Enrico Peterlunger

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.

Development of a standard set of microsatellite reference alleles for identification of grape cultivars

In order to investigate the comparability of microsatellite profiles obtained in different laboratories, ten partners in seven countries analyzed 46 grape cultivars at six loci (VVMD5, VVMD7, VVMD27, VVS2, VrZAG62, and VrZAG79). No effort was made to standardize equipment or protocols. Although some partners obtained very similar results, in other cases different absolute allele sizes and, sometimes, different relative allele sizes were obtained. A strategy for data comparison by means of reference to the alleles detected in well-known cultivars was proposed. For each marker, each allele was designated by a code based on the name of the reference cultivar carrying that allele. Thirty-three cultivars, representing from 13 to 23 alleles per marker, were chosen as references. After the raw data obtained by the different partners were coded, more than 97% of the data were in agreement. Minor discrepancies were attributed to errors, suboptimal amplification and visualization, and misscoring of heterozygous versus homozygous allele pairs. We have shown that coded microsatellite data produced in different laboratories with different protocols and conditions can be compared, and that it is suitable for the identification and SSR allele characterization of cultivars. It is proposed that the six markers employed here, already widely used, be adopted as a minimal standard marker set for future grapevine cultivar analyses, and that additional cultivars be characterized by means of the coded reference alleles presented here. The complete database is available at http://www.genres.de/eccdb/vitis/. Cuttings of the 33 reference cultivars are available on request from the Institut National de la Recherche Agronomique Vassal collection (didier.vares@ensam.inra.fr).

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.

Water stress and rootstock influence on leaf gas exchange of grafted and ungrafted grapevines

Abstract In order to investigate the effects of rootstocks on plants of the genus Vitis subjected to gradually increased levels of water stress we studied the links between abscisic acid in leaves and the major leaf physiological parameters. Cuttings of Vitis vinifera cv. Muller Thurgau were grafted onto three hybrid rootstocks (H1, H8, H26) and grown in pots. Own-rooted plants of V. vinifera and own-rooted hybrids were also cultivated in an identical manner. Vines were tested during 7 and 14 days of water stress (i.e. daily water supply reduced to 66% of well-watered plants). Water stress reduced net photosynthesis and stomatal conductance with large differences between grafted and ungrafted vines. Significant differences were observed after only 14 days of water stress. In particular two grafting combinations, V. vinifera grafted on H1 (V/H1) and H8 (V/H8), did not show net photosynthesis reduction after 14 days of water stress. Water use efficiency increased with water stress whereas carboxylation efficiency was reduced. Net photosynthesis, stomatal conductance and carboxylation efficiency were affected by the rootstock genotype combination, so that under water stress only some rootstock genotypes transferred drought tolerance to the scion. Abscisic acid in leaves increased significantly from Control to 7 and 14 days of water stress but the relations with CO 2 assimilation rate, stomatal conductance and carboxylation efficiency appeared to be affected by the genotype of the vegetative apparatus. Our data show that the interaction between scion and rootstock with regard to production performance under water deficits may be an important consideration in vineyard planting strategies.

Isolation of (AC)n-microsatellites in Vitis vinifera L. and analysis of genetic background in grapevines under marker assisted selection

Primers were developed for 118 microsatellites isolated from grape (Vitis vinifera) genomic libraries enriched for (AC)n repeats. Only one microsatellite sequence matched other grape SSR-sequences in the GeneBank database. Genotyping was carried out in the parental lines and four offspring of two pseudo-test-cross populations, ‘Cabernet Sauvignon’ x ‘Seyval’ and ‘Chardonnay’ x ‘Bianca’, and a further six other grape genotypes (V. vinifera ‘Sultanina’, ‘Merlot’, ‘Syrah’, ‘Müller-Thurgau’, Vitis ‘Regent’ and V. riparia ‘Gloire de Montpellier’). A total of 108 microsatellites showed easily scorable alleles and 100 of them segregated according to a configuration suitable for mapping in either cross. A further 8 SSRs, although unsuitable for mapping in those crosses, showed polymorphism in the other genotypes tested. This set of markers was used, along with 75 microsatellites of other repeat-types, to fingerprint 46 offspring of the cross ‘Chardonnay’ x ‘Bianca’. For each full-sib, individual heterozygosity and distance in repeat units between pairs of alleles at each locus (mean d2) were calculated as a tool for predicting ‘highly outbred’ recombinant individuals. Six microsatellites with segregation ratios significantly distorted towards the lack of homozygous sibs were identified and mapped to linkage groups LG 3 and LG 5. Estimation of heterozygosity at genome-wide level and genotyping at loci for which homozygous sibs are discriminated against are discussed for marker-assisted background selection in outcrossing grapevines.

Transcriptome and metabolite profiling reveals that prolonged drought modulates the phenylpropanoid and terpenoid pathway in white grapes (Vitis vinifera L.)

