genotype different from the norm may belong to another variety or ecotype, which has not yet been analysed, and are simply cases of misnaming. Cluster analysis performed for the 36 most important accessions produced an UPGMA dendrogram depicting the genetic relationships within the studied accessions (Fig. 6). Gabiana and Travisò accessions were grouped in cluster I. Almost all accessions named Marrone clustered whit most Italian cultivars in the large cluster II. The interesting data observed was the existence of a unique genetic profile among these Marroni accessions that are cultivated in different Italian regions. Finally, all Euro-Japanese hybrids analysed, except Bouche de Bétizac, were placed in group III together with some C. sativa accessions. Genet Resour Crop Evol (2012) 59:1727–1741 1739 Gabiana Travisò Marrone Gemonio Marrone Roccamonfina Marrone Castel del Rio M. Caprese Michelangelo Marrone San Mauro Marrone Segni Marrone Val Susa Monte Zocca Contessa Bouche de Bètizac Verdeisa Pugnenga Bracalla Neirana Primitiva Roccamonfina Grossulee Yeuillaz Ruiana Rossera Castagno 100 cavalli Canepina Tempuriva Madonna - Lazio Capannaccia Garfa gnana Monte Scarello 2 Monte Arso 3 Marrone Etna Colossal Bonosora Garfagnana Pontecosa Garfagnana Peiosoia Precoce Migoule Maraval Marigoule I II III 0 Fig. 6 UPGMA dendrogram of 36 chestnut accessions based on alleles at 7 SSR loci Various studies (Botta et al. 2001, Marinoni et al. 2003) have demonstrated the usefulness of microsatellite (SSR) analysis in discriminating between genotypes. Both approaches—morphological and molecular—are considered useful in characterising some aspects of the germplasm (Beccaro et al. 2009), however differing information is given from the results. Ex situ conservation The DNA results confirm that in one site alone a huge amount of still to be implemented Castanea spp. Biodiversity has been amassed. To date, more than 300 Castanea spp. trees have been grafted into the arboretum with accessions coming from all over the world. The arboretum currently includes several European ecotypes and cultivars from Italy, Portugal, Spain (including the Canary Islands), France and Switzerland, and accessions from the USA, China, and Japan. The collection also includes 17 Euro-Japanese hybrids (C. sativa 9 C. crenata) obtained from different countries. The majority of hybrids were obtained from in France in the ‘80s, by INRA (Institut National de la Récherche Agronomique) in order to create ink disease and canker blight resistant genotypes (Bounous et al. 2001) and are actually used both on ‘‘their own roots’’ or as rootstocks for superior varieties. On the one hand, hybridization increases the commercial viability of the species, thus increasing the amount of material grown and ‘‘safeguarding’’ Castanea spp. germplasm taken in general. However, it also has the potentially negative effect of genetically contaminating native populations (coppices). According to the convention on biological diversity (CBD) signed in 1992, which gave ownership of biodiversity to national governments and required equitable benefit sharing for commercial use of biodiversity with the country of origin, the aim of the arboretum is to promote Castanea conservation by putting a value on biodiversity. As the arboretum will give the next generation of researchers a new opportunity to further assess the genetic variability of the species, genetic material will be provided to growers and nurseries on request, in order to improve the role of horticulture in the conservation of resources. Future plans include increasing the number of genotypes in the arboretum, including important areas still not represented, such as Turkey and Greece, and establishing networks with other germplasm collections, e.g. the National Chestnut Germplasm Repository of China (Li Guo-tian et al. 2009). 123 1740 Conclusions In the past, the high horticultural value of many Castanea spp. genotypes allowed the biodiversity of the species to be maintained by the human populations, representing an excellent case for the intersection of conservation and horticulture. However, today all the Castanea species have serious conservation problems and despite the current efforts to protect them, their genetic variability is critically endangered. 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