A sample of 48 accessions belonging to important autochthonous grapevine varieties from Friuli Venezia Giulia (North-Eastern Italy) was analyzed using two sets of microsatellite markers. One marker set, based on di-nucleotide core repeats, was compared with a recently developed set of markers based on tri-, tetra-, and penta-nucleotide repeats in order to determine genetic identities, estimate genetic diversity, and establish the power of discrimination of the two sets. A total of 20 di-nucleotide SSR markers and 19 tri-, tetra-, and penta-nucleotide SSR markers were used to screen the accessions. All 39 primers produced PCR amplicons that were polymorphic and easily scorable in all of the accessions studied. Both datasets allowed the discrimination of all cultivars but two ('Refosco di Runcis' and 'Refoscone'). The observed heterozygosity ranged from 0.21 to 1.0 for the di-nucleotide markers, and from 0.21 to 0.88 for the tri-, tetra-, and penta-nucleotide repeat motif microsatellites, respectively. The population structure of the 48 autochthonous accessions was evaluated, together with 8 international cultivars included as references, providing evidence for significant population structure within the sample. Microsatellites composed of tri-, tetra-, and penta-nucleotide repeats, characterized by low ‘stuttering’ and larger relative size differences between incremental alleles, are now available for grape fingerprinting.

The function of monomeric GTPases of the RAS superfamily in fruit development and ripening has been partially characterized. Here the identification of peach (Prunus persica) small GTPases of the RAS superfamily expressed in fruit and the characterization of their expression profiles during fruit development are described. Extensive searches on expressed sequence tag (EST) databases led to the selection of a total of 24 genes from peach encoding proteins with significant similarity to Arabidopsis small GTPases. Sequence similarity analyses and identification of conserved motifs, diagnostic of specific RAS families and subfamilies, enabled bona fide assignment of fourteen PpRAB, seven PpARF/ARL/SAR, two PpROP and one PpRAN GTPases. Transcriptional expression profiles of peach monomeric GTPases, analysed by real-time quantitative reverse transcription-PCR, were obtained for mesocarp samples, collected in two consecutive years. Reproducible patterns of expression could be identified for five peach RAB-encoding genes (PpRABA1-1, PpRABA2, PpRABD2-1, PpRABD2-2, and PpRABC2), two ARFs (PpARFA1-1 and PpARLB1), and two ROPs (PpROP3 and PpROP4). Interestingly, the transient transcriptional up-regulation of PpARF genes and of PpRAB genes of the A and D clades, putatively controlling the exocytic delivery of cell wall components and modifying enzymes, appeared to coincide with peaks of growth speed and sugar accumulation and with the final phases of ripening. To our knowledge, this is the first description of the co-ordinated differential expression of a set of genes encoding small GTPases of the ARF and RAB families which takes place during key moments of fruit development and maturation.

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.

The first peach oligo-based microarray has been constructed by the Italian Consortium for Genomics in Prunus (ESTree, www.itb.cnr.it/ESTree) starting from Expressed Sequence Tags (ESTs) isolated in peach fruit. Four cDNA libraries have been synthesized from mesocarp of cv. 'Fantasia', 'Redhaven', 'OroB' and 'Bolero' and 1261 and 1133 ESTs were generated from S3 (second exponential growth) and S4 (maturation and ripening) stages, respectively. These 2394 sequences have been processed and combined with other 8807 ESTs retrived from NCBI GenBank and GDR (Genome Database for Rosaceae) at Clemson University Genomics Institute (CUGI) to create a gene dataset of 11201 entries. After sequence assembling, an unigene set composed of 4818 independent sequences has been obtained. Of these, 1442 contigs contain 2 or more ESTs, while the others 3376 represent single sequences (singletons). About 40% of the 4818 unigenes showed homology higher than 1.0e(-10) with Arabidopsis proteome and have been grouped according to MIPS functional categorization. 70-mer oligo-probes corresponding to 4806 unigenes expressed in peach fruit have been designed, synthesised, and deposited onto glass slides to create a microarray (mu PEACH 1.0). The first trials performed with mu PEACH 1.0 revealed that more than 70% of the probes give a positive signal when hybridized with RNA extracted from peach ripe fruits.

We have isolated 44 SSRs from almond and 99 SSRs from apricot from genomic libraries enriched for AC- and AG-repeats, respectively. The efficiency of the isolation procedure was higher in apricot (99 SSRs from 127 positive plaques) than in almond (44 SSRs from 93 positive plaques) although both libraries had a high percentage of positive plaques. The reason for the low number of markers recovered from almond was the presence of duplicates (26 cases) and the shortness of the cloned inserts that did not allow for primer design (23 cases). Primers were synthesized and twenty SSRs from each library were tested on eight different Prunus species, i.e. almond, peach, nectarine, apricot, European plum, Japanese plum, sweet cherry, sour cherry, and Malus x domestica, the apple. A wide transportability across the different Prunus was observed in both sets of SSRs.

