A. Akkak

Development and characterization of microsatellite markers in Castanea sativa (Mill.)

Thirty-three simple sequence repeat (SSR) markers were isolated andcharacterized in Castanea sativa (Mill.) from the cultivarGarrone Nero. For the identification of SSR loci, primers were designed on eachside flanking the repeat region and they were initially tested on 5 chestnutsamples using chemiluminescence detection. Twenty four loci where shown to bepolymorphic and the number of alleles detected per locus varied from 2 to 7.Fourteen loci were chosen for the analysis of 20 cultivars grown in North Italyusing the semi-automatic system ABI PRISM 377. These 14 markers showed a highlevel of genetic polymorphism with a total of 90 alleles; the number of allelesranged from 4 to 10 per locus, with an average level of 6.4. The mean expectedand observed heterozygosity were 0.724 (range: 0.649–0.835) and 0.793(range: 0.350–0.950) respectively. The estimated frequency of nullallelesshowed a positive value for 3 loci, but except for 1 locus, the values wereverylow. The total value for the probability of identity was 7.04 ×10−11. Paternity exclusion probability was very high (0.999),sufficiently high to study pollen flow.

Development and evaluation of microsatellite markers in Phoenix dactylifera L. and their transferability to other Phoenix species

Forty one simple sequence repeats were isolated from two microsatellite enriched libraries of date palm (Phoenix dactylifera L.). After screening, 17 selected microsatellite loci were characterized and evaluated on a set of 31 cultivars and clones from Algerian and Californian germplasm. All primer pairs produced an amplification product of the expected size and detected high polymorphism among the analysed samples. These nuclear simple sequence repeat (SSR) markers are expected to be a very effective tool for evaluating genetic diversity in date palm germplasm. Acrosstaxa amplification showed the usefulness of most SSR markers in 14 other species across the genus Phoenix.

Genetic and morphological characterization of chestnut ( Castanea sativa Mill.) germplasm in Piedmont (north-western Italy)

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.

Genetic traceability of Asti Spumante and Moscato d’Asti musts and wines using nuclear and chloroplast microsatellite markers

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 (north-western 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.