Genotyping of Vitis vinifera L. within the Slovak national collection of genetic resources

Microsatellites were used as a very effective tool for genetic diversity analysis and characterization of 51 grapevine (Vitis vinifera L.) accessions from the national collection of genetic resources. Genetic diversity was relatively high, 8.91 alleles were detected per analysed microsatellite locus in average, and fifty-one accessions were distinguished into 45 groups. Distribution of recent Slovak cultivars across the dendrogram accented both their genetic diversity and the effectiveness of the national breeding program in maintaining genetic diversity and generating new genetic variants. Each cultivar was different from the others and twelve of them contained 77.6% of the total genetic diversity of the whole analysed set. Microsatellite patterns were also able to confirm parentage in selected Slovak cultivars. An unusual phenomenon of triallelism was also detected in one of the analysed accessions. The present study has initiated molecular characterization within the national grapevine genetic resource collection and their comparison with well-established international cultivars.     

Relative Susceptibility of Vitis vinifera Cultivars to Vector-Borne Xylella fastidiosa through Time

Understanding the interactions between pathogen, crop and vector are necessary for the development of disease control practices of vector-borne pathogens. For instance, resistant plant genotypes can help constrain disease symptoms due to infections and limit pathogen spread by vectors. On the other hand, genotypes susceptible to infection may increase pathogen spread owing to their greater pathogen quantity, regardless of their symptom status. In this study, we evaluated under greenhouse conditions the relative levels of resistance (i.e. relatively lower pathogen quantity) versus tolerance (i.e. less symptom severity) of 10 commercial grapevine (Vitis vinifera) cultivars to Pierce’s disease etiological agent, the bacterium Xylella fastidiosa. Overall, no correlation was detected between pathogen quantity and disease severity, indicating the existence of among-cultivar variation in plant response to infection. Thompson Seedless and Barbera were the two most susceptible among 10 evaluated cultivars. Rubired showed the least severe disease symptoms and was categorized as one of the most resistant genotypes in this study. However, within each cultivar the degree of resistance/tolerance was not consistent across sampling dates. These cultivar and temporal differences in susceptibility to infection may have important consequences for disease epidemiology and the effectiveness of management protocols.     

Genetic diversity and parentage of Tunisian wild and cultivated grapevines (Vitis vinifera L.) as revealed by single nucleotide polymorphism (SNP) markers

Based on 261 single nucleotide polymorphism (SNP) markers, we analyzed 57 grapevine genotypes, consisting of 29 wild grapevines (Vitis vinifera subsp. sylvestris) prospected from the northwest part of Tunisia and 28 cultivated accessions (V. vinifera subsp. vinifera) maintained in the repository of the Arid Land Institute of Medenine (Tunisia). Pair-wise multilocus comparison with the ICVV SNP database allowed the identification of 13 cultivated genotypes, including ten synonymous groups with known Mediterranean or international varieties, three cases of color sports, and two misnomers. Genotypic analysis showed a high level of genetic diversity for both wild and cultivated groups. Multivariate and structure analyses clearly differentiated wild from cultivated grapevines and showed high average posterior probabilities of assignment to their group of origin. The clustering results largely supported the perceived classification and reflect that most of the present Tunisian cultivated varieties do not derive directly from the local wild populations but could correspond to materials introduced from different locations during historical times. Parentage analysis allowed the determination of the genetic origin of four Tunisian cultivars, “Garai”, “Jerbi” (from Kerkennah), “Mahdoui”, and “Reine de Vignes faux”, and showed that “Heptakilo” and “Planta Fina”, two old and widely distributed varieties in the Mediterranean basin, had an important role in the origin of Tunisian grapevines. The present study demonstrates the efficacy of SNP makers for germplasm characterization and genetic studies in grapevine.     

