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The analysis of simple sequence repeats (SSRs) and of bulk ground samples by droplet digital polymerase chain reaction (ddPCR) was investigated as an alternative to individual kernel testing for assessing the presence of local cultivars in common wheat (Triticum aestivum L.) varietal blends. The recent Protected Geographical Indication (PGI) of ‘Pan Galego’ (Galician bread) requires that the flour comprise a minimum 25% local wheat cultivars. As a test for compliance with this minimum level, wheat flours were prepared by mixing commercial flours with 0%, 5%, 20%, 25% and 30% ‘Caaveiro’ and 100% ‘Caaveiro’ and 100% commercial flours were used as controls. A second analysis was performed with a second set of wheat flours with 5% and 25% ‘Caaveiro’. These were mixed with two different commercial flours to assess the potential ability of five SSRs to identify the percentage of ‘Caaveiro’, constituting the first reference of the use of SSRs in the traceability of specific autochthonous cultivars in flour blends. ddPCR using the QX200 system platform was used to the targeted proportions across the simulated range with two out of five SSRs, indicating that they can be used in the traceability of ‘Caaveiro’ in mixed flours and breads SI

In Switzerland, chestnut forests cover about 27,100 ha, plus some 6800 ha of mixed stands. Due to environmental and historical reasons, most of these still existing forests are located in the Swiss Southern Alps, whereas in the northern parts of the country the chestnut cultivation and the related knowledge strongly regressed since the Little Ice Age period. Nevertheless, Switzerland still hosts valuable genetic resources of the sweet chestnut tree. The present genetic study bases on a nationwide inventory, identification and precise localisation of old and/or grafted chestnut trees for conservation purposes. The main objectives were: (1) to evaluate the genetic diversity and the genetic structure of Castanea sativa in Switzerland, and (2) to define a program of conservation including the proposal of a defined core collection. We genetically analysed a pre-selection of 962 accessions (out of 14,165 inventoried trees throughout Switzerland), profiling them with 24 microsatellites. We identified 675 different genotypes out of 962 accessions with a 29.8% of repetitiveness due to clonality. A structural analysis based on a Bayesian method allowed to identify two main clusters, one mostly related to the genetic group from southern Europe (Reconstructed Panmictic Population RPP1) and a second one (RPP2) which revealed to be independent and genetically different from other European groups of chestnut cultivars. The Swiss RPP2 represents a new genetic group, and consequently a complement to genetic resources of chestnut tree in Europe. Genetic analysis allowed defining a core collection of 46 genotypes, which should be used in priority for the Swiss conservation program.

For more than 50 years, the CIAM-AGACAL (Agricultural Research Centre of Mabegondo, Xunta de Galicia, A Coruña, Spain) has been carrying out the important task of conserving the phytogenetic resources of ecotypes and natural populations of grassland species from northwestern Spain. The CIAM-AGACAL’s germplasm bank has 57 populations of red clover (Trifolium pratense Lam.), one of the most cultivated forage legumes in the world. The goal of the present study was to evaluate the diversity among cultivars and natural clover populations at morphological and molecular level. Twelve polymorphic SSR loci revealed 241 microsatellite alleles with an average of 20.08 alleles per locus. Two main groups were detected by the Structure software, one of them including local populations and the second clustering cultivars and related populations. Intra-specific variability was found among cultivars and natural populations. A moderate genetic differentiation of Spanish red clover cultivars was observed (Fst = 0.08) between the two main clusters. Finally, a certain relationship between phenotypic and genotypic variation was detected.

The domesticated apple (Malus × domestica Borkh.) is one of the most relevant cultivated fruit crops worldwide, in particular in temperate zones, and the fourth most economically important. In order to avoid loss of genetic diversity, germplasm banks have been established to conserve proper genetic diversity, among them we have evaluated five that covers western Iberian Peninsula (the one from Galicia and three from northern to southern Portugal) and Canary Islands.A total of 634 accessions composed of old and local autochthonous apple germplasm, as well as commercial and reference varieties, plus 16 accessions that were collected as wild apples, were studied. Of the total, 484 local accessions were obtained from Spain and 67 from Portugal. The objective of the present study was to analyse the genetic diversity and genetic structure, and define core collection (a small group of cultivars that preserve the allele diversity of the set), by using 13 microsatellite markers (SSRs). Of the total of 634 accessions studied, corresponding to 370 unique genotypes, were grouped into two reconstructed panmictic populations or RPPs; the first RPP included 506 accessions (304 unique genotypes), which belonged to local and autochthonous varieties; and the second RPP clustered the remaining 128 (66 distinct genotypes), which were commercial and reference varieties. In total, 29 new synonyms (74 accessions) were identified between Galicia and the Canary Islands, which indicated the close historical relationship between these two regions located 2000 km apart. A core collection to preserve the whole allelic diversity of the west of the Iberian Peninsula (Galicia and Portugal) and the Canary Islands was defined keeping 10% of the genotypes (63) with respect to total accessions of the complete study, 14% of them (54) from Galicia with wild accessions or 15% (55) from Galicia without wild accessions, 40% (30) from the Canary Islands and 36% (24) from Portugal. The results obtained will allow update of the germplasm banks with correct identification of cultivars, differentiating the autochthonous ones from those derived from commercial cultivars, with the elimination of repetitions (42%), and proposal of the core collection necessary to preserve allele diversity.

