Sharka is a worldwide serious disease with a severe impact on the productivity and fruit qual- ity of Prunus species. It is caused by Plum Pox Virus (PPV), a virus against which no chemical or biological curative treatments are available. PPV is easily trans- mitted by aphids and by grafting; so, despite the con- siderable efforts made in many countries, PPV has spread in all the most important Prunus growing areas. The current short-term solution is the eradica- tion of infected trees and the plantation of virus-free material. In Italy, the virus presence was first identi- fied in 1973 on apricot trees in Val Venosta area (Northern Italy). Since then, and despite the precau- tionary measures put in place, it has been spreading in almost all Italian regions, posing a serious threat to the stone fruit industry. In Emilia Romagna region (southern Po valley) it has been found since the early '80s, but the situation has worsened over the past decade, when outbreaks of the M strain of the virus have been identified. The M strain is characterized by a fast spreading by aphid vectors. The Italian Ministry of Agriculture sponsored the National Project PPV- CON, aimed to fight the virus threat through breeding actions in order to increase tolerance or introduce resistance into peach and improve knowledge on the virus biology and host interaction as well. No source of resistance has yet been found in peach, thus the isolation and introgression into P. persica of genes coding for the resistance to Sharka disease would be of significant economic and environmental benefit. In France, the resistance was found in P. davidiana (clone P1908), a species related to peach. In the off- spring obtained from crossing 'Summergrand' (yellow- flesh nectarine) and P1908, six QTL (Quantitative Trait Loci) were identified. One of them, highly corre- lated to the resistance, appears to be homologous to a QTL found in apricot. High level of resistance was also observed in several almond cultivars and the potential role of this species as a source of resistance to PPV in peach breeding programs has been demon- strated (Gradziel, 2002; Martinez-Gomez et al., 2004). As an alternative or complementary approach, geneti- cally engineered resistance by transgenes obtained from the virus itself is being tested. Transgenic PPV resistant Prunus domestica plants containing the PPV-CP (coat protein) gene have been obtained . Field tests performed on 'C5' selection, one of these transgenic clones (now patented as 'Honey Sweet'), clearly demonstrated to be resistant to PPV infection either through aphid vectors and by graft inoculation. Despite ethical-legislative limitations to the use of genetically modified organisms (GMO) in the European Union, at present, the goal of obtaining

Una attenzione crescente viene rivolta alla conservazione della biodiversita vegetale al di fuori degli ambienti naturali, sia per le specie di interesse agronomico, sia per la flora spontanea, anche in attuazione agli obblighi previsti dalla Convenzione sulla Diversita Biologica (Rio de Janeiro, 1992). Quest’ultima, infatti, all’articolo 9 “Conservazione ex situ” indica una serie di misure da adottare per il recupero, la ricostituzione e la reintroduzione di specie minacciate a completamento delle strategie di conservazione in situ. Inoltre, il quarto report dell’Intergovernmental Panel on Climate Change (2007) indica la conservazione ex situ tra le principali azioni di adattamento degli ecosistemi ai cambiamenti climatici in corso. La conservazione in situ (aree di origine) e quella on farm (nelle aree di coltivazione) sono prioritarie, ma quella ex situ (banche genetiche, collezioni, orti botanici, ecc.) si rende indispensabile in quei casi, e sono tanti, in cui le prime due, per motivi diversi, sono difficili da realizzare. Attualmente, infatti, le molteplici pressioni che agiscono sugli habitat possono in alcuni casi minacciare la sopravvivenza di una o piu specie o l’integrita e la funzionalita di interi ecosistemi, tanto da rendere difficile attuare strategie di conservazione in situ. In questi casi, solo le tecniche ex situ possono garantire la conservazione della variabilita genetica del germoplasma (semi, polline, parti di pianta, spore, ecc.) e quindi la rigenerazione, riproduzione e/o moltiplicazione delle specie da conservare. La conservazione ex situ ha inoltre un ruolo indispensabile per la ricerca e il miglioramento genetico al fine di promuovere un utilizzo sostenibile del germoplasma disponibile.Solo pochi anni fa, nel 2004, la maggior parte dei Paesi del mondo ha ratificato il Trattato Internazionale della FAO sulle risorse fitogenetiche per l’alimentazione e l’agricoltura che, inter alia, considera la conservazione ex situ come uno dei pilastri essenziali per assicurare a lungo termine la sostenibilita e la sicurezza alimentare e ambientale.