Unravelling genetic diversity and cultivar parentage in the Danish apple gene bank collection

Characterization of apple germplasm is important for conservation management and breeding strategies. A set of 448 Malus domestica accessions, primarily of local Danish origin, were genotyped using 15 microsatellite markers. Ploidy levels were determined by flow cytometry. Special emphasis was given to pedigree reconstruction, cultivar fingerprinting and genetic clustering. A reference set of cultivars, mostly from other European countries, together with a private nursery collection and a small set of Malus sieversii, Malus sylvestris and small-fruited, ornamental Malus cultivars, was also included. The microsatellite markers amplified 17–30 alleles per loci with an average degree of heterozygosity at 0.78. We identified 104 (23%) duplicate genotypes including colour sports. We could infer first-degree relationships for many cultivars with previously unknown parentages. STRUCTURE analysis provided no evidence for a genetic structure but allowed us to present a putative genetic assembly that was consistent with both PCA analysis and parental affiliation. The Danish cultivar collection contains 10% duplicate genotypes including colour sports and 22% triploids. Many unique accessions and considerable genetic diversity make the collection a valuable resource within the European apple germplasm. The findings presented shed new light on the origin of Danish apple cultivars. The fingerprints can be used for cultivar identification and future management of apple genetic resources. In addition, future genome-wide association studies and breeding programmes may benefit from the findings concerning genetic clustering and diversity of cultivars.     

Relationships among durum wheat accessions. II. A comparison of molecular and pedigree information.

The study of direct ancestry relationships provides information with which to determine essential derivation. SSR profiles were used to determine the pattern of relatedness among 134 durum wheat accessions, representing the most important modern durum wheat gene pools. Simple sequence repeat (SSR)- and amplified fragment length polymorphism (AFLP)-based genetic similarities among cultivars with accurate pedigrees were compared with pedigree-based coefficients of parentage. Sizeable departures of molecular similarities from the expected ones were observed, indicating the unreliability of inferring the pattern of genetic relatedness from the coefficient of parentage. Case studies consisting of parent-progeny cultivar trios and pairs, identified on the basis of their registered pedigree, were studied to evaluate the probability of ancestry of each progeny cultivar, compared with all the remaining accessions. Rare alleles and haplotype sharing were also explored. When the results did not agree with the registered parentages, SSR markers provided information with which to identify the most probable parents (or the corresponding "breeding lineages") in the collection.     

High throughput analysis of grape genetic diversity as a tool for germplasm collection management

Using 20 SSR markers well scattered across the 19 grape chromosomes, we analyzed 4,370 accessions of the INRA grape repository at Vassal, mostly cultivars of Vitis vinifera subsp. sativa (3,727), but also accessions of V. vinifera subsp. sylvestris (80), interspecific hybrids (364), and rootstocks (199). The analysis revealed 2,836 SSR single profiles: 2,323 sativa cultivars, 72 wild individuals (sylvestris), 306 interspecific hybrids, and 135 rootstocks, corresponding to 2,739 different cultivars in all. A total of 524 alleles were detected, with a mean of 26.20 alleles per locus. For the 2,323 cultivars of V. vinifera, 338 alleles were detected with a mean of 16.9 alleles per locus. The mean genetic diversity (GDI) was 0.797 and the level of heterozygosity was 0.76, with broad variation from 0.20 to 1. Interspecific hybrids and rootstocks were more heterozygous and more diverse (GDI?=?0.839 and 0.865, respectively) than V. vinifera cultivars (GDI?=?0.769), Vitis vinifera subsp. sylvestris being the least divergent with GDI?=?0.708. Principal coordinates analysis distinguished the four groups. Slight clonal polymorphism was detected. The limit between clonal variation and cultivar polymorphism was set at four allelic differences out of 40. SSR markers were useful as a complementary tool to traditional ampelography for cultivar identification. Finally, a set of nine SSR markers was defined that was sufficient to distinguish 99.8% of the analyzed accessions. This set is suitable for routine characterization and will be valuable for germplasm management.     