The nature and structure of genetic diversity in the Spanish apple germplasm preserved at the national level was widely unknown, since studies performed to date on this topic have been exclusively carried out at the regional scale. Here, 1453 accessions from Spanish collections of Malus × domestica were evaluated with a common set of 13 SSR (Simple Sequence Repeats) markers in order to estimate genetic diversity, to identify the underlying genetic structure and to unravel the relationships among them and among a wide set of international cultivars for reference. In total, 737 unique genotypes were identified, 581 diploids and 156 triploids. Using a model-based Bayesian clustering procedure, two reconstructed populations were obtained for diploid genotypes; one retaining only Spanish cultivars (42% of genotypes), and a second containing all foreign cultivars the latter exhibiting evidence supporting the existence of a secondary sub-structure. Similarly, analysis performed on the 156 triploid genotypes also revealed two reconstructed populations; one exclusively associated with local Spanish genotypes (44%). The Jaccard coefficient allowed clustering by UPGMA (Unweighted Pair Group Method) diploid and triploid genotypes, and remarkable differences in allelic composition among the different partitioning levels were found. AMOVA analyses showed moderate but significant differentiation among the main groups (0.08 ≤ FST ≤ 0.12). Our results highlight an important fraction of the Spanish apple germplasm that constitutes a differentiated genepool with respect to the international and commercial apple cultivars. Moreover, the extent of the Spanish genetic diversity was spatially distributed along the northern Iberian Peninsula, suggesting an extensive migration of genotypes along the country. This study is the first valuable action for genetic conservation of apple at the national scale, and constitutes a decisive step towards the definition of a Spanish core collection that will be useful for further studies in dissecting the genetic control of important horticultural traits through genome-wide association analysis in apple.

This study presents the results of the first genetic analysis of ancient chestnut trees (Castanea sativa Mill.) in Italy and in the Iberian Peninsula to better understand the effect of grafting on the domestication process of chestnut and to investigate the impacts of early selection and improvement on the genetic diversity retained. We evaluated 105 giant ancient trees from Italy, Spain and Portugal and compared them with the European Union (EU) database of chestnut cultivars by using a set of 24 simple sequence repeats (SSRs; microsatellite markers). We measured the perimeter (girth) at the diameter at breast height (DBH). Samples from both the canopy and the roots of each tree were analysed to distinguish which trees were self-rooted and which were grafted. Diversity was compared using standard metrics and model-based approaches based on the expected heterozygosity (He) at equilibrium. We could differentiate 91 new genotypes; 9.6% matched known chestnut cultivars. We found the first evidences of cultivation, that is, grafting to produce “instant domestication” in Galicia and in the Douro Valley in trees of 14-m perimeter (15th century) and in the Basque Country (first report in that area) in a tree of 11.5-m perimeter (16th century). In Italy, the cultivar “Marrone Fiorentino” was found in some giant trees with perimeters of 8 and 9 m (17th-18th centuries) in the Toscana and Umbria. Those findings matched with written references in Portugal from the 16th century and from the 18th century in Spain. “Instant domestication” could be dated back to the 15th century and was related to the wild populations existing in the same areas where cultivars are being propagated, without a different genetic structure for wild chestnut trees and with a high diversity maintained through the initiation of domestication.

Apple breeding has been extremely successful in providing a highly diverse fruit crop. Recent (>50 millions years ago) genome-wide duplication (GWD) resulted in the 17 chromosomes in the Pyreae and confirmed the origin of cultivated apple on Malus sieversii being the same species as M. × domestica. Malus, as many other species in the family Rosaceae, shows gametophytic self-incompatibility (GSI), which forces outcrossing. GSI at the pistil is regulated by extacellular ribonuclease, S-RNase, which is encoded by S locus. Growers and agronomists have provided multiple cultivars with different colors, shapes, resistances, climatic adaptation or industrial aptitudes. The aim in apple breeding was the combination of different kinds of resistance and good fruit quality to produce dessert cultivars and cultivars for processing. Some of the best of these cultivars display resistance to scab (Venturia inaequalis), mildew (Podosphaera leucotricha), fire blight (Erwinia amylovora), bacterial canker (Pseudomonas syringae), red spider mite (Panonychus ulmi), winterfrost and good fruit quality. Different scab resistance sources of wild species (Vf, Vr, VA) were combined in the new series of cultivars. Multiple efforts worldwide have conserved most of that variation, the pillar for the traditional and new techniques profiting from the analysis of the apple genome, the genome-wide association studies (GWAS), identifying SNPs and genes, the analysis of genes differentially expressed (GDE) identified by qRT-PCR and microarray analysis, and the recent molecular genetic tool CRISPR/Cas9 to edit and correct the genome.

