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The cultivation of nut species, such as hazelnut and chestnut, still largely relies on traditional cultivars that were selected across centuries. Up to date, only a few breeding programs are active, yielding a limited number of new accessions, due to the particularly long time required for the selection process and the high heterozygosity of these species. There is an urgent need for improved breeding techniques based on marker-assisted selection (MAS) and biotechnological tools, to face new threats, such as the spread of alien species and the climatic changes. In this research we present the results of 10 years of activity addressed to the development of genetic maps in hazelnut (Corylus avellana L., progeny 'Tonda Gentile delle Langhe' × 'Merveille de Bollwiller' syn. 'Hall's Giant') and chestnut (progeny Castanea sativa × (C. sativa × C. crenata)) aimed at the detection of markers putatively useful for MAS. Saturated maps for the parent cultivars of the two progenies, accounting about 180 individuals each, were constructed using both SSR markers and SNP markers obtained by genotyping by sequencing, using the reference genomes as template. In hazelnut, phenological traits and fruit characters of commercial and technological relevance were mapped as QTLs; in chestnut, a trait of resistance to Dryocosmus kuriphilus Yasumatsu was mapped as a simple Mendelian character, and its genomic region is under deep investigation to find putative genes of resistance. Further breeding strategies are being carried out for hazelnut using in vitro techniques aimed at developing efficient systems of genetic transformation and regeneration for the modification or introduction of genes, such as those related to self-incompatibility and resistance to major pathogens.

Castanea sativa is an important multipurpose species in Europe for nut and timber production as well as for its role in the landscape and in the forest ecosystem. This species has low tolerance to chestnut gall wasp (Dryocosmus kuriphilus Yasumatsu), which is a pest that was accidentally introduced into Europe in early 2000 and devastated forest and orchard trees. Resistance to the gall wasp was found in the hybrid cultivar &lsquo, Bouche de B&eacute, tizac&rsquo, (C. sativa &times, C. crenata) and studied by developing genetic linkage maps using a population derived from a cross between &lsquo, and the susceptible cultivar &lsquo, Madonna&rsquo, (C. sativa). The high-density genetic maps were constructed using double-digest restriction site-associated DNA-seq and simple sequence repeat markers. The map of &lsquo, consisted of 1459 loci and spanned 809.6 cM, the map of &lsquo, consisted of 1089 loci and spanned 753.3 cM. In both maps, 12 linkage groups were identified. A single major QTL was recognized on the &lsquo, map, explaining up to 67&ndash, 69% of the phenotypic variance of the resistance trait (Rdk1). The Rdk1 quantitative trait loci (QTL) region included 11 scaffolds and two candidate genes putatively involved in the resistance response were identified. This study will contribute to C. sativa breeding programs and to the study of Rdk1 genes.

Eighty-two European chestnut populations, from Greece, Italy, France, Spain and the UK were studied using 73 ISSR and 16 isoenzyme loci in a total exceeding 2100 trees and 25000 genotypic designations. A multivariate analysis was used in order to detect locus interactions in space, not directly observable with averaging techniques such as genetic distances, after data vector transformation. Discussion elaborates on the major gene pools of chestnut in Europe and on the differences and similarities observed between different populations, regions, and countries. Within-stand population dynamics were investigated via a gene flow analysis of 15 populations from six sites (two from Spain, two from Italy and two from Greece) using 12 SSR and 26 isoenzyme loci, through paternity analysis and spatial analysis of genetic diversity patterns. Within each stand, 26-43 trees were genotyped and 7-9 selected mother trees provided the half-sib progenies (16-22 seeds per tree) analysed (about 58000 genotypic designations). Cryptic and outcoming gene flow appeared reasonably high for a predominantly outcrossing species (about 64%), ensuring a number of incoming migrants per generation large enough to counterbalance loss of diversity by inbreeding and drift, while selfing was very low (

