Linkage maps of grapevine displaying the chromosomal locations of 420 microsatellite markers and 82 markers for R-gene candidates.

Genetic maps functionally oriented towards disease resistance have been constructed in grapevine by analysing with a simultaneous maximum-likelihood estimation of linkage 502 markers including microsatellites and resistance gene analogs (RGAs). Mapping material consisted of two pseudo-testcrosses, 'Chardonnay' x 'Bianca' and 'Cabernet Sauvignon' x '20/3' where the seed parents were Vitis vinifera genotypes and the male parents were Vitis hybrids carrying resistance to mildew diseases. Individual maps included 320-364 markers each. The simultaneous use of two mapping crosses made with two pairs of distantly related parents allowed mapping as much as 91% of the markers tested. The integrated map included 420 Simple Sequence Repeat (SSR) markers that identified 536 SSR loci and 82 RGA markers that identified 173 RGA loci. This map consisted of 19 linkage groups (LGs) corresponding to the grape haploid chromosome number, had a total length of 1,676 cM and a mean distance between adjacent loci of 3.6 cM. Single-locus SSR markers were randomly distributed over the map (CD = 1.12). RGA markers were found in 18 of the 19 LGs but most of them (83%) were clustered on seven LGs, namely groups 3, 7, 9, 12, 13, 18 and 19. Several RGA clusters mapped to chromosomal regions where phenotypic traits of resistance to fungal diseases such as downy mildew and powdery mildew, bacterial diseases such as Pierce's disease, and pests such as dagger and root-knot nematode, were previously mapped in different segregating populations. The high number of RGA markers integrated into this new map will help find markers linked to genetic determinants of different pest and disease resistances in grape.

Microsatellite and AFLP markers in the Prunus persica [L. (Batsch)]xP. ferganensis BC(1)linkage map: saturation and coverage improvement.

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 persicaxP. ferganensis)xP. persica BC(1) 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 >or=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.

Comparative study of the discriminating capacity of RAPD, AFLP and SSR markers and of their effectiveness in establishing genetic relationships in olive.

RAPDs, AFLPs and SSRs were compared in terms of their informativeness and efficiency in a study of genetic diversity and relationships among 32 olive cultivars cultivated in Italy and Spain. SSRs presented a higher level of polymorphism and a greater information content, as assessed by the expected heterozygosity, than AFLPs and RAPDs. The lowest values of expected heterozygosity were obtained for AFLPs, which, nevertheless were the most efficient marker system due to their capacity to reveal the highest number of bands per reaction and because of the high values achieved for a considerable number of indexes. All three techniques discriminated the genotypes very effectively, but only SSRs were able to discriminate the cultivars Frantoio and Cellina. The correlation coefficients of similarity were statistically significant for all three marker systems used but were lower for the SSR data than for RAPDs and AFLPs. For all markers a high similarity in dendrogram topologies was obtained although some differences were observed. All the dendrograms, including that obtained by the combined use of all the marker data, reflect some relationships for most of the cultivars according to their geographic diffusion. AMOVA analysis detected greater genetic differentiation among cultivars within each country than it did between the two countries.

A set of simple-sequence repeat (SSR) markers covering the Prunus genome.

A set of 109 microsatellite primer pairs recently developed for peach and cherry have been studied in the almond x peach F(2) progeny previously used to construct a saturated Prunus map containing mainly restriction fragment length polymorphism markers. All but one gave amplification products, and 87 (80%) segregated in the progeny and detected 96 loci. The resulting Prunus map contains a total of 342 markers covering a total distance of 522 cM. The approximate position of nine additional simple sequence repeats (SSRs) was established by comparison with other almond and peach maps. SSRs were placed in all the eight linkage groups of this map, and their distribution was relatively even, providing a genome-wide coverage with an average density of 5.4 cM/SSR. Twenty-four single-locus SSRs, highly polymorphic in peach, and each falling within 24 evenly spaced approximately 25-cM regions covering the whole Prunus genome, are proposed as a 'genotyping set' useful as a reference for fingerprinting, pedigree and genetic analysis of this species.

Microsatellite markers isolated in olive ( Olea europaea L.) are suitable for individual fingerprinting and reveal polymorphism within ancient cultivars.

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 Tsp509I 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.

Microsatellite DNA in peach (Prunus persica L. Batsch) and its use in fingerprinting and testing the genetic origin of cultivars.

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.

Restriction-site variation of PCR-amplified chloroplast DNA regions and its implication for the evolution and taxonomy of Actinidia.

Twenty six restriction sites from five PCR-amplified chloroplast DNA sequences (rbcL, psbA, rpoB, and two spacers flanking the trnL gene) were mapped and analysed in 20 Actinidia taxa, encompassing all four sections into which the genus is divided. At least three species out of the 20 examined have been found to have originated through natural interspecific hybridisation on the basis of the discrepancy between morphological and biochemical traits and the cpDNA profiles of pairs of species. A widely reticulate evolution has therefore been postulated in Actinidia. Wagner and weighted parsimony analysis produced consensus trees that did not match the traditional taxonomy based on morphological characters. The molecular data clearly showed that some taxa, such as A. rufa and A. kolomikta, occupy a wrong position and most, if not all, of the traditional groups represented by sections and series are weakly supported, since they appear as polyphyletic. A. chinensis and A. deliciosa were confirmed to be very closely related. Since chloroplast DNA is paternally inherited in Actinidia, A. chinensis is a paternal progenitor, if not the only one, of A. deliciosa, the domesticated kiwifruit.

Paternal inheritance of plastids in interspecific hybrids of the genus Actinidia revealed by PCR-amplification of chloroplast DNA fragments.

RFLPs (restriction fragment length polymorphisms) of PCR (polymerase chain reaction) -amplified fragments were used to trace the pattern of plastid DNA inheritance in the genus Actinidia. A total of 51 progeny originating from interspecific crosses between three A. arguta cultivars and A. deliciosa, the kiwifruit, and 12 progeny originating from the cross between A. kolomikta and A. chinensis were analysed together with their parents. No reciprocal crosses could be tested since they all failed to set viable seeds. Attempts to rescue immature embryos failed in all cases as well. The A. argutaXA. deliciosa crosses were checked for the RFLP patterns of a sequence encoding part of the Rubisco large subunit (rbcL), using either AluI or MseI, and for a sequence encoding part of the photosystem II D1 protein (psbA), using HinfI. The A. kolomiktaXA. chinensis cross was checked for the RFLP patterns of sequences encoding the spacers between trnT and the 5'-trnL exon (a-b spacer DNA) and the trnL 3' exon and trnF (e-f spacer DNA), respectively. The first spacer revealed a natural polymorphism between the two parent species due to a large deletion occurring in A. kolomikta detectable without further restriction enzyme treatment. The e-f spacer DNA was digested with HinfI. The comparison of the RFLP patterns in the parents and their progeny showed a strictly paternal inheritance of chloroplast DNA in Actinidia, with no exception found in any of the crosses examined. As the reciprocal crosses were not available, we do not know whether paternal inheritance of plastids is restricted to the crosses we analysed or if this is the general rule for plastid inheritance in the genus Actinidia. Actinidia is dioecious and is the first purely outbreeding species for which a paternal plastid inheritance has so far been documented.