We have evaluated 271 accessions corresponding to 118 European cultivars, 96 from Spain, 16 from Italy, four from France and two from Portugal with the following objectives: (1) to provide a European database based on reference simple sequence repeats (SSRs) and (2) to define a core collection. A set of 24 highly polymorphic SSRs were used for the genetic analysis. Two main clusters were identified using a model-based Bayesian procedure, which correspond to Spanish and Italian cultivar clusters, with the latter showing a higher genetic diversity. An additional genetic substructure was observed among five different groups of cultivars. A core collection with a minimum of 37 cultivars was selected. We provided a database including 132 European accessions with unique genotypes evaluated with 24 SSRs as a reference for distinction, registering and traceability. Finally, we found that a core collection based on 14% of the total accessions conserves all allelic diversity.

The main grapevine germplasm bank of Vitis spp. at the Estacion de Viticultura y Enologia de Galicia in northwestern Spain includes 272 accessions. Eight SSRs differentiated 66 different genotypes, with 27 groups of synonyms and six homonyms. Brancellao Blanco (white berries) could not be differentiated from Brancellao (black berries). Eleven genotypes had not been previously described. The eight SSRs used in this study confirmed the existence of two groups of cultivars from Galicia: the Caino group in the west and the Merenzao group in the east.

The grapevine germplasm bank from Estacion de Viticultura e Enoloxia de Galicia contains 110 accessions collected from more than 100-year-old grapevines from northwestern Spain. We identified 60 different genotypes (among which ‘Pinot Noir’ and ‘Shyrah’ were included as references) with 32 polymorphic microsatellites (SSRs), 21 of which were unlinked SSRs and without null alleles, which were used to detect genetic structure. We found four reconstructed populations that were highly differentiated by analysis of molecular variance (18.3%): (i) western Galician group (‘Caino Bravo’); (ii) eastern Galician group (‘Merenzao’); (iii) Castilian group (‘Garnacha’) and (iv) southern Spanish group (‘Moscatel’). The greatest genetic differentiation was found between the western Galician/eastern Galician groups and the southern Spanish/Castilian groups, with Fst > 0.13. Western and eastern Galician groups contained most of the ancient Galician cultivars (86%) and included ancestors from central Europe, such as ‘Pinot Noir’ and ‘Shyrah’ in the western Galician group and the introgressant ‘Merenzao’ (French ‘Trousseau’) in the eastern Galician group; the Castilian and southern Spain groups included traditional cultivars from central and southern Spanish and suggest a further introduction of those cultivars in northwestern plantations. Genetic structure corresponded with East-to-West geographical differentiation in the northern Iberian Peninsula, following the old path to Santiago de Compostela from France, with the southern Spanish and Castilian groups in the East (Castilian plateau and southern Spain), the eastern Galician group and the western Galician group in the West. Five new relationships were discovered: ‘Caino Bravo’/‘Caino Longo2’, ‘Albarin Tinto’/‘Torrontes1’, ‘Treixadura’/‘Treixadura Francesa’, ‘Moscatel’/‘Moscatel de Bago Miudo’ and ‘Moscatel’/‘Moscatel de Hamburgo’, indicating that hybridisation and further selection by growers have been the origin of most of the genetic diversity found in northwestern Spain.

The germplasm pear bank in north-western Spain includes 221 accessions collected in the region between 1978 and 1981. Four reconstructed populations (RPPs) were detected by simple sequence repeats (SSRs) using a Bayesian method, two of which included French and English cultivars introduced to north-western Spain after reconquest by the Muslims in the 10th century. We studied 22 morphological and 15 phenological characteristics. We used principal component and cluster analysis to identify and classify the main origins of variability, these being flowering time, size and shape of fruit, time of harvesting, percentage of russeting and firmness of the flesh. Genetic differentiation between reconstructed populations, detected by a Bayesian method using SSRs, was also found in morphology, though with a great diversity inside each RPP that overlapped the main RPPs. This was consistent with the excellent quality of the reference cultivars apparently involved in the origin of the two main RPPs, ‘Mantecosa Hardy’ and ‘Williams’, suggesting that the contribution of foreign cultivars and subsequent selection by farmers have led to common phenotypic characteristics in both main groups. The introduction of cultivars from France and the United Kingdom could have occurred before 1746, when the first references to local cultivars related to both main RPPs were made. Furthermore, hybridisation with local species could explain part of the variability detected, which can be exploited for breeding programmes and local productions, nowadays focused on a reduced number of cultivars.