The genus Pistacia of the Anacardiaceae family consists of at least eleven species with dioecious plants. Some cases of monoecism have been reported for P. vera, P. atlantica and P. terebinthus. P. vera is an important crop that produces commercially valuable nuts. Dioecism represents an inconvenience to pistachio breeding and the long juvenile period (5-8 years) hampers sex determination until flowering. In this outline a molecular marker linked to sex could facilitate breeding allowing early seedling selection, saving time and economic resources. Very little work has been done at molecular level on the Pistacia genus and no linkage map and only very few codominant markers (Simple Sequence Reapeats, SSRs) are available so far. In the last years most of the research has been based on RAPDs (Random Amplified Polymorphic DNA) for the identification of a marker able to determine plant gender early and on AFLPs (Amplified Fragment Length Polymorphism) for genetic diversity studies. In this study a genetic analysis was carried out on 82 accessions among P. vera, P. terebinthus, P. atlantica subsp. mutica, P. mutica x P. khinjuk and P. integerrima. Microsatellite markers developed from a genomic library of the P. vera cultivar (Ahamad et al., 2003) were used with a capillary electrophoresis apparatus. The analyses were performed with NTSYS-pc 2.11X. A similarity matrix was constructed according to Jaccard's index and the dendrogram was obtained by the UPGMA method. The cluster analysis revealed species-based grouping, with all the P. vera accessions clustering independently from their gender and forming one cluster separated from all the remaining species. All the P. terebinthus were gathered in the same subcluster; P. mutica accessions were divided in three different subclusters showing a high level of genetic variability. The P. vera cultivars analysed, though separated in several different subclusters, displayed a low level of genetic variability probably due to the narrow genetic pool of cultivated materials. The microsatellite markers analysed in the present work, though less polymorphic than AFLPs or RAPDs, are more robust and repeatable.

Twenty-one expressed sequence tag-simple sequence repeat (EST-SSR) markers were developed in peach from a mesocarp cDNA library. Eighteen of them gave successful amplification in 22 peach genotypes and produced one to three alleles each with an average of 1.8 alleles per locus. The average value of expected and observed heterozygosities was 0.24 and 0.20, respectively. All the primers gave successful amplification in other six Prunus species (almond, apricot, sweet cherry, Japanese plum, European plum and Prunus ferganensis).

Blueberry is becoming a high value crop in Northern Italy (Trentino area) and several germplasm collections are now available. In the last few years, attention has been paid to varietal evaluation for high quality production. Previous studies, based on a total of 27 morphological, phenological and floral characteristics, revealed a possible misidentification of 4 clones out of 38 accessions, two of which are extensively cultivated.Up to now, the characterization of cultivars was mainly performed by the evaluation of a large set of phenotypic data, which can be difficult to assess and often requires the complete development of the plant. Phenotypic traits are also influenced by the genotype/environment interaction and their evaluation can become complex, subjective and sometimes inaccurate. Molecular markers can be used as tools to efficiently identify or reclassify misidentified germplasm. In order to characterize the accessions we performed a molecular marker analysis using RAPDs. Nine primers were tested on 38 accessions. The varieties Darrow, Brigitta and Duke were represented by two accessions each, coming from different geographical areas.A set of 3 primers (OPA05, OPA07 and OPA20), was enough to discriminate all the accessions except Northblue and Northland.The analysis pointed out a mismatching between the two accessions of Darrow and between those of Duke, confirming suspicions that had arisen after morphological evaluation in the field. Concerning the mismatching of the two Brigitta accessions the analysis showed a different pattern for several loci despite identical morphological and phenological descriptions.Similarity among the accessions ranged from 40.0% to 97.4% as expected in an outcrossing species subjected to inbreeding depression.

