Daniela Torello Marinoni

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.

Transgene silencing in grapevines transformed with GFLV resistance genes: analysis of variable expression of transgene, siRNAs production and cytosine methylation

Eight transgenic grapevine lines transformed with the coat protein gene of Grapevine fanleaf virus (GFLV-CP) were analyzed for a correlation between transgene expression, siRNAs production and DNA methylation. Bisulphite genome sequencing was used for a comprehensive analysis of DNA methylation. Methylated cytosine residues of CpG and CpNpG sites were detected in the GFLV-CP transgene, in the T7 terminator and in the 35S promoter of three grapevines without transgene expression, but no detectable level of siRNAs was recorded in these lines. The detailed analysis of 8 lines revealed the complex arrangements of T-DNA and integrated binary vector sequences as crucial factors that influence transgene expression. After inoculation with GFLV, no change in the levels of cytosine methylation was observed, but transgenic and untransformed plants produced short siRNAs (21–22 nt) indicating that the grapevine plants responded to GFLV infection by activating a post-transcriptional gene silencing mechanism.

Currants and strawberries as bioactive compounds source: determination of antioxidant profile with HPLC-DAD/MS system

*summary Among plant foods, berry fruit shows a high antioxidant capacity. Medical research has pointed out the medicinal properties of certain pigmented polyphenols, such as flavonoids, anthocyanins, tannins and other phytochemicals, which are mainly found in the skin and seeds of the berries. The aim of this work was to contribute to the study of the nutraceutical features of some berry fruit (currants, gooseberries and strawberries). The different antioxidant compound contents of the fresh fruit of different cultivars and selections of Ribes spp. and Fragaria x ananassa Duch have been analyzed. The fruit of 29 cultivars from 3 different species of Ribes spp. and 5 strawberry cultivars have been analysed by High Performance Liquid Chromatography coupled to a UV/Vis detector and a mass detector (MS) to identify and quantify the main antioxidant compounds. As far as the Ribes spp. cultivars are concerned, the presence of a high content of phenolic compounds has been confirmed, and they represent therefore an important source of antioxidant compounds. Moreover, the results have shown that the considered cultivars and selections of strawberries are good sources of bioactive compounds, especially phenolic substances. The results of this study could contribute to offer new insights into the nutraceutical aspects of the considered berry fruit species.

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.

Early embryo achievement through isolated microspore culture in Citrus clementina Hort. ex Tan., cvs. ‘Monreal Rosso’ and ‘Nules’

Microspore embryogenesis is a method of achieving complete homozygosity from plants. It is particularly useful for woody species, like Citrus, characterized by long juvenility, a high degree of heterozygosity and often self-incompatibility. Anther culture is currently the method of choice for microspore embryogenesis in many crops. However, isolated microspore culture is a better way to investigate the processes at the cellular, physiological, biochemical, and molecular levels as it avoids the influence of somatic anther tissue. To exploit the potential of this technique, it is important to separate the key factors affecting the process and, among them, culture medium composition and particularly the plant growth regulators and their concentration, as they can greatly enhance regeneration efficiency. To our knowledge, the ability of meta-Topolin, a naturally occurring aromatic cytokinin, to induce gametic embryogenesis in isolated microspores of Citrus has never been investigated. In this study, the effect of two concentrations of meta-Topolin instead of benzyladenine or zeatin in the culture medium was investigated in isolated microspore culture of two genotypes of Citrus. After 11 months of isolated microspore culture, for both genotypes and for all the four tested media, the microspore reprogramming and their sporophytic development was observed by the presence of multinucleated calli and microspore-derived embryos at different stages. Microsatellite analysis of parental and embryo samples was performed to determine the embryo alleles constitution of early embryos produced in all tested media, confirming their origin from microspores. To our knowledge, this is the first successful report of Citrus microspore embryogenesis with isolated microspore culture in Citrus, and in particular in Citrus clementina Hort. ex Tan, cvs. ‘Monreal Rosso’ and ‘Nules.’