Spain is among the ten largest European pear (Pyrus communis L.) producers. Pear production is based on foreign cultivars. However, local cultivars are still grown in the old orchards in northwestern Spain. Between 1978 and 1981, the Centro de Investigaciones Agrarias de Mabegondo (CIAM, Xunta de Galicia) in Galicia established a germplasm bank in order to conserve genetic resources including local pear cultivars. In the current study, 216 accessions from CIAM and 19 commercial Pyrus cultivars were analyzed and compared with 15 polymorphic microsatellites, each marker being located at a different locus. We differentiated 130 genotypes out of the 216 accessions of the germplasm bank (60%). 86 of the accessions were duplications due to clonality (40%). A total of eight accessions of the germplasm bank (4%) were identified as commercial cultivars, those being: 'Castell' (1), 'Mantecosa Hardy' (2), 'Roma' (1), 'Blanquilla' (1) and 'Williams' (3). 33 genotypes were identified as duplications, some homonyms and synonyms that had not previously been described. The most frequent duplicated genotypes were those corresponding to 'Mantecosa', 'Urraca', 'Sevillana' and 'Donguindo', all of them previously reported as important pear cultivars in northwestern Spain. These results will allow the correct identification of unique genotypes and elimination of duplications in the CIAM germplasm bank. Some of these local cultivars can be used to diversify Spanish production.

The grapevine germplasm bank EVEGA contains 135 accessions of 15 Galician cultivars. Analysis with 33 polymorphic microsatellites (SSRs) indicate that Albarino and Loureira could be related to the Caino group in western Galicia, Spain. Likelihood ratios support the hypothesis of significant parentage relationship between Albarino and Caino Bravo as parents of Caino Blanco. Loureira shared alleles for each locus with Caino Bravo, indicating close relationship by hybridization. Caino Bravo was related directly with four cultivars: Caino Blanco, Caino Longo, Castanal and Loureira, as well as with two others (Caino Redondo1 and Souson) but with one less loci out of 33, indicating that it could be the origin of this lineage, being the key cultivar of the Caino group.

This is the first known large-scale molecular study of simple sequence repeats loci based on pear cultivars from the northwestern Iberian Peninsula. Most of the Spanish pear crop (one of the largest in Europe) is based on the Spanish cultivar Blanquilla and various other foreign cultivars. However, local cultivars can still be found in old orchards in northwestern Spain. Between 1978 and 1981, the Centro de Investigaciones Agrarias de Mabegondo (Xunta de Galicia) established a Germplasm Bank of local pear cultivars containing 221 accessions. In the current study, these were analysed and compared with 20 commercial cultivars of Pyrus spp. using 19 microsatellites. We identified 127 genotypes out of 221 accessions with an average of 43% clonality in this collection. Genotypes were analysed using a model-based Bayesian procedure (Structure), factorial correspondence analysis and molecular variance analysis, and Jaccard coefficients were estimated. Four reconstructed populations were identified by Structure, one related to Asian cultivars, two to French and English cultivars and one to Galician cultivars. The four identified groups of pears had evolved independently. This study explains the diversification process in pear cultivars from northwestern cultivars based on hybridization (16%), selection of triploids (38%) and the introgression of commercial cultivars in the collection (4%).

Chestnut tree in Spain not only produces nuts and timber, but it is also part of the landscape and Spanish heritage. Chestnut is present in 169,572 ha, but only in 27,511 ha were orchards in 2006 (www.mma.es). Commercialised nut production is about 20,000 t according to the FAO while the Spanish Government estimates 59,907 t (www.mma.es). Main producing areas are, in decreasing order, Galicia, Castilla-Leon (El Bierzo), Central Spain (Salamanca, Caceres and Avila), Andalucia and Canary Islands (Tenerife, La Palma, Gomera and El Hierro). Timber production is important in Northern Spain (Asturias and Galicia), Extremadura and Cataluna. Nuts are sold fresh in Spain and abroad (50% approximately) and the rest are transformed after peeling and freezing. In Spain, there are six main industries that transform chestnuts, the biggest ones are located in Galicia, more than 20 chestnut traders and six cooperatives. They produce and sell frozen and peeled nuts, "marron glace", "marron natural", cream, marmalade, flour, bonbons and dried nuts. Worst nuts are used as fodder for pigs in Andalucia and Galicia. Nuts are collected by growers and sold to industries, central markets and platforms by intermediaries. Minimum postharvest treatments are disinfestations, cleaning and packaging. Cooperatives located in the main producing areas commercialize more than 10% of total production. Only big producers, mostly in Central Spain, have enough production to sell by themselves. Timber production is coming from coppices in Asturias, Central Spain and Cataluna, and from grafted trees in Galicia. Best timber quality is not only for furniture making, plain-slice veneer, but also for construction. Chestnut timber is also used for making barrels (Cataluna), fences (most of the regions) and baskets (Central Spain). Chestnut contributes to the landscape everywhere, some countryside paths have been improved in Galicia to visit our centenary trees and some rural festivals are based on chestnuts, as the so-called "magostos". Some old trees are protected by authorities, since they are considered as natural monuments. Chestnut is referred to in Galician literature in medieval manuscripts since 10 th century and some painters, as Castelao, have reflected their relevance in our culture. These values have encouraged research programs to promote chestnut in our country, and the increase of chestnut plantations in North Spain is noticeable. The Protected Geographical Indication (PGI) "Castana de Galicia" has been approved recently to improve regional productions linked to the origin and the genetic singularity of chestnut cultivars. Contracts regulated by the Government, between growers and industrials, are being signed with prices ranging from 0.4 to 1.4 euros per kg depending on the nut size and the cultivar (in order of importance, 'Famosa', 'Negral', 'Longal', 'Garrida' and 'Parede'). Chestnut research in Spain begun in the fifties with hybridisation programs, which produced hybrids resistant to ink disease commercialized nowadays. During the last years, chestnut research has been focused in forestry and nut production, propagation, landscape and genetics.