We have isolated 44 SSRs from an AC-enriched genomic library from almond (Prunus amygdalus Batsch.). Twenty SSRs were screened for their polymorphism in 16 cultivars and for their transportability in seven different Prunus species (peach, nectarine, apricot, European plum, Japanese plum, sweet cherry, sour cherry) and in apple. The expected heterozygosity ranged from 0.62 to 0.89. About 30% of primers gave successful amplification in seven different Prunus species; in two cases amplifications were obtained also in apple.

We report 99 simple sequence repeats (SSRs) newly isolated from an apricot (Prunus armeniaca L.) genomic library enriched for AG/CT repeats. Twenty SSRs were screened for their polymorphism in 16 apricot cultivars. The number of alleles ranged from two to nine, whereas the expected heterozygosity (H-E) ranged from 0.26 to 0.82. The same SSRs showed also an appreciable transportability across different Prunus species, such as peach, nectarine, almond, European plum, Japanese plum, sweet cherry and sour cherry, with 20% of primers giving successful amplifications in all Prunus species assayed. None gave amplification in apple.

A breeding program aiming to introgress genes of resistance to several diseases, such as downy mildew, powdery mildew and Botrytis into the European grapevine has been initiated in 1998 at the University of Udine. Susceptible (S) and resistant (R) grapevine breeding lines were crossed according to a mating plan designed to produce S x R and R x R families. The R-lines included genotypes carrying resistances from American and/or Asian species. For each class of cross, several genotypes were crossed in all combinations irrespective of the way and without reciprocals. Selfing of S- and R-lines was also included. Twenty-eight crosses have already been made, the family size ranging from a few to 136 individuals. At the same time, molecular markers (anonymous SSRs and SNPs derived from Resistance Gene Analogs) were isolated to produce a transportable and functional linkage map. Beside 371 SSRs isolated together with other partners of the Vitis Microsatellite Consortium, further 101 new SSRs from AC-repeat enriched libraries were isolated. New libraries enriched in di- and tri-nucleotide repeats have been produced and isolation of new SSRs is underway. SNPs are being produced from 80 RGAs isolated in grape up to now. Through recurrent cycles of assessment of classes of phenotypic resistance/susceptibility and QTL analysis, we expect to elucidate the genetic control of resistance to several diseases and to identify genes/QTLs, which control such diseases. In the present paper, we give an overview of the whole research plan, the activities carried out, the results achieved in the first three years, and the strategy adopted to develop new grapevine cultivars suitable for the Friuli-Venezia Giulia region.

We have isolated and sequenced 52 microsatellites or simple sequence repeats (SSRs) from nearly 60 positive clones obtained from two ’Frantoio’ olive genomic libraries enriched in (AC/GT) and (AG/CT) repeats, respectively. The repeat-containing fragments obtained from genomic DNA restricted with Tsp509 I were separated using a biotinylated probe bound to streptavidin-coated paramagnetic beads. Fragments were then cloned into lambda ZAPII vector and sequenced. Thirty of the 36 primer pairs which gave correct re-amplification in the source genome were used to assay the polymorphism of 12 olive cultivars, namely four well-known cultivars (’Coratina’, ’Frantoio’, ’Leccino’, ’Pendolino’) and eight ancient cultivars grown locally near Lake Garda (’Casaliva’, ’Favarol’, ’Fort’, ’Grignan’, ’Less’, ’Raza’, ’Rossanel’, ’Trep’). The local cultivars were each re- presented by two to four long-lived individuals. The analysis was carried out using 33 P-labelled primers and 6% polyacrylamide sequencing gels. All except two microsatellites showed polymorphism, the number of alleles varying from 1 to 5. The average genetic diversity ( H ) was 0.55. The power of discrimination ( PD ) was 0.60. All cultivars, including the local ones, were easily separated from each other. Variations in the SSR pattern were observed among individual plants of the same cultivar in four out of the eight local cultivars analysed. Several primer pairs (17%) amplified more than one locus.

We have isolated and sequenced 52 microsatellites or SSR from nearly sixty positive clones obtained from a "Frantoio" olive genomic library enriched in (AC/GT) and (AG/CT) repeats. The repeat-containing fragments obtained from genomic DNA restricted with Tsp5091 were separated using a biotinylated probe bound to streptavidin-coated paramagnetic beads. Fragments were then cloned into lambda ZAPII vector and sequenced. Thirty of the 36 primer pairs, which gave correct re-amplification in the source genome, were used to assay the polymorphism of 28 olive genotypes, representing five Italian cultivars ("Coratina", "Frantoio", "Leccino", "Maurino", "Pendolino") and eight local cultivars grown in the lake Garda area ("Casaliva", "Favarol", "Fort", "Grignan", "Less", "Raza", "Rossanel", "Trep"). The local cultivars were each represented by 2 to 4 long-living individuals. The analysis was carried out using P-33-labelled primers and 6 % acrylamide sequencing gels. All microsatellites except two showed polymorphism, the number of alleles varying from 1 to 7. All cultivars, including the local ones, were easily separated from each other. Variations in the SSR pattern were observed among individuals of the same variety. This was the case of "Casaliva", "Fort", "Raza", and "Rossaanel". We were able to solve the identity of "Casaliva", which is probably a sport mutation of "Frantoio"; whereas "Leccino" which is sometimes said to be similar to the local variety "Less", showed patterns largely different from those of the local cultivar. Interestingly, several primer pairs (17 %) of the 30 assayed amplified more than one locus, resulting in patterns of three of more alleles in numerous individuals. This would agree with the hypothesis that domesticated olive (Olea europaea L.) is an ancient polyploid, as postulated since 1954.