Genetic variation and differentiation within and between species can be investigated using microsatellite or simple sequence repeats (SSRs). This can be done using primer sequences identified in one species, which can be amplified across species depending on whether or not the microsatellite flanking regions are conserved between species. This study provides an insight into the distribution of SSRs in related genera of the Fagaceae, Betulaceae, and Nothofagaceae families. Cross-species amplification of 17 SSR loci isolated in Castanea sativa Mill. were evaluated across 16 different species [C. sativa Mill., C. crenata Sieb. et Zucc., C. dentata Borkhausen (Marshall), C. mollissima Blume, C. seguinii Dode, Quercus robur L., Q. cerris L., Q. ilex L., Q. petraea (Mattuschka) Liebl., Q, suber L., Fagus sylvatica L., Betula pendula Roth, B. pubescens Ehrh., Carpinus betulus L., Corylus avellana L., Nothofagus antarctica (Forster) Oerst.] belonging to 7 genera. Moreover, 6 Q. petraea and 3 Q. robur SSR loci were evaluated only in the Betulaceae species. Among the C. sativa SSR loci, 13 of them generated one or more amplification products, in the expected size range, in Castanea species other than C. sativa, and 4 loci failed to amplify C., seguinii, C. mollissima or C. dentata. Surprisingly 5 C. sativa and 2 Q. robur primer pair amplified in the Betulaceae family. The apparent broad cross-species/genus/family transferability are being confirmed by cloning and sequencing the amplified alleles. The results indicate a high level of nucleotide sequence conservation within the primer region of 16 different species and have potential in phylogenetic and genetic variation studies and open up new interesting prospects for comparative genetic.

Establishing genetic origin of food products allows verification of the authenticity of valuable foods and discourages adulteration with material of lower cost and value. This is particularly important for food products that have obtained European recognition. The use of molecular markers could be a solution for species and cultivar identification and for the genetic traceability. The development of efficient DNA-extraction protocols is an essential step for the procedure. In this work, a method for total DNA isolation was developed for hazelnut, almond and walnut seeds. The efficiency and reliability of the method was tested by assessing quantity and quality of the extracted DNA, and by Polymerase Chain Reaction (PCR) amplification, using two decamer primers and three universal primer pairs designed on the chloroplast DNA. The success of amplifications confirmed the presence of both nuclear and chloroplast DNA in the extracted sample. PRACTICAL APPLICATIONS Cultivar identification of nuts, based on morphological traits only, is often difficult, and adulterations with seeds of lower cost and quality are easy, above all when they are sold as shelled kernel, as is common for hazelnut. The genetic identification of cultivars is nowadays a routine practice, because of the development of DNA-typing techniques based on molecular markers. An efficient DNA-extraction procedure for hazelnut, almond and walnut seeds is a preliminary step required for enabling the recognition of the cultivar of origin of the nuts and fighting commercial frauds. It will also be useful in marker-assisted selection, applied in breeding.

In this work, 18 microsatellite loci were developed in the European hazelnut ( Corylus avellana L.) using three enriched genomic libraries. They were evaluated on a set of 20 accessions of this species on the basis of number of alleles (mean: 7.1), expected heterozygosity (mean: 0.67), power of discrimination (mean: 0.77) and polymorphism information content (mean: 0.64). Cross-species transferability was evaluated using seven other Corylus species. All primer pairs amplified in all species, except for CaT-C505 in Corylus ferox and CaT-A114 in Corylus californica .

The final characteristics of a wine are strongly influenced by must varietal composition. Further, wine quality and value can be heavily modified if grape varieties other than those expected/allowed are used, especially in the case of monovarietal wines. 'Moscato bianco', which is one of the main grape varieties grown in Piedmont (northwestern Italy), is used for the production of two renowned monovarietal sparkling wines: Asti Spumante and Moscato d'Asti. Here, the genetic traceability of these wines was assessed using a simple sequence repeat (SSR or microsatellite) DNA-based method. Must and wine samples from two local wineries were collected at different winemaking steps: after grape crushing and pressing, without the skins (must sample 1, M1); after static clarification or flotation (M2); halfway through fermentation (M3); and finished wines. A DNA extraction protocol was developed, and samples were analysed using a set of 9 nuclear (nSSR) and 7 chloroplast (cpSSR) markers. The application of nSSR markers was successful for M1 and M2, but was inadequate for M3 and wines. CpSSR gave better results as amplifications were achieved using DNA extracted from M1, M2 and wines, despite the lack of amplification in M3. Furthermore, the amplified cpSSR loci showed high polymorphism, allowing the identification of 5 distinct chlorotypes among 7 muscat-flavoured and 2 non-aromatic grapevines. Altogether, these results suggest that this technique could be extended to wine quality and authenticity control, as well as origin protection.