International audience; This is the first known large-scale molecular study of simple sequence repeats (SSR) loci based on samples from grafted trees found in the Iberian Peninsula and Canary Islands and hybrids. Interspecific hybrids resistant to ‘ink disease’ (Phytophthora spp.) were obtained in France, Portugal and Spain, although difficult to distinguish by morphology. This study focuses on genetic variation using 10 SSRs (11 unlinked loci) from clonally propagated cultivars (574 accessions) of European chestnut (Castanea sativa) and hybrids (71 accessions). They were compared with a representative sample of exotic chestnut species present in the Atlantic area, 47 accessions of Castanea crenata, 37 of Castanea mollissima and 33 of Castanea henryi. Accessions were analysed using a model-based Bayesian procedure (Structure), factorial correspondence analysis and analysis of molecular variance. The main chestnut species, hybrids and alien introgressions were differentiated. Two main origins of variability in European cultivated chestnut were found in the Iberian Peninsula, one in the North and a second in the Centre. Andalusian and Canary Island accessions could be assigned to both of these zones, which indicate that they could have been colonised with cultivars originating from either zone, in the case of the Canary Islands from the sixteenth century on.

Apple cultivars from Canary Islands can possibly be valuable genetic resources for subtropical areas. We localised 31 accessions considered by growers to be local, and confirmed by historical references that apple crop was introduced in XV century. These accessions were compared with 77 Spanish and 26 commercial cultivars in order to detect synonyms. A set of 10 SSRs were studied, and 2 of them presented 2 loci. Cultivars from La Palma (Canary Islands) presented five specific alleles not found in other Spanish regions. Those polymorphisms allowed detecting one introgressant in La Palma from non-native cultivars, and the other 30 accessions were classified into 14 genotypes. Some accessions derived from non-native cultivars such as Golden Delicious. A main cultivar could be detected, Del Pais, with 14 accessions. Secondary ones were Camuesa and Pero. Genetic differentiation was small between regions (Fst = 0.057) but significant, confirmed by analysis of molecular variance (AMOVA). Major genetic differentiation was found between non-native cultivars and cultivars from Asturias and Basque Country. Bayesian method and admixture analysis reconstructed three ancestral groups (RPP), Asturian and Basque cultivars grouped in RPPI (mainly those used for cider production), a mixture of cultivars from Galicia and La Palma in RPPII and non-native cultivars were in RPPIII. This genetic differentiation was also confirmed by factorial correspondence analyses (FCA). AMOVA over RPPs increased the genetic differentiation. Allelic variation found in this study showed that Spanish local cultivars represent a differentiated genetic pool that will provide original genotypes to diversify the reduced number of cultivars used in commercial production. In addition, differentiated genotypes localised in La Palma will be preserved in the local Germplasm Bank.