Genomic analysis of Grapevine Retrotransposon 1 (Gret1) in Vitis vinifera

The complete sequence of the first retrotransposon isolated in Vitis vinifera, Gret1, was used to design primers that permitted its analysis in the genome of grapevine cultivars. This retroelement was found to be dispersed throughout the genome with sites of repeated insertions. Fluorescent in situ hybridization indicated multiple Gret1 loci distributed throughout euchromatic portions of chromosomes. REMAP and IRAP proved to be useful as molecular markers in grapevine. Both of these techniques showed polymorphisms between cultivars but not between clones of the same cultivar, indicating differences in Gret1 distribution between cultivars. The combined cytological and molecular results suggest that Gret1 may have a role in gene regulation and in explaining the enormous phenotypic variability that exists between cultivars.     

Genomics Assisted Ancestry Deconvolution in Grape

The genus Vitis (the grapevine) is a group of highly diverse, diploid woody perennial vines consisting of approximately 60 species from across the northern hemisphere. It is the world’s most valuable horticultural crop with ~8 million hectares planted, most of which is processed into wine. To gain insights into the use of wild Vitis species during the past century of interspecific grape breeding and to provide a foundation for marker-assisted breeding programmes, we present a principal components analysis (PCA) based ancestry estimation method to calculate admixture proportions of hybrid grapes in the United States Department of Agriculture grape germplasm collection using genome-wide polymorphism data. We find that grape breeders have backcrossed to both the domesticated V. vinifera and wild Vitis species and that reasonably accurate genome-wide ancestry estimation can be performed on interspecific Vitis hybrids using a panel of fewer than 50 ancestry informative markers (AIMs). We compare measures of ancestry informativeness used in selecting SNP panels for two-way admixture estimation, and verify the accuracy of our method on simulated populations of admixed offspring. Our method of ancestry deconvolution provides a first step towards selection at the seed or seedling stage for desirable admixture profiles, which will facilitate marker-assisted breeding that aims to introgress traits from wild Vitis species while retaining the desirable characteristics of elite V. vinifera cultivars.     

Genetic analysis of the cultivated potato Solanum tuberosum L. Phureja Group using RAPDs and nuclear SSRs

The Solanum tuberosum L. Phureja Group consists of potato landraces widely grown in the Andes from western Venezuela to central Bolivia, and forms an important breeding stock due to their excellent culinary properties and other traits for developing modern varieties. They have been distinguished by short-day adaptation, diploid ploidy (2n = 2x = 24), and lack of tuber dormancy. This nuclear simple sequence repeat (nSSR or microsatellite) study complements a prior random amplified polymorphic DNA (RAPD) study to explore the use of these markers to form a core collection of cultivar groups of potatoes. Like this prior RAPD study, we analyzed 128 accessions of the Phureja Group using nuclear microsatellites (nSSR). Twenty-six of the 128 accessions were invariant for 22 nSSR markers assayed. The nSSR data uncovered 25 unexpected triploid and tetraploid accessions. Chromosome counts of the 102 accessions confirmed these nSSR results and highlighted seven more triploids or tetraploids. Thus, these nSSR markers (except 1) are good indicators of ploidy for diploid potatoes in 92% of the cases. The nSSR and RAPD results: (1) were highly discordant for the remaining 70 accessions that were diploid and variable in nSSR, (2) show the utility of nSSRs to effectively uncover many ploidy variants in cultivated potato, (3) support the use of a cultivar-group (rather than a species) classification of cultivated potato, (4) fail to support a relationship between genetic distance and geographic distance, (5) question the use of any single type of molecular marker to construct core collections.     

Genetic diversity of wild grapevine populations in Spain and their genetic relationships with cultivated grapevines

The wild grapevine, Vitis vinifera L. ssp. sylvestris (Gmelin) Hegi, considered as the ancestor of the cultivated grapevine, is native from Eurasia. In Spain, natural populations of V. vinifera ssp. sylvestris can still be found along river banks. In this work, we have performed a wide search of wild grapevine populations in Spain and characterized the amount and distribution of their genetic diversity using 25 nuclear SSR loci. We have also analysed the possible coexistence in the natural habitat of wild grapevines with naturalized grapevine cultivars and rootstocks. In this way, phenotypic and genetic analyses identified 19% of the collected samples as derived from cultivated genotypes, being either naturalized cultivars or hybrid genotypes derived from spontaneous crosses between wild and cultivated grapevines. The genetic diversity of wild grapevine populations was similar than that observed in the cultivated group. The molecular analysis showed that cultivated germplasm and wild germplasm are genetically divergent with low level of introgression. Using a model‐based approach implemented in the software structure , we identified four genetic groups, with two of them fundamentally represented among cultivated genotypes and two among wild accessions. The analyses of genetic relationships between wild and cultivated grapevines could suggest a genetic contribution of wild accessions from Spain to current Western cultivars.     