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.

Isolation of a gene encoding for a class III peroxidase in female flower of Corylus avellana L.

Hazelnut is a monoecious species characterized by mid-winter blooming and sporophytic incompatibility. The molecular mechanisms at the basis of the female flower development and of the pollen-stigma interaction are little known, although pollination in this species is a critical factor to ensure good yield. Differential display technique was used to study genes expressed during the female flower development, comparing styles before emergence from the bud and styles at full bloom. The full-length cDNA clone, designated CavPrx (Corylus avellana peroxidase) and isolated in mature styles, was characterized as a sequence encoding for a 330 amino acids protein, containing all the conserved features of class III peroxidases. CavPrx resulted expressed only in styles, with a peak in mature styles pollinated with compatible pollen. Class III peroxidases are expressed in several different plant tissue types and are involved in a broad spectrum of physiological processes. Until now, four peroxidases expressed in the stigma were identified in Arabidopsis thaliana and Senecio squalidus: they were assumed to be possibly involved in pollen–pistil interaction, pollen tube penetration/growth and/or in defence against pathogens. CavPrx is the first gene for a floral peroxidase isolated in hazelnut and its expression pattern suggests a possible role in the pollination process.

Genetic Characterization of Grape Cultivars from Apulia (Southern Italy) and Synonymies in Other Mediterranean Regions

Forty-five grape accessions, traditional and historically mentioned in Apulia (southeastern Italy), were genotyped at 13 microsatellite (SSR) markers and observed for their morphological features with the aim of characterizing and identifying the local grape diversity relevant for economic or historical significance and for endangered germplasm conservation. Twelve of the 45 accessions examined were found to be synonyms or somatic mutants, leaving 33 distinct genotypes. Attempts were then made to verify the true identities of the accessions investigated and to determine their appropriate denominations. This entailed comparing them with published allelic profiles and morphological features of cultivars from Apulia and from surrounding areas linked historically to the region. While confirming the identity of the major Apulian cultivars, further matches with varieties from other Mediterranean regions were revealed. Approximately half of the Apulian cultivars investigated were found to have a foreign counterpart mainly along the Adriatic Sea (Croatia), in Greece, or in other southern Italian regions. The new synonymies found with cultivars traditional to other areas shed light on the migration of cultivars following the settlement of colonies and the historical establishment of Mediterranean trade routes.

High density SNP mapping and QTL analysis for time of leaf budburst in Corylus avellana L.

The growing area of European hazelnut (Corylus avellana L.) is increasing, as well as the number of producing countries, and there is a pressing need for new improved cultivars. Hazelnut conventional breeding process is slow, due to the length of juvenile phase and the high heterozygosity level. The development of genetic linkage maps and the identification of molecular markers tightly linked to QTL (quantitative trait loci) of agronomic interest are essential tools for speeding up the selection of seedlings carrying desired traits through marker-assisted selection. The objectives of this study were to enrich a previous linkage map and confirm QTL related to time of leaf budburst, using an F1 population obtained by crossing Tonda Gentile delle Langhe with Merveille de Bollwiller. Genotyping-by-Sequencing was used to identify a total of 9,999 single nucleotide polymorphism markers. Well saturated linkage maps were constructed for each parent using the double pseudo-testcross mapping strategy. A reciprocal translocation was detected in Tonda Gentile delle Langhe between two non-homologous chromosomes. Applying a bioinformatic approach, we were able to disentangle ‘pseudo-linkage’ between markers, removing markers around the translocation breakpoints and obtain a linear order of the markers for the two chromosomes arms, for each linkage group involved in the translocation. Twenty-nine QTL for time of leaf budburst were identified, including a stably expressed region on LG_02 of the Tonda Gentile delle Langhe map. The stability of these QTL and their coding sequence content indicates promise for the identification of specific chromosomal regions carrying key genes involved in leaf budburst.