Genetic stability of regenerants is a fundamental feature when somatic embryogenesis is used for regeneration after genetic transformation, as well as for germplasm cryopreservation and for virus eradication. On the contrary, somatic embryogenesis has been used in breeding strategies based on the exploitation of somaclonal variations. In order to assess the genetic fidelity of regenerants and the possibility of off-type somaclones we applied molecular typing on a number of anther and ovary-derived grapes. Plantlets were obtained by indirect somatic embryogenesis from anthers and ovaries of Grignolino and Dolcetto (Vitis vinifera L.). Thirty-seven lines of Grignolino and 9 of Dolcetto were evaluated through SSR analysis using 20 nuclear microsatellite loci for Grignolino and 9 for Dolcetto. All checked samples generated the same SSR profile of the cultivar and clone of the original explant. AFLP assays were also performed on 14 lines of Grignolino using two primer combinations for selective amplification. None of AFLP bands produced showed polymorphism among the lines. In addition, an experimental vineyard was planted in spring 2005 with plants from the same lines. No major difference between regenerants and control plants was observed so far. Further analyses are in progress.

European chestnut is grown extensively for its fruit and timber and is important not only for rural diversification, local economies and cultures, but also for its contribution to the landscape and environment. The species is threatened across Europe by human activities and by the ravages of fungal attacks such as Phytophtora spp. causing ink disease. European chestnut is therefore proposed as a model species for the development of long-term strategies combining conservation of evolutionary and adaptive potential, exploitation of breeding stocks and preservation of Mediterranean landscapes. The results of a four year EU funded project (CASCADE-EVK2-CT-1999-00006) are reported. The main objective of the project was to develop a strategy for long-term gene conservation and management of European chestnut. It was a multidisciplinary project involving scientists from 11 research institutions in five European countries covering areas such as ecology, ecophysiology, pathology, molecular genetics, population genetics, quantitative genetics, gene conservation, tree breeding, environmental economics and forest management. Both results obtained in different fields of investigation as well as synthetic considerations and recommendations related to gene conservation programs to be applied to this complex multipurpose tree species are given.

The general objective of the CASCADE project was to develop a strategy for long-term gene conservation and management of European Chestnut. The distribution area of chestnut was described by climate and edaphic conditions. Eighty-two populations, naturalised - coppice - fruit orchards, were analysed with respect to genetic markers to describe diversity and gene flow. Six naturalised populations from a broad span of climate conditions were included in studies of early growth, drought tolerance, and tolerance against Phytophthora cambivora. Six field trials were established. To identify the socio-economic impact of commodities from chestnut forests and orchards enquiries were distributed to regions, in which chestnut plays a role in the local economy. It was noted that chestnut is demanding with respect to edaphic conditions. A xerothermic index considering precipitation and mean temperature during the growth period was developed. European chestnut stands retain a fairly large amount of marker-based genetic variation. The effective number of alleles is fairly abundant in southern European regions, decreasing toward north and west. Mating probability dropped below 0.01 for distances larger than 2 km, and increased up to about 10% for distances closer than 300 meters according to the gene flow study. Genetic variability among and within populations was observed for juvenile growth, phenology, carbon isotope discrimination, and Phythophthora cambivora susceptibility. The latter variability was lower between domestication levels than within populations. Based on an inventory a distribution map for different Phytophthora species was provided. The enquiries disclosed that restoration of high forests was a top-ranked preference and so was the willingness to pay. Based on the data collected conservation values were derived for adaptive traits and markers. One suggestion for gene conservation was developed in the form of a network of gene resource populations covering the different climatic conditions of the sweet chestnut distribution area.