Chloroplast Genome Diversity in Portuguese Grapevine (Vitis vinifera L.) Cultivars

Grapevine chloroplast (cp) DNA diversity was analysed for the first time through amplification and digestion of fragments of the large single copy (LSC) region by polymerase chain reaction–restriction fragment length polymorphism methodology and also by amplification of three microsatellite loci, previously described as polymorphic in grapevine. Thirty-eight grapevine cultivars collected mainly in the North of Portugal, including some neglected cultivars, four international cultivars (Chasselas, Muscat of Alexandria, Muscat of Hamburg and Pinot) and Vitis riparia and Vitis rupestris, were used in this study with the main goal of finding out their cpDNA diversity and compare the obtained results with previously published data on cultivars from other regions to ascertain their possible origin. Two different alleles were found in each of the three cpSSR loci. Allele variants of the three loci combined in a total of three different haplotypes (A, B and D). The most frequent haplotype, A, was previously reported as the most frequent in Iberian Peninsula and Occidental Europe. Haplotype B was unique to Rabigato, Muscat of Alexandria, V. riparia and V. rupestris. This haplotype was previously proposed to be an ancestral haplotype. Twenty-seven fragments of the LSC region of Vitis vinifera cpDNA were amplified and then digested with HinfI and TaqI restriction enzymes. Polymorphisms were found in the trnT-psbC (TC) and orf184-petA (OA) fragments. In the TC fragment, the polymorphism corresponds to a point mutation in a restriction site of TaqI and is only present in all cultivars with cpSSR haplotype D. In the OA fragment, a short deletion exclusive to the Rabigato cultivar was found. In this case, one sequence tagged site-based marker was developed and will be very useful in future phylogenetic and fingerprinting studies in a broader number of cultivars and in wild grapevine populations. Inference about the progenitors of the Touriga Franca cultivar is done. The present work supports and completes its origin as a descendent of the female and male parents, Marufo and Touriga Nacional.     

Microsatellite repeats in grapevine reveal DNA polymorphisms when analysed as sequence-tagged sites (STSs)

Microsatellite repeat sequences were investigated as sequenced-tagged site (STS) DNA markers to determine the potential for genetic analysis of the grapevine genome. The PCR-generated markers detect codominant alleles at a single locus or site in the genome. The marker type is very informative detecting high heterozygosity (69%–88%) within individual grapevine cultivars and high genetic variation between cultivars, making it a useful marker type for plant genome mapping and genome typing. For five loci a screening of 26 V. vinifera cultivars found 13, 12, 8, 5, and 4 different length alleles respectively with some alleles more common than others. The genomic DNA sequences surrounding microsatellite sequences were conserved within the genus permitting STS primers to amplify STSs from other Vitis species. These Vitis species were found to have some unique alleles not present in V. vinifera.     

