Raffaele Testolin

The high-quality draft genome of peach (Prunus persica) identifies unique patterns of genetic diversity, domestication and genome evolution

Rosaceae is the most important fruit-producing clade, and its key commercially relevant genera (Fragaria, Rosa, Rubus and Prunus) show broadly diverse growth habits, fruit types and compact diploid genomes. Peach, a diploid Prunus species, is one of the best genetically characterized deciduous trees. Here we describe the high-quality genome sequence of peach obtained from a completely homozygous genotype. We obtained a complete chromosome-scale assembly using Sanger whole-genome shotgun methods. We predicted 27,852 protein-coding genes, as well as noncoding RNAs. We investigated the path of peach domestication through whole-genome resequencing of 14 Prunus accessions. The analyses suggest major genetic bottlenecks that have substantially shaped peach genome diversity. Furthermore, comparative analyses showed that peach has not undergone recent whole-genome duplication, and even though the ancestral triplicated blocks in peach are fragmentary compared to those in grape, all seven paleosets of paralogs from the putative paleoancestor are detectable.

AC/GT and AG/CT microsatellite repeats in peach [Prunus persica (L) Batsch]: isolation, characterisation and cross-species amplification in Prunus

Abstract 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.

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

Abstract  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 33P-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.

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

Abstract. 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.

Mapping With a Few Plants: Using Selective Mapping for Microsatellite Saturation of the Prunus Reference Map

The concept of selective (or bin) mapping is used here for the first time, using as an example the Prunus reference map constructed with an almond × peach F2 population. On the basis of this map, a set of six plants that jointly defined 65 possible different genotypes for the codominant markers mapped on it was selected. Sixty-three of these joint genotypes corresponded to a single chromosomal region (a bin) of the Prunus genome, and the two remaining corresponded to two bins each. The 67 bins defined by these six plants had a 7.8-cM average length and a maximum individual length of 24.7 cM. Using a unit of analysis composed of these six plants, their F1 hybrid parent, and one of the parents of the hybrid, we mapped 264 microsatellite (or simple-sequence repeat, SSR) markers from 401 different microsatellite primer pairs. Bin mapping proved to be a fast and economic strategy that could be used for further map saturation, the addition of valuable markers (such as those based on microsatellites or ESTs), and giving a wider scope to, and a more efficient use of, reference mapping populations.

Conservation of microsatellite loci within the genus Vitis

Abstract Eleven microsatellites isolated from grapevine (Vitis vinifera) were used to study the degree of conservation of these sequences across different Vitis species. Nine microsatellites were newly isolated, the remaining two (VVS2 and VVS5) came from the literature. A preliminary assay on the conservation of priming sites was carried out on 14 non-V. vinifera species, including relevant taxa for breeding. Parthenocissus quinquefolia was added as representative of a related genus. Cross-species amplification was obtained in 94% of the 176 genotype×locus tested combinations. Three microsatellite loci were then cloned and sequenced in ten species. The microsatellite repeat was found present in all cases. The repeat region was often longer in V. vinifera than in the other species. Furthermore the non-source species showed interruptions in the repeat. In spite of these constraints, which could reduce the polymorphism of microsatellites in non-source species, the results demonstrate the possibility of extending the use of microsatellite markers to wild germplasm and inter-specific hybrids. Point mutations have been found in microsatellite flanking regions and these variations have been used to investigate the genetic relationship among taxa. The Neighbor-joining tree that was obtained on the basis of ten nucleotide variations, showed that there is not a clear cut difference between American, Asian and European species and that the actual taxonomy which reflects the geographical distribution of species must most likely be revised. Moreover, in general, nucleotide variations which occur in microsatellite flanking regions provide new molecular tools for investigating the evolution of species.

A consensus list of microsatellite markers for olive genotyping

Cultivar identification is a primary concern for olive growers, breeders, and scientists. This study was aimed at examining the SSR markers retrieved from the literature and currently used in olive study, in order to select those most effective in characterizing the olive accessions and to make possible the comparison of data obtained by different laboratories. Olive microsatellite profiles were assessed by four independent laboratories, which analyzed 37 pre-selected SSR loci on a set of 21 cultivars. These SSR markers were initially tested for their reproducibility, power of discrimination and number of amplified loci/alleles. Independent segregation was tested for each pair of SSRs in a controlled cross and the allelic error rate was quantified. Some of them were finally selected as the most informative and reliable. Most of the alleles were sequenced and their sizes were determined. Profiles of the reference cultivars and a list of alleles with their sizes obtained by sequencing are reported. Several genetic parameters have been analysed on a larger set of cultivars allowing for a deeper characterization of the selected loci. Results of this study provide a list of recommended markers and protocols for olive genotyping as well as the allelic profile of a set of reference cultivars that would be useful for the establishment of a universal database of olive accessions.