International audience; Simple sequence repeat (SSR) markers from Quercus and Castanea were used for comparative mapping between Quercus robur (L.) and Castanea sativa (Mill.). We tested the transferability of SSRs developed in Quercus to Castanea and vice-versa. In total, 47% (25) of the Quercus SSRs and 63% (19) of the Castanea SSRs showed a strong amplification product in the non-source species. From these 44 putative comparative anchor tags, 19 (15 from Quercus and 4 from Castanea) were integrated in two previously established genetic linkage maps for the two genera. SSR loci were sequenced to confirm the orthology of the markers. The combined information from both genetic mapping and sequence analysis were used to determine the homeology between seven linkage groups, aligned on the basis of pairs or triplets of common markers, while two additional groups were matched using a single microsatellite marker. Orthologous loci identified between Q. robur and C. sativa will be useful as anchor loci for comparative mapping studies within the Fagaceae family

Castanea sativa Mill. is an important multipurpose tree species for north-western Italy, and specially for Piedmont Region. The preservation of its germplasm from the genetic erosion due to the changes in socio-economic structure of rural areas and specific pathogen attacks is critical. The principal aims of this work were to characterize the chestnut germplasm grown in Piedmont and investigate its genetic structure. Sixty-eight grafted chestnut trees were evaluated using 10 SSRs (simple sequence repeats) loci and 20 morphological descriptors. Thirty-six different genotypes were identified; the analysis of the genetic structure of this germplasm revealed that four gene pools contributed to the formation of the population sampled. In general, cultivars tended to group into a main gene pool on the basis of their prevalent use and growing area. These results are substantially in agreement with those of the cluster analysis that was carried out to estimate the genetic relationships among the cultivars. Morphological analyses showed large variation of traits among the individuals, related with the market destination of the nuts and useful for cultivar and clonal selection. Discriminant analysis was applied to find a correlation between genetic and morphological data: nut and leaf shape, nut hairiness and male flower type resulted to be the most discriminant traits associated with the genetic structure. In the end, this work clarified the genetic structure of the cultivated germplasm in Piedmont describing the main cultivars of the region, giving useful information for conservation and breeding purposes.

Sequence Tagged Microsatellite Sites (STMSs) and morphological trait markers were used to evaluate 33 rhododendron germplasm for genetic diversity assessment and discrimination power. The average genetic diversity estimates were 0.724 (morphological traits) and 0.174 (STMSs) marker datasets. The Shannon index was higher for morphological traits (1.797) than STMS (0.302). The correlation coefficients obtained by the Mantel matrix correspondence test, which was used to compare the cophenetic matrices for the two markers, showed that estimated values of relationships given for morphological and STMS were not significantly related (p > 0.05). The dataset from STMS, supported by the total probability of identity (1.13 × 10−9) and total paternity exclusion probability (0.9999), allowed all accessions to be uniquely identified. In summary, STMS marker proved to be an efficient tool in assessing the genetic variability among old broad leaf rhododendron genotypes. The pattern of variation appeared to be consistent, and it can be used for germplasm conservation and management for restoration of historical genetic resources.

Recent increase in the availability of expressed sequence tag (EST) data has facilitated the development of microsatellite markers in a number of plant species, including grapevine. We identified 23 ESTs containing perfect simple sequence repeats (EST-SSR) of di-, tri-, tetra-, and penta-nucleotide type from 506 expressed sequence tags (EST) transcribed during berry development in ‘Cabernet Sauvignon’. Sixteen ESTs that contain SSRs at least 14 bp long were selected, and specific primers were developed and tested on five accessions. Thirteen produced a clear and strong amplification. Eight primer pairs revealed higher variation and were further studied on 19 grapevine accessions, including Vitis vinifera cultivars and different Vitis species. These eight loci showed transferability across the studied germplasm and good polymorphism both at cultivar level and among Vitis spp. Our results indicated that EST-SSR are valuable genetic markers for Vitis vinifera and have high potential use for comparative mapping.