A set of 146 single sequence repeats (SSRs) and 14 amplified fragment length polymorphism (AFLP) primer combinations were used to enrich a previously developed linkage map obtained from a (Prunus persica×P. ferganensis)×P. persica BC1 progeny. Forty-one SSR primer pairs gave polymorphic patterns detecting 42 loci. The restriction/selective primer AFLP combinations produced a total of 79 segregating fragments. The resulting map is composed of 216 loci covering 665 cM with an average distance of 3.1 cM. Novel regions were covered by the newly mapped loci for a total of 159 cM. Eight linkage groups were assembled instead of the earlier 10 as two small groups (G1a and G8b), previously independent, were joined to their respective major groups (G1b and G8a). Several gaps were also reduced resulting in an improved saturation of the map. Twelve gaps ≥10 cm are still present. A comparative analysis against the Prunus reference map (71 anchor loci) pointed out an almost complete synteny and colinearity. Six loci were not syntenic and only two were not colinear. Genetic distances were significantly longer in our map than in the reference one.

Simple sequence repeats (SSRs) were developed in target regions of a peach linkage map using the peach bacterial artificial chromosome (BAC) resource available at Clemson University. The map was obtained using a BC, progeny [peach x (peach x P ferganensis)] and was composed of 109 loci covering 521 cM of the peach genome. Two regions of the map were chosen to develop SSRs. One was located in G5. This group was composed of only 4 loci covering 16 cM. An RFLP (FG202), localized on the homologous region of the anchored map (Texas x Earlygold), was chosen for this purpose. The other region was located in G7 and contained the QTL 'powdery mildew resistance'. The RFLP AG104, closely linked to this QTL, was used. Of the five SSRs obtained, only one was polymorphic in our mapping population and localized on the targeted G7 region. A set of 24 SSRs previously obtained at Clemson University and which includes three SSRs linked to the evergrowing gene (evg), were also tested and mapped. In this way, an additional SSR was obtained for the QTL for mildew resistance, while evg could be approximately located on the distal part of G1b.

The same Restriction Fragment Length Polymorphism (RFLP) and microsatellite markers made it possible to construct and saturate genomic maps for several Prunus species. Here, we compare the maps build from peach, sweet cherry and almond intraspecific populations and maps build from almond x peach, myrobalan plum x (almond x peach) and peach x Prunus ferganensis interspecific populations. As the order of the markers was highly conserved, it was possible to deduce the approximate position of RFLP and microsatellite markers that were not previously use for the construction of the reference European Prunus map, almond 'Texas' x peach 'Earlygold'. Based on the anchor loci, the locations of 31 genes from four species (peach, almond, cherry and myrobalan plum) were established on the framework of a single Prunus map. In addition, among the RFLP and microsatellite markers, several have sequences homologous to genes with known function. The homology between Prunus species should then allow a rapid transfer of the results obtained in one species to the others, especially for genes controlling agronomical characters.

The purpose of the present study was to identify tight linkage between important agronomic traits and molecular markers with the aim of using them in Marker Assisted Selection (MAS) in the peach breeding programs at ISF. Quantitative trait loci (QTLs) influencing several agronomic traits were identified in a BC, population issued from a cross between a selection of Prunus persica (IF 7310828) used as female recurrent parent and an accession of the related wild species P. ferganensis carrying a source of resistance to powdery mildew. One hundred and nine molecular markers (RFLPs, SSRs and RAPDs), spaced through the genome, were used to analyze 70 random progeny of this population. Fourteen characters, including blooming time, ripening time, disease resistance and traits related to tree architecture and fruit quality, were analyzed for 3-5 years on each tree of the BC, progeny. QTLs were identified for internode length, blooming time, ripening time, skin color, soluble solids content and Sphaerotheca pannosa resistance. Most of the QTLs were consistent through the years showing that the expression of the genes is largely independent of the environmental conditions. All of them displayed the same effect as the parental phenotype. For each trait the detected QTLs explained up to 42% of the total variance.

