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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.

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.

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.

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%).

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.

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.