Inter- and Intra-Varietal Genetic Variability in Malvasia Cultivars

The DNA molecular analyses together with ampelography, ampelometry, and biochemistry are essential for grapevine identification and investigation of genetic differences among the Vitis vinifera L. cultivars and clones. Ten Malvasia cultivars (i.e., Istrian Malvasia; M. delle Lipari; M. bianca di Candia; M. di Candia Aromatica; M. del Lazio; M. bianca lunga, also known as Malvasia del Chianti; M. nera di Brindisi/Lecce; M. di Casorzo; M. di Schierano, and M. nera di Bolzano) were analyzed using molecular approaches to study the genetic inter-varietal variability. Thirty Istrian Malvasia genotypes (i.e., 8 Italian clones, such as ISV 1, ISV F6, VCR 4, VCR 113, VCR 114, VCR 115, ERSA 120, ERSA 121, and 22 autochthonous grapevine accessions grown in Istrian Peninsula, Croatia) were investigated to evaluate the morphological and genetic intra-varietal variability. DNA analysis allowed discrimination of all Malvasia genotypes at molecular level using AFLP, SAMPL, and M-AFLP markers. Italian clones and autochthonous Croatian accessions of Istrian Malvasia were grouped according to their different geographic origins. These results showed the great genetic variability of Malvasia genotypes suggesting the need for the preservation of autochthonous grapevine biotypes found on different areas to approve the correct choice and selection of the grape multiplication materials.     

Dimorphism in inflorescence scent of dioecious wild grapevine

Wild grapevine (Vitis vinifera subsp. sylvestris) is the dioecious ancestral form of grapevine, from which the domesticated cultivars have derived (V. vinifera subsp. vinifera). Little is known about the floral scent compounds of wild grapevine that is considered as being partly insect pollinated. The knowledge of volatiles released by male and female inflorescence may contribute to the understanding of the pollination biology of this endangered taxon. Inflorescence scents of male and female individuals were collected by dynamic headspace and analysed by thermal desorption-GC/MS. A total of 17 compounds of C5-branched chain alcohols, aliphatics, aromatics, and terpenoids were identified with benzyl alcohol being most abundant in both sexes. Eight of the compounds were sex-specific and differences in 1,2-dimethoxybenzene were most obvious. This aromatic compound was a main constituent in the scent of females (30%), but it did not occur in males. Some of the main compounds of the scent samples are known to be detected by beetles (Cerambycidae) or attract sweat bees (Halictidae) and honey bees (A. mellifera), all well-known inflorescence visitors in wild grapevine. The data presented here are an important step in understanding the chemical communication between wild grapevine and its inflorescence visitors/potential pollinators.     

Microsatellite variability in grapevine cultivars from different European regions and evaluation of assignment testing to assess the geographic origin of cultivars

Nine microsatellite markers (VVMD5, VVMD7, VVS2, ssrVrZAG21, ssrVrZAG47, ssrVrZAG62, ssrVrZAG64, ssrVrZAG79 and ssrVrZAG83) were chosen for the analysis of marker information content, the genetic structure of grapevine cultivar gene pools, and differentiation among grapevines sampled from seven European vine-growing regions (Greece, Croatia, North Italy, Austria and Germany, France, Spain and Portugal). The markers were found to be highly informative in all cultivar groups and therefore constitute a useful set for the genetic characterization of European grapevines. Similar and high levels of genetic variability were detected in all investigated grapevine gene pools. Genetic differentiation among cultivars from different regions was significant, even in the case of adjacent groups such as the Spanish and Portuguese cultivars. No genetic differentiation could be detected between vines with blue and white grapes, indicating that they have undergone the processes of cultivar development jointly. The observed genetic differentiation among vine-growing regions suggested that cultivars could possibly be assigned to their regions of origin according to their genotypes. This might allow one to determine the geographical origin of cultivars with an unknown background. The assignment procedure proved to work for cultivars from the higher differentiated regions, as for example from Austria and Portugal. Received: 10 April 1999 / Accepted: 25 May 1999     

A reference genetic map of Muscadinia rotundifolia and identification of Ren5, a new major locus for resistance to grapevine powdery mildew