Fifty-two peach genotypes, nine nectarines, one almond (Ferragnes) and four peach x almond hybrids were tested using RAPDs. Thirty-nine of the peach genotypes were also tested using 28 RFLP probes from two almond libraries (genomic and cDNA) obtained by IRTA of Cabrils (Spain). A total of 115 primers were screened for their ability to produce strongly amplified products. Out of 79 (68.7%) primers, with a strong amplification, 42 (53.2%) showed polymorphisms among the tested genotypes. Eight of these were considered as the most informative producing 24 polymorphic reliable bands. A set of five primers (OPA9, OPA14, OPK19, OPJ13 and OPP9), for a total of 16 RAPDs, proved to be enough to discriminate 39 genotypes (59.1%). Eleven additional patterns characterized more than one genotype. The remaining polymorphic bands did not add further information for distinguishing those genotypes showing the same profile. Using RFLPs, from nine polymorphic probes (out of 28 tested) we obtained 17 markers. They permitted to discriminate 16 (41.0%) genotypes, out of the 39 tested, and nine patterns characterizing more than one genotype. The undiscriminated genotypes were the same in both trials. The complete set of 61 RAPD bands (24 polymorphic) and of 55 RFLP bands (17 polymorphic) were separately analyzed by cluster analysis using NTSYS version 1.80. The similarity indexes obtained from RAPD and RFLP are quite the same varying from about 80% to 100%. The results confirmed the high degree of inbreeding within the peach genotypes tested.

A linkage map was obtained using a BC1 progeny (Prunus persica x (P. persica x P ferganensis)). The map is composed of 109 loci (74 RFLPs, 17 SSRs, 16 RAPDs, and two morphological traits) distributed in 10 linkage groups. Loci, segregating in five different ratios, were integrated in the map with JoinMap 2.0 software. The map covers 521 cM of the peach genome. The average distance between adjacent loci is 4.8 cM. Two monogenic traits, flesh adhesion (F/f) and leaf glands (E/e), were placed on the map. Thirty-two loci in common with a saturated linkage map of Prunus allowed a comparative analysis to be made between the two maps. Homologies were found among the respective linkage groups. No relevant differences were observed in the linear order of the common loci.

We isolated and sequenced 26 microsatellites from two genomic libraries of peach cultivar 'Redhaven', enriched for AC/GT and AG/CT repeats, respectively. For 17 of these microsatellites, it was possible to demonstrate Mendelian inheritance. Microsatellite polymorphism was assayed in 50 peach and nectarine cultivars. Of the 1300 PCRs carried out, all but two produced amplified products of the expected size. All microsatellites were polymorphic, showing 2-8 alleles per locus. Heterozygosity ranged from 0.04-0.74 (mean 0.47); the discrimination power (PD) ranged from 0.04-0.84 (mean 0.60). Cultivar heterozygosity varied greatly, with one cultivar ('Independence') being homozygous at all loci. The set of microsatellites discriminated all cultivars investigated, except several sport mutations, i.e., 'Dixitime' vs. 'Springcrest', 'Compact Redhaven' vs. 'Redhaven', and two pairs of cultivars, 'Venus' vs. 'Orion' and 'Elegant Lady' vs. 'Rome Star', whose pedigrees are controversial. We were able to analyze the paternity of several cultivars. In most cases, the parenthood was confirmed. The comparison of three long-living 'Redhaven' accessions supplied by different repositories did not provide any evidence of somatic instability of microsatellites. Hence, microsatellites, ranked according to their information content, are recommended as markers of choice for peach fingerprinting and suggestions are provided for interpreting band profiles and the correct sizing of alleles.

