R. Botta

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.

Development, characterization, segregation, and mapping of microsatellite markers for European hazelnut (Corylus avellana L.) from enriched genomic libraries and usefulness in genetic diversity studies

Eighty-six new microsatellite loci were developed for European hazelnut (Corylus avellana L.) by screening two genomic libraries enriched for dinucleotide repeats. The loci, 73 developed from a GA-enriched library and 13 from a CA-enriched library, showed a high level of polymorphism in 50 accessions. The number of alleles per locus ranged from five to 21, with a mean of 10.55. Mean values for expected heterozygosity, observed heterozygosity, and polymorphism information content were high, averaging 0.76, 0.69, and 0.73, respectively. In a mapping population, loci segregated 1:1, 1:2:1, and 1:1:1:1. Segregation distortion and null alleles were observed at some loci. Eighty-one of the 86 loci were assigned to linkage groups. A neighbor-joining dendrogram reflected great diversity among the 50 accessions and showed clustering by geographic origin.

Molecular and morphological diversity of on-farm hazelnut (Corylus avellana L.) landraces from southern Europe and their role in the origin and diffusion of cultivated germplasm

Hazelnut (Corylus avellana L.) is a traditional nut crop in southern Europe. Germplasm exploration conducted on-farm in five countries (Portugal, Spain, Italy, Slovenia, and Greece) identified 77 landraces. The present work describes phenotypic variation in nut and husk traits and investigates genetic relationships using ten simple sequence repeat (SSR) markers among these landraces, 57 well-known references cultivars, and 19 wild accessions. Among the 77 landraces, 42 had unique fingerprints while 35 showed a SSR profile identical to a known cultivar. Among the 42 unique landraces, morphological observations revealed high phenotypic diversity, and some had characteristics appreciated by the market such as nut round and caliber. Analysis of genetic relationships and population structure allowed investigation of the origin and spread of the cultivated germplasm in southern Europe. Our results indicate the existence of three primary centers of diversity in the Mediterranean basin: northwestern Spain (Tarragona) and southern Italy (Campania) in the West and Black Sea (Turkey) in the East. Moreover, the data suggest the existence of secondary gene pools in the Iberian (Asturias) and Italian (Liguria and Latium) Peninsulas, where local varieties were recently domesticated from wild forms and/or from introduced ancient domesticated varieties.

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.

Nuclear and chloroplast microsatellite markers to assess genetic diversity and evolution in hazelnut species, hybrids and cultivars

The US Department of Agriculture, Agricultural Research Service, National Clonal Germplasm Repository in Corvallis, Oregon, preserves more than 800 accessions of hazelnut (Corylus), including C. avellana cultivars and representatives of 10 other recognized shrub and tree species. Characterization and study of genetic diversity in this collection require cross-transferable markers, such as trinucleotide microsatellite or simple sequence repeat (SSR) markers and universal chloroplast SSR markers. We developed new SSR markers and evaluated 114 Corylus accessions representing 11 species and 44 interspecific hybrids. Eight of 23 SSRs generated easy-to-score alleles in all species and seven were highly polymorphic. For those seven, the average heterozygosity was moderate at 0.49, and mean allele number, genetic diversity and polymorphism information index were high at 11.71, 0.79 and 0.76, respectively. The three most polymorphic SSRs were CaC-C008, CaC-C040 and CaC-C118. Neighbor-joining (NJ) clustering and structure analysis agreed with classical taxonomic analysis and supported inclusion of C. maxima within the large polymorphic species, C. avellana. Analysis also indicated that C. californica is a distinct species rather than a botanical variety of C. cornuta. Six universal cpSSRs were polymorphic in Corylus and generated 21 distinct chlorotypes with an average of 3 alleles per locus. Diversity at these cpSSRs was high and ranged from 0.33 to 0.64, with an average of 0.54. Incongruence in NJ topologies between the nuclear and chloroplast markers could be attributed to chloroplast capture related to hybridization during the ancestral diversification of the genus, or to homoplasy. The phylogeographical relationships among the 21 chlorotypes in the 11 Corylus species support Asia as a refugium where several hazelnut lineages survived during glaciation and from which they continued to evolve after dispersal from Asia through the Mediterranean to Europe, and across the Atlantic and/or the Bering land bridge to North America.

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.’

Food safety and nutritional quality for the prevention of non communicable diseases: the Nutrient, hazard Analysis and Critical Control Point process (NACCP)

BackgroundThe important role of food and nutrition in public health is being increasingly recognized as crucial for its potential impact on health-related quality of life and the economy, both at the societal and individual levels. The prevalence of non-communicable diseases calls for a reformulation of our view of food. The Hazard Analysis and Critical Control Point (HACCP) system, first implemented in the EU with the Directive 43/93/CEE, later replaced by Regulation CE 178/2002 and Regulation CE 852/2004, is the internationally agreed approach for food safety control. Our aim is to develop a new procedure for the assessment of the Nutrient, hazard Analysis and Critical Control Point (NACCP) process, for total quality management (TMQ), and optimize nutritional levels.MethodsNACCP was based on four general principles: i) guarantee of health maintenance; ii) evaluate and assure the nutritional quality of food and TMQ; iii) give correct information to the consumers; iv) ensure an ethical profit. There are three stages for the application of the NACCP process: 1) application of NACCP for quality principles; 2) application of NACCP for health principals; 3) implementation of the NACCP process. The actions are: 1) identification of nutritional markers, which must remain intact throughout the food supply chain; 2) identification of critical control points which must monitored in order to minimize the likelihood of a reduction in quality; 3) establishment of critical limits to maintain adequate levels of nutrient; 4) establishment, and implementation of effective monitoring procedures of critical control points; 5) establishment of corrective actions; 6) identification of metabolic biomarkers; 7) evaluation of the effects of food intake, through the application of specific clinical trials; 8) establishment of procedures for consumer information; 9) implementation of the Health claim Regulation EU 1924/2006; 10) starting a training program.Results and discussionWe calculate the risk assessment as follows: Risk (R) = probability (P) × damage (D). The NACCP process considers the entire food supply chain “from farm to consumer”; in each point of the chain it is necessary implement a tight monitoring in order to guarantee optimal nutritional quality.

FRUIT SET AND EARLY BERRY DEVELOPMENT IN TWO GRAPEVINE CULTIVARS

The growth of the ovary/fruitlet of the beiry of two grapevine cultivars (‘Barbera’ and ‘Freisa’) was investigated at the karyological and histochemical level, in order to study the relationship between mitosis and cell-wall development and the synthesis of polysaccharides. The experiment started when flower buds appeared and continued throughout the formation of fruitlets until early July. In general, the peak of mitosis occurred a few days after fruit set, and the nucellar tissues were affected by the mi- totic processes longer than any other ovary tissues. The growth of the berry was mainly due to the mesocarp cell enlargement It seems that the main components of the cell-wall were pectins, which appeared to be increasing during the mitotic phase of tissue growth. Afterwards, stretching of cell-walls proceeded at a faster pace than pectin synthesis. Differences between the varieties were small but definite, indicating that they may be genotypical.

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.