BackgroundSecondary metabolism contributes to the adaptation of a plant to its environment. In wine grapes, fruit secondary metabolism largely determines wine quality. Climate change is predicted to exacerbate drought events in several viticultural areas, potentially affecting the wine quality. In red grapes, water deficit modulates flavonoid accumulation, leading to major quantitative and compositional changes in the profile of the anthocyanin pigments; in white grapes, the effect of water deficit on secondary metabolism is still largely unknown.ResultsIn this study we investigated the impact of water deficit on the secondary metabolism of white grapes using a large scale metabolite and transcript profiling approach in a season characterized by prolonged drought. Irrigated grapevines were compared to non-irrigated grapevines that suffered from water deficit from early stages of berry development to harvest. A large effect of water deficit on fruit secondary metabolism was observed. Increased concentrations of phenylpropanoids, monoterpenes, and tocopherols were detected, while carotenoid and flavonoid accumulations were differentially modulated by water deficit according to the berry developmental stage. The RNA-sequencing analysis carried out on berries collected at three developmental stages—before, at the onset, and at late ripening—indicated that water deficit affected the expression of 4,889 genes. The Gene Ontology category secondary metabolic process was overrepresented within up-regulated genes at all the stages of fruit development considered, and within down-regulated genes before ripening. Eighteen phenylpropanoid, 16 flavonoid, 9 carotenoid, and 16 terpenoid structural genes were modulated by water deficit, indicating the transcriptional regulation of these metabolic pathways in fruit exposed to water deficit. An integrated network and promoter analyses identified a transcriptional regulatory module that encompasses terpenoid genes, transcription factors, and enriched drought-responsive elements in the promoter regions of those genes as part of the grapes response to drought.ConclusionOur study reveals that grapevine berries respond to drought by modulating several secondary metabolic pathways, and particularly, by stimulating the production of phenylpropanoids, the carotenoid zeaxanthin, and of volatile organic compounds such as monoterpenes, with potential effects on grape and wine antioxidant potential, composition, and sensory features.

Characterization of major ripening events during softening in grape: turgor, sugar accumulation, abscisic acid metabolism, colour development, and their relationship with growth

Highlight The earliest events in ripening are decreases in turgor, softening, and increases in abscisic acid. Later events integral to regulating colour development include growth, further increases in abscisic acid, and sugar accumulation.

Multi-Omics and Integrated Network Analyses Reveal New Insights into the Systems Relationships between Metabolites, Structural Genes, and Transcriptional Regulators in Developing Grape Berries (Vitis vinifera L.) Exposed to Water Deficit

Grapes are one of the major fruit crops and they are cultivated in many dry environments. This study comprehensively characterizes the metabolic response of grape berries exposed to water deficit at different developmental stages. Increases of proline, branched-chain amino acids, phenylpropanoids, anthocyanins, and free volatile organic compounds have been previously observed in grape berries exposed to water deficit. Integrating RNA-sequencing analysis of the transcriptome with large-scale analysis of central and specialized metabolites, we reveal that these increases occur via a coordinated regulation of key structural pathway genes. Water deficit-induced up-regulation of flavonoid genes is also coordinated with the down-regulation of many stilbene synthases and a consistent decrease in stilbenoid concentration. Water deficit activated both ABA-dependent and ABA-independent signal transduction pathways by modulating the expression of several transcription factors. Gene-gene and gene-metabolite network analyses showed that water deficit-responsive transcription factors such as bZIPs, AP2/ERFs, MYBs, and NACs are implicated in the regulation of stress-responsive metabolites. Enrichment of known and novel cis-regulatory elements in the promoters of several ripening-specific/water deficit-induced modules further affirms the involvement of a transcription factor cross-talk in the berry response to water deficit. Together, our integrated approaches show that water deficit-regulated gene modules are strongly linked to key fruit-quality metabolites and multiple signal transduction pathways may be critical to achieve a balance between the regulation of the stress-response and the berry ripening program. This study constitutes an invaluable resource for future discoveries and comparative studies, in grapes and other fruits, centered on reproductive tissue metabolism under abiotic stress.

Grape Metabolic Response to Postveraison Water Deficit Is Affected by Interseason Weather Variability

Postveraison water deficit is a common strategy implemented to improve fruit composition in many wine-growing regions. However, contrasting results are often reported on fruit size and composition, a challenge for generalizing the positive impact of this technique. Our research investigated the effect of water deficit (WD) imposed at veraison on Merlot grapevines, during two experimental seasons (2014-2015). In both years WD resulted in reduced carbon assimilation rates and leaf shedding. However, the treatment effect on the analyzed berry parameters varied between seasons. Modification of skin metabolites was more evident in 2015 than in 2014, despite the similar soil water content and water stress physiological parameters (gas exchange, water potential) recorded in the two experimental years. Higher solar radiation and air temperature in 2015 than in 2014 hint for the involvement of atmospheric parameters in fulfilling the potential effect of WD. Our results suggest that the interaction between water availability and weather conditions plays a crucial role in modulating the grape berry composition.