A set of 87 apple accessions, located in three Portuguese apple germplasm collections, plus eight reference cultivars, were analyzed using 16 SSRs with the aim of assessing their genetic diversity and structure and evaluating relationships among them. Among the accessions studied, 64 unique genotypes were identified, 51 diploids and 13 putative triploids, revealing 19 groups of synonyms and 4 of homonyms. The genetic analyses performed by Bayesian model based clustering (Structure) revealed a clear differentiation of two major groups (RPP1 and RPP2), one of them (RPP1) corresponding to old local Portuguese accessions, some of them putatively derived from ancient hybridization with Reineta, and the second one (RPP2) including all reference accessions and local Portuguese accessions considered to be of more recent origin. These results were confirmed by factorial correspondence analysis (FCA) and molecular variance analysis (AMOVA) and are in accordance with the Jaccard coefficients. Results indicate that the Portuguese apple germplasm includes an important and distinct gene pool of cultivars with respect to reference international cultivars, being a relevant source of genetic diversity for present and future breeding programs, originated at least 200 years ago, when some of these cultivars were firstly named. Moreover, this study will be useful to improve the apple germplasm management and in the definition of a preservation strategy concerning the Portuguese apple landraces. This work was supported by European Investment Funds by FEDER/COMPETE/POCI–Competitiveness and Internationalization Operational Programme, under the Project POCI-01-0145-FEDER-006958, National Funds by FCT–Portuguese Foundation for Science and Technology, under the project UID/AGR/ 04033/2013 and the scholarship SFRH/BD/96400/2013 and Galicia Norte Portugal European Grouping of Territorial Cooperation (GNP-EGTC) funds supporting an internship at University of Santiago de Compostela, under the IACOBUS program. The authors acknowledge the “Research Institute of Horticulture and Seeds” INRA, Agrocampus Ouest, University of Angers, for providing the eight reference DNA samples.

The population of apple, Malus pumila Mill., indigenous to northwest Spain shows a wide range of ripening periods and fruit quality and is an unexploited resource for breeding programmes. The main ...

4 Pags.- 2 Tabls. Articles derived from XIV EUCARPIA Symposium on Fruit Breeding and Genetics (Bologna,Italy. June 14-18 2015) . The definitive version is available at: http://www.actahort.org/index.htm, The description of phenotypic traits in apple cultivars is generally performed using internationally agreed descriptors such as UPOV guidelines, which defines for each trait several states of expression. However, it is not always possible to classify a cultivar unambiguously using those guidelines, because in practice the states are not clearly enough defined or the example cultivars are not always available in the collections. This work presents the results of a harmonization project performed by the teams responsible of the main apple germplasm collections in Spain. The objective was to develop a standardized method for the 57 traits included in the TG/14/9 UPOV guidelines for apple characterization, defining their states of expression in a clear and unambiguous way for Spanish germplasm. Phenotypic data collected for more than 1,600 accessions from Spanish collections were used and the method to define each state depended on the type of expression. For quantitative traits the number of states and their limits were defined according to the variability that exists within and between accessions. For qualitative traits, high-resolution images clearly depicting each state were selected. A standardized characterization protocol for the 57 traits of apple germplasm has been provided, enabling to comparing properly the phenotypes of Spanish genetic resources., This Project has been funded by the Spanish Ministry of Science and Innovation/National Institute for Agricultural and Food Research and Technology (RF2011- 00017-C05-00).

A collection of 114 local cultivars of Malus × domestica Borkh. from the Northwest of Spain and a group of 26 non-native commercial cultivars, were studied in order to determine the level of genetic differentiation between them, to know the population genetic structure of the three main production regions in North Spain, and to detect possible duplications, misidentifications, and intrusions of foreign cultivars into the present day Spanish apple gene pools. Ten primer pairs of microsatellite loci were selected from previous studies on apple. Allelic frequencies were used to estimate the average expected heterozygosity (Hexp) and F-statistics. A Principal Component Analysis and a Cluster Analysis were conducted in order to determine the relationships among cultivars. Considering all populations (Spanish and commercial cultivars) we found 119 alleles with the 10 microsatellites; 10 out of 119 were rare (with frequencies lower than 0.01). Spanish cultivars can be used as references for those alleles for further studies since they are available in Germplasm banks. Triploid cultivars were quite frequent, 39 out of 140, with the highest number occurring in Galicia (29 cultivars). North Spain presented a high variability for apple according to heterozygosity levels. Microsatellites have provided useful information about the singularity of Spanish apple cultivars, revealed six groups of synonymies and five introgressions of commercial cultivars.

Morphological characters (six traits) and isozymes (four systems, fi ve loci) were used to discriminate between Spanish chestnut cultivars (Castanea sativa Mill.) from the Iberian Peninsula. A total of 701 accessions (representing 168 local cultivars) were analyzed from collections made between 1989 and 2003 in the main chestnut growing areas: 31 were from Andalucia (12 cultivars), 293 from Asturias (65 cultivars), 25 from Castilla-Leon (nine cultivars), four from Extremadura (two cultivars) and 348 from Galicia (80 cultivars). Data were synthesized using multivariate analy- sis, principal component analysis, and cluster analysis. A total of 152 Spanish cultivars were verifi ed: 58 cultivars of major importance and 94 of minor importance, of which 18 had high intracultivar variation. Thirty-seven cultivars were clustered into 14 synonymous groups. Six of these were from Galicia, one from Castilla-Leon (El Bierzo), four from Asturias, one from Asturias and Castilla-Leon (El Bierzo), and two from Asturias, Castilla-Leon (El Bierzo), and Galicia. The chestnut cultivars from Galicia and Asturias were undifferentiated in genetic terms, indicating that they are not genetically isolated. Overall, chestnut cultivars from southern Spain showed the least variation. Many (58%) of Spanish cultivars produced more than 100 nuts/kg; removing this low market-value character will be a high priority. The data obtained will be of use in chestnut breeding programs in Spain and elsewhere. Growers have selected chestnut cultivars since Roman times. Traditionally, chestnut stands are derived by grafting selected cultivars onto planted seedlings. In the Middle Ages huge plan- tations were established for forestry with the dual purpose of timber and nut production (Pereira-Lorenzo and Ramos-Cabrer, 2004). Some of these plantations can still be found in Tuscany (Italy), Galicia (Spain), Tras-Os-Montes (Portugal), and Aveyron and Cevennes (France). Monasteries established most of these extensive chestnut orchards. There are 187 cultivars documented in Spain (Pereira-Lorenzo et al., 2001a, 2001b). There is considerable need to defi ne precisely chestnut cultivar variation for improved planting regimes and chestnut breeding. Breviglieri (1955) made the fi rst comprehensive study of chestnut variation using morphological characteristics in the "Scheda Castanografi ca." Later, the International Union for the Protection of New Varieties of Plants (UPOV) published a chestnut guide for distinguishing chestnut cultivars and suggested some