Microsatellite DNA in Actinidia chinensis: isolation, characterisation, and homology in related species

Abstract We have isolated and sequenced 263 microsatellite-containing clones from two small insert libraries of Actinidia chinensis enriched for (AC/GT) and (AG/CT) repeats, respectively. Primer pairs were designed for 203 microsatellite loci and successfully amplified from both plasmid and A. chinensis genomic DNA. In this paper we report the sequences of 40 primer pairs for which we have demonstrated Mendelian segregation in the progeny from controlled crosses. The polymorphism of ten microsatellites of each type was evaluated in four diploid and six tetraploid genotypes of A. chinensis. All microsatellites proved to be polymorphic, the number of alleles per locus detected in polyacrylamide sequencing gels ranging from 9 to 17. The high degree of polymorphism in Actinidia renders these markers useful either for mapping in A. chinensis or for fingerprinting cultivars of both domesticated kiwifruit species (A. chinensis and A. deliciosa). While most primer pairs produced single amplification products, about 20% generated banding patterns consistent with the amplification of two different loci. This supports the hypothesis that diploid species of Actinidia (2n=2x=58) are polyploid in origin with a basic chromosome number x=14/15 and that chromosome duplication may have occurred during the evolution of the genus. Finally, we have assayed the cross-species transportability of primer pairs designed from A. chinensis sequences and have found extensive cross-species amplification within the genus Actinidia; 75% of primer pairs gave successful amplification in the eight species assayed (A. arguta, A. rufa, A. polygama, A. chrysantha, A. callosa, A. hemsleyana, A. eriantha, and A. deliciosa), which are representative of the four sections into which the genus is currently split.

A set of microsatellite markers with long core repeat optimized for grape (Vitis spp.) genotyping.

BackgroundIndividual fingerprinting based on molecular markers has become a popular tool for studies of population genetics and analysis of genetic diversity in germplasm collections, including the solution of synonymy/homonymy and analysis of paternity and kinship.Genetic profiling of individuals is nowadays based on SSR (Simple Sequence Repeat) markers, which have a number of positive features that make them superior to any other molecular marker developed so far. In humans, SSRs with core repeats three to five nucleotides long are preferred because neighbour alleles are more easily separated and distinguished from each other; while in plants, SSRs with shorter repeats, namely two-nucleotides long, are still in use although they suffer lower separation of neighbour alleles and uncomfortable stuttering.ResultsNew microsatellite markers, containing tri-, tetra-, and penta-nucleotide repeats, were selected from a total of 26,962 perfect microsatellites in the genome sequence of nearly homozogous grapevine PN40024, assembled from reads covering 8.4 X genome equivalents.Long nucleotide repeats were selected for fingerprinting, as previously done in many species including humans. The new grape SSR markers were tested for their reproducibility and information content in a panel of 48 grape cultivars. Allelic segregation was tested in progenies derived from two controlled crosses.ConclusionA list of 38 markers with excellent quality of peaks, high power of discrimination, and uniform genome distribution (1–3 markers/chromosome), is proposed for grape genotyping. The reasons for exclusion are given for those that were discarded. The construction of marker-specific allelic ladders is also described, and their use is recommended to harmonise allelic calls and make the data obtained with different equipment and by different laboratories fully comparable.

Breakdown of resistance to grapevine downy mildew upon limited deployment of a resistant variety

BackgroundNatural disease resistance is a cost-effective and environmentally friendly way of controlling plant disease. Breeding programmes need to make sure that the resistance deployed is effective and durable. Grapevine downy mildew, caused by the Oomycete Plasmopara viticola, affects viticulture and it is controlled with pesticides. Downy mildew resistant grapevine varieties are a promising strategy to control the disease, but their use is currently restricted to very limited acreages. The arising of resistance-breaking isolates under such restricted deployment of resistant varieties would provide valuable information to design breeding strategies for the deployment of resistance genes over large acreages whilst reducing the risks of the resistance being defeated. The observation of heavy downy mildew symptoms on a plant of the resistant variety Bianca, whose resistance is conferred by a major gene, provided us with a putative example of emergence of a resistance-breaking isolate in the interaction between grapevine and P. viticola.ResultsIn this paper we describe the emergence of a P. viticola isolate (isolate SL) that specifically overcomes Rpv3, the major resistance gene carried by Bianca at chromosome 18. We show that isolate SL has the same behaviour as two P. viticola isolates avirulent on Bianca (isolates SC and SU) when inoculated on susceptible plants or on resistant plants carrying resistances derived from other sources, suggesting there is no fitness cost associated to the virulence. Molecular analysis shows that all three isolates are genetically closely related.ConclusionsOur results are the first description of a resistance-breaking isolate in the grapevine/P. viticola interaction, and show that, despite the reduced genetic variability of P. viticola in Europe compared to its basin of origin and the restricted use of natural resistance in European viticulture, resistance-breaking isolates overcoming monogenic resistances may arise even in cases where deployment of the resistant varieties is limited to small acreages. Our findings represent a warning call for the use of resistant varieties and an incentive to design breeding programmes aiming to optimize durability of the resistances.