We have isolated and sequenced microsatellites in several fruit crop species, such as grape (Vitis vinifera L.), kiwifruit (Actinidia, chinensis Planch), olive (Olea europea L.), and peach (Prunus persica (L.) Batsch), using microsatellite repeat-enriched genomic libraries. The efficiency of the enrichment procedure was variable (about 50 % of positive plaques in kiwifruit and peach, lower values in grape and olive). The re-amplification from the source genomic DNA was obtained in most cases. The polymorphism was high in all species assayed (5 to 15 alleles/locus in grape, 9 to 17 alleles/locus in kiwifruit, 2 to 8 alleles/locus in peach, data for olive are still being scrutinised). The Mendelian segregation was demonstrated for 8/10 SSR analysed in grape, 99/233 SSR analysed in kiwifruit, and 17/26 SSR analysed in peach. All microsatellites segregated as a single locus in peach, whereas in kiwifruit, grape, and olive several primer pairs amplified two loci each, in agreement with the suspected polyploid origin of the last species. The somatic stability was assayed in peach and olive, using three long-living accessions of cv. 'Redhaven' of different origin and 2-4 centuries-old plants of olive for each of the eight cultivars assayed. No pattern variation of the 26 microsatellites analysed was found in peach, whereas in olive rare intra-variety polymorphisms were observed in several of the 35 microsatellites analysed up to now. The assays on cross-species transportability were carried out in species representative of each genus (14 species of Vitis, 8 species of Actinidia, and 7 species of Prunus) and gave positive amplifications in high percentages (91 % of microsatellites amplified in all Vitis species, 52 % in all Prunus species, and 75 % in all Actinidia species). In some cases, successful amplifications were obtained also in related genera such as Parthenocissus, a genus close to Vitis, and Malus, which together with the genus Prunus belong to the Rosaceae family.

We have isolated and sequenced twenty-six microsatellites from two genomic libraries of peach (Prunus persica (L) Batsch) cv. 'Redhaven', enriched for AC/CT and AG/CT repeats respectively. The enrichment procedure allowed to have as much as 50 % of plaques containing the tal get repeat.We have assessed the cross-species transportability in Prunus, using DNA samples from seven different taxa out of peach (P. persica): nectarine (P. persica var. laevis), almond (P. dulcis), apricot (P. armeniaca), European plum (P. domestica), Japanese plum (P. salicina), sweet cherry (P. avium), and sour cherry (P. cerasus). Apple (Malus x domestica) was also included in the sample.More than half (52 %) of the microsatellites gave apparently correct amplification in all Prunus species surveyed, with bands within the range of expected sizes and compatible in the number with the known levels of ploidy. The remaining microsatellites gave less-extensive amplifications. The frequencies of lack of amplification were higher for species phylogenetically less related to peach, such as plums and cherries.The comparison of DNA sequences of the microsatellite UDP96-005, a complex microsatellite containing either AC/GT or AG/CT repeats, obtained by cloning and sequencing PCR products of all species but almond and sweet cherry, showed an almost perfect conservation across species of primer sequences designed on microsatellite flanking regions and the presence in all species of both CA and CT repeats.

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.

We report the sequences of 17 primer pairs of microsatellite loci, which we have cloned and sequenced from two genomic libraries of peach [ Prunus persica (L) Batsch] ‘Redhaven’, enriched for AC/GT and AG/CT repeats respectively. For ten of these microsatellite loci we were able to demonstrate Mendelian inheritance in a segregating back-cross population; the remainder did not segregate. The polymorphism of the microsatellites was evaluated in a panel of ten peach genotypes, including true-to-type peaches, nectarines and one canning-peach. Fifteen microsatellites (88%) were polymorphic showing 2–4 alleles each. The mean heterozygosity, averaged over all loci, was 0.32 and significantly higher than that reported in the literature for isozymes and molecular markers, such as RFLPs and RAPDs. We have also assayed the cross-species transportability and found that ten microsatellite (59%) gave apparently correct amplification in all Prunus species surveyed, namely P. domestica (European plum), P. salicina (Japanese plum), P. armeniaca (apricot), P. dulcis (almond), P. persica var. vulgaris (peach), P. persica var. laevis (nectarine), P. avium (sweet cherry) and P. cerasus (sour cherry), with three of them also being amplified in Malus (apple). The remaining microsatellites gave less-extensive amplification. Because of their appreciable polymorphism and wide cross-species transportability, most of these new markers can be integrated into the linkage maps which are currently being constructed in peach, as well as in other stone fruit crops, such as almond, apricot, cherry and plum.