Chestnut cultivation and production in Spain has employed grafted seedlings from selected local cultivars. Previously, we have characterised the Spanish cultivars by morphological and molecular markers. We are presenting in this paper the proximate analysis and mineral content for the main Spanish cultivars. A total of 131 samples were collected from 47 cultivars in six important Spanish chestnut production regions; located in the North such as Asturias, Castilla-Leon (El Bierzo) and Galicia; in the Central such as Extremadura and in the South such as Andalucia; as well as the Canary Islands, the southermost part of Spain near to North Africa. High variability in chemical composition between cultivars and regions corresponded to the high genetic variability between cultivars. Correlations with environmental parameters were low, indicating that differences found between regions were probably reflecting the differences between cultivars. In Central and Southern Spain, some cultivars presented lowest moisture content due to the low summer rainfall in these regions. Differences in starch and total sugar contents were high and were negatively correlated with each other. There was no negative correlation between nut size and total sugar content. Lowest values of fibre content and ease of digestibility were found in cultivars from Galicia and Extremadura. No significant differences in Fe, Zn and Cu were found although Zn content is twice the value reported for European chestnuts. This work would be a valuable reference to chestnut quality for the food processing industry, nutritionists, breeders and growers alike.

This study is focused in the main chestnut growing areas from north- to south-western Spain. We studied 50 accessions corresponding to 22 cultivars named by local growers; 11 from Andalucia, 9 from Castilla-Leon, and 2 from Extremadura. These were compared with 22 types classified previously in Galicia (82 accessions). Ten morphological traits and five isoenzyme systems were used to classify them and establish genetic relationships. A lower genetic variability has been found in Extremadura (middle Spain) and Andalucia (south) than in Galicia (north western) which may be because of the lower importance of the crop in those areas and the geographic barriers between them. Galicia could be the origin of the types found in Castilla-Leon, Extremadura, and south-western Andalucia, and the types found in Central Andalucia were closer to those found in Castilla-Leon and Extremadura. Most accessions studied (117/132) could be classified positively, making 37 out of 44 cultivars found from northern to southern Spain. Three groups of synonymies were found in Castilla-Leon: ‘Brava’ and ‘Negral’, ‘Gallego’ and ‘Injerto’, and ‘Rapega’ and ‘Verdello’. Most of the Spanish types have been propagated as a principal clone. Variability within the types is important and must be considered to select the best clones as cultivars.

ADDITIONAL INDEX WORDS. Malus ×domestica, isoenzymes, pomology, fruit breeding, principal component analysis, cluster analysis ABSTRACT. Spain is the 15 th largest apple (Malus ×domestica Borkh.) producer in the world with production depending mainly on foreign cultivars. During the 1970s, a germplasm bank of local cultivars was established in Galicia with the aim of preserving the local resources of northwestern Spain. A total of 408 accessions were studied using 89 morphological characters, with 15 corresponding to phenology, 46 to fruit, 7 to flowers, 11 to leaves, 6 to pests and 4 to diseases. Three variable isoenzymes, PGM E.C.5.3.1.9, PGI E.C.2.7.5.1 and EST E.C.2.7.5.1, were analyzed for 405 accessions and 27 commercial cultivars. The main objectives of this work were 1) to evaluate the inter- and intracultivar variability using morphological characters and isoenzymes, 2) to classify the accessions according to the main sources of variability, and 3) to identify repetitions in the germplasm bank. Principal component analysis (PCA) revealed six main sources of variability in the following order: size of fruit, color of skin, acidity, sweetness, harvest time, and attractiveness. The PCA analysis across 350 accessions produced 42 morphological groups. The 3 isoenzymes produced 190 genotype clusters. Combining morphological classification with the isoenzyme genotypes, we found 31 groups of synonyms involving 82 accessions and 8 more possible groups involving 17 accessions. This result allows the elimination of 53 repetitive accessions from the germplasm bank. Six commercial cultivars were identified as the progenitors of eighteen accessions: 'Reineta Blanca' of seven, 'Reina de Reinetas' of two, 'Reineta de Caux' of eight and 'Golden Delicious', 'Golden 4187' or 'Ozark Gold' of one each. Because inter- and intracultivar variability was high and names given by the growers were not reliable, the suggested selection strategy is to select individual clones among and within cultivars to exploit both the inter- and intracultivar genetic variability.

Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699; International audience; This is a large-scale molecular study based on simple sequence repeat (SSR) loci of the diversification process in chestnut cultivars from Portugal and Spain, from the northern Iberian Peninsula to the Canary Islands and the Azores. A total of 593 grafted chestnut trees (Castanea sativa Mill.) were analysed with 10 SSRs: 292 from Portugal and 301 from Spain. Some of the trees studied were more than 300 years old. Accessions were analysed using a model-based Bayesian procedure to assess the geographical structure and to assign individuals to reconstructed populations based on the SSR genotypes. We found 356 different genotypes with a mean value of clonality of 33% owing to grafting. Mutations accounted for 6%, with hybridization being the main diversification process that can explain the great diversity found. Ten main cultivar groups were detected: four in northern Spain, five in the centre of the Iberian Peninsula, and one in southern Spain related to the centre of the Iberian Peninsula. This work demonstrated that cultivar origin and the diversification process was a combination of clonal propagation of selected seedlings, hybridization, and mutations, which allowed high levels of diversity to be maintained with respect to selected clones for fruit production. Furthermore, seedlings and graft sticks facilitated the transport to new destinations in the colonization process, transporting sometimes more than 3000 km if we consider the Azores and the Canary Islands.; Les auteurs ont réalisé, à l’aide de marqueurs SSR, une étude moléculaire à grande échelle sur le processus de diversification des cultivars de châtaignier du Portugal et de l’Espagne, l’étude couvre une zone allant du nord de la Péninsule Ibérique jusqu’aux iles Canaries et aux Açores. Au total, 593 châtaigniers (Castanea sativa Mill.) greffés ont été analysés avec 10 SSR : 292 provenaient du Portugal et 301 de l’Espagne. Certains de ces arbres étaient vieux de plus de 300 ans. Une analyse basée sur un modèle bayésien a été réalisée pour déterminer la structure géographique des accessions et attribuer les individus au sein des différentes populations établies sur la base des génotypes SSR. Les auteurs ont trouvé 356 génotypes distincts dont en moyenne 33 % relèvent d’un état clonal attribuable au greffage. Les mutations permettent d’expliquer 6 % des génotypes observés alors que l’hybridation apparaît comme le principal processus de diversification et permettrait d’expliquer la grande diversité observée. Dix groupes principaux de cultivars ont été détectés, quatre dans le nord de l’Espagne, cinq autres au centre de la Péninsule Ibérique et un dans le sud de l’Espagne proche des groupes du centre. Ce travail montre que l’origine des cultivars et le processus de diversification sont le produit d’une combinaison de propagation clonale de plantules sélectionnées, d’hybridation et de mutations. Ces deux dernières permettent de maintenir une importante diversité au sein de clones sélectionnés pour la production de fruits. En outre, les plantules et les greffons ont facilité le transport vers de nouvelles destinations au cours du processus de colonisation, et ce parfois sur plus de 3000 km si l’on considère les Açores et les Îles Canaries.

The apple tree is a hybrid originating from a combination of wild species (Malus sieversii is supposed to be the main contributor). Growers at first selected the best specimens by seedlings, but when grafting was discovered as a mean of vegetative propagation, improvement in fruit quality became faster. Apple is cultivated in most of the temperate regions due to the fruit’s quality, its easiness to propagate, and its natural aptitude to bear. Apples are considered a healthy fruit, as the saying goes ‘an apple a day keeps the doctor away’. An apple tree can reach up to 10 m height above its own roots, having a globose canopy and the longevity between 60 and 100 years. Depending on the rootstock and the age of the tree, the roots can occupy between 2 and 104 m, although most frequently they range between 10 and 30 m (Atkinson 1980).

Chestnut (Castanea sativa Mill.) is an important crop in Spain. This inventory of chestnut cultivars complements previous studies. We have located 152 chestnut cultivars in 131 municipalities covering 108.6 ha, with 72 new cultivars in addition to the 80 previously found. Fewer than 50% of these cultivars are extensively cultivated. Chestnuts in Spain are grown from sea level to 1100 m, but are more frequent between 200 and 800 m on northern-facing slopes. Most of the chestnuts are harvested from 25 Oct. to 10 Nov.