A genetic linkage map of peach was constructed using 75 individuals of a BC1 population from a cross between the semidwarf peach selection IF7310828 (tolerant to powdery mildew) as female recurrent parent and an accession of Prunus ferganensis carrying a source of powdery mildew (Sphaerotheca pannosa) resistance.The current map, consisting mainly of RFLP markers, covers 460cM of the peach genome. Markers have been assigned to nine linkage groups. Twenty-one markers were in common with the saturated peach x almond map obtained by the European "Prunus mapping" group.Several morphological and agronomical traits of this population were studied during three years. The monogenic traits, leaf glands and flesh adhesion, were assigned respectively to group 7 and to group 4, both tightly linked to an RFLP marker.The polygenic traits, such as powdery mildew resistance, tree architecture, blooming and ripening time and fruit quality were analyzed for QTLs.Ten QTLs were identified by using interval-mapping analysis; some of them were consistent through the years showing that the expression of the genes influencing the traits was largely independent on the environmental conditions. The additive effect of each QTL was consistent through the years and in agreement with the parents' phenotype. In particular a major gene for powdery mildew resistance was detected in group 7 tightly linked to the marker Ag 104 and to the monogenic trait "leaf glands".

Detection of polymorphisms among the parents of the ISF segregating populations IF7310828 x P. ferganensis (F1) and IF7310828 x P72 (BC1) was performed using probes from different genomic and cDNA libraries provided by IRTA, Cabrils (Spain), Clemson University (USA) and ISF itself. Of the 725 probe/restriction enzyme combinations tested, 107 (14.8%) showed polymorphism among the parents and, of these, 73 (68.2%) were segregating in the F1 and 95 (88.8%) in the BC1 populations. Of the 154 probes tested, 58 (37.7%) showed polymorphism with at least one restriction enzyme. Morphological and agronomical traits, such as internode length, glandular foliage, blooming and ripening time, fruit size and flesh adhesion, were recorded in the field on the BC1 segregating population.

A progeny of 76 BC1 seedlings, resulting from a cross between the semidwarf selection IF7310828 as female recurrent parent and a seedling of Prunus ferganensis used as donor, carrying a potential source of resistance to powdery mildew, was studied in order to construct a genetic linkage map of peach nuclear genome.Thirty-seven RFLPs, detected by genomic and cDNA clones, 3 morphological traits and 8 RAPDs were analyzed for inheritance and linkage. Two of the cDNA clones correspond to known genes (phosphoglyceromutase and peptide induced by jasmonic acid). Thirty-three loci segregated in a 1:1 ratio (28 were heterozygous in the F1 parent and 5 in the recurrent parent), 8 segregated in a 1:2:1 ratio and 7 in a 3:1 ratio. Nine loci showed a distorted segregation ratio. A genetic linkage map was elaborated with a total of 36 loci using JoinMap. Eight linkage groups covering 257 cM of the peach genome were found. Twelve loci remained unlinked. Linkage between leaf glands, Sphaerotheca pannosa resistance, flesh adhesion and molecular markers were found.

A map with 246 markers (11 isozymes and 235 RFLPs) was constructed using an interspecific F 2 population between almond (cv Texas) and peach (cv Earlygold). RFLPs were obtained using 213 probes from the genomic and cDNA libraries of different species (almond, peach, P. ferganensis , cherry, plum and apple), including 16 almond probes which correspond to known genes. All markers were distributed in eight linkage groups, the same as the basic chromosome number of the genus, covering a total distance of 491 cM. The average map density was 2.0 cM/marker and only four gaps of 10 cM or more were found; the two largest gaps were 12cM each. This map was compared with one constructed previously with an intraspecific almond population sharing 67 anchor loci. Locus order was nearly identical and distances were not significantly different. A large proportion of the mapped loci (46%) had skewed segregations; in approximately half of them, the distortion was due to an excess of heterozygotes. One of the distorted regions could be associated with the position of the self-incompatibility gene of almond.

The use of DNA-based molecular markers allowed to develop linkage map for identifying and localizing important genes in several plant species. The final purpose of this work is to develop a saturated genetic linkage map in peach in order to identify ergonomically important traits, such as disease resistance and fruit quality, for use in marker assisted selection programs.