Muscadinia rotundifolia, a species closely related to cultivated grapevine Vitis vinifera, is a major source of resistance to grapevine downy and powdery mildew, two major threats to cultivated traditional cultivars of V. vinifera respectively caused by the oomycete Plasmopara viticola and the ascomycete Erisyphe necator. The aim of the present work was to develop a reference genetic linkage map based on simple sequence repeat (SSR) markers for M. rotundifolia. This map was created using S1 M. rotundifolia cv. Regale progeny, and covers 948?cM on 20 linkage groups, which corresponds to the expected chromosome number for muscadine. The comparison of the genetic maps of V. vinifera and M. rotundifolia revealed a high macrosynteny between the genomes of both species. The S1 progeny was used to assess the general level of resistance of M. rotundifolia to P. viticola and E. necator, by scoring different parameters of pathogen development. A quantitative trait locus (QTL) analysis allowed us to highlight a major QTL on linkage group 14 controlling resistance to powdery mildew, which explained up to 58?% of the total phenotypic variance. This QTL was named ‘Resistance to Erysiphe Necator 5’ (Ren5). A microscopic evaluation E. necator mycelium development on resistant and susceptible genotypes of the S1 progeny showed that Ren5 exerts its action after the formation of the first appressorium, and acts by delaying, and then stopping, mycelium development.     

Identification of grapevine clone genotypes by use of microsatellite markers

An SSR-analysis of rootstock, technical, and table grapevine cultivar clones was performed. The allelic characteristics of grapevine cultivar clones were obtained at microsatellite loci; this characteristic can be used to identify and sertificate grapevine clone genotypes. A high level of mutation variability among the rootstock and technical cultivars was discovered. DNA passports for prospective clones were created. The genotyping results can be used for the registration of clones and the protection of breeders’ rights.     

AFLP analysis of genetic relationships among aromatic grapevines (<Emphasis Type="Italic">Vitis vinifera</Emphasis>)

Genotypic diversity has been detected among aromatic grapevines (Vitis vinifera) by molecular markers (AFLPs). The 22 primer-pairs generated a total of 1,331 bands of which 564 (40%) were polymorphic over all the genotypes. The bootstrap analysis pointed out that a large number of polymorphic bands (200–400) has to be used for a better estimation of the genetic distances among genotypes; 383 polymorphic AFLP bands were used for the cluster and the principal coordinate analyses because they did not present missing data across all the genotypes. The cluster analysis (UPGMA), based on polymorphic AFLP markers, revealed no relationship between the Moscato and Malvasia grapevines. The Malvasias, unlike the Moscatos distinguished by their distinct muscat aroma, have to be considered a more complex group because it includes muscat and non-muscat grapevines. The principal coordinate analysis (PCO) confirmed the pattern of the cluster analysis only for those varieties which presented a low coefficient of dissimilarity, while for the other varieties there was no correspondence between the two analyses. The pattern of aggregation among aromatic grapevines in the cluster and principal coordinate analyses does not support any classification that might include an aromatic grapevine group in V. vinifera. Even though some synonyms and homonyms are present among aromatic grapevines (V. vinifera), genetic diversity exists among genotypes in AFLP markers.Communicated by H.F. Linskens     

Mapping of crown gall resistance locus Rcg1 in grapevine

Agrobacteria are efficient plant pathogens. They are able to transform plant cells genetically resulting in abnormal cell proliferation. Cultivars of Vitis vinifera are highly susceptible to many virulent Agrobacterium strains but certain wild Vitis species, including Vitis amurensis have resistant genotypes. Studies of the molecular background of such natural resistance are of special importance, not only for practical benefits in agricultural practice but also for understanding the role of plant genes in the transformation process. Earlier, crown gall resistance from V. amurensis was introgressed into V. vinifera through interspecific breeding and it was shown to be inherited as a single and dominant Mendelian trait. To develop this research further, towards understanding underlying molecular mechanisms, a mapping population was established, and resistance-coupled molecular DNA markers were identified by three different approaches. First, RAPD makers linked to the resistance locus (Rcg1) were identified, and on the basis of their DNA sequences, we developed resistance-coupled SCAR markers. However, localization of these markers in the grapevine genome sequence failed due to their similarity to many repetitive regions. Next, using SSR markers of the grapevine reference linkage map, location of the resistance locus was established on linkage group 15 (LG15). Finally, this position was supported further by developing new chromosome-specific markers and by the construction of the genetic map of the region including nine loci in 29.1?cM. Our results show that the closest marker is located 3.3?cM from the Rcg1 locus that may correspond to 576?kb.