Repetitive DNA of grapevine: classes present and sequences suitable for cultivar identification

Summary Repetitive DNA sequences present in the grapevine genome were investigated as probes for distinguishing species and cultivars. Microsatellite sequences, minisatellite sequences, tandemly arrayed genes and highly repetitive grapevine sequences were studied. The relative abundance of microsatellite and minisatellite DNA in the genome varied with the repeat sequence and determined their usefulness in detecting RFLPs. Cloned Vitis ribosomal repeat units were characterised and showed length heterogeneity (9.14–12.15 kb) between and within species. A highly repetitive DNA sequence isolated from V. vinifera was found to be specific only to those species classified as Euvitis. DNA polymorphisms were found between Vitis species and between cultivars of V. vinifera with all classes of repeat DNA sequences studied. DNA sequences suitable for DNA fingerprinting gave genotype-specific patterns for all of the cultivars and species examined. The DNA polymorphisms detected indicates a moderate to high level of heterozygosity in grapevine cultivars.On leave from the Biochemical Research Institute, Nippon Menard Cosmetic Co, Ltd, Ogaki Gifuken, 503 Japan     

Nuclear DNA content of Vitis species,cultivars, and other genera of the Vitaceae

The nuclear DNA content was analyzed in Vitis species, hybrid cultivars, and genera of the Vitaceae using flow cytometry. Significant variation was found among Vitis species, hybrids, and other genera of the Vitaceae (Ampelopsis and Parthenocissus). DNA content was estimated to range from 0.98 to 1.05 pg/2C within V. labrusca (ns) and 0.86 to 1.00 pg/2C within V. vinifera (ns). Genotypes from Vitis and Parthenocissus were similar in nuclear DNA content (approximately 1.00 pg/2C) whereas they differed significantly from Ampelopsis (1.39 pg/2C). No correlation between DNA content and the center of origin of genotypes of the Vitaceae was noted. Based on the present study, the Vitis genome size is 475 Mbp, 96% of which is non-coding. Knowledge of DNA content is useful in order to understand the complexity of the Vitis genome and to establish a relationship between the genetic and physical map for map-based cloning.     

Characterization of sequence polymorphisms from microsatellite flanking regions in <Emphasis Type="Italic">Vitis</Emphasis> spp

Single nucleotide polymorphisms or SNPs are the most abundant form of genetic variation in the genome of plants and animals. Microsatellites are hypervariable regions of genome, while their flanking regions are assumed to be as conserved as the average of the genome. In the present study, flanking sequences of 10 microsatellite loci were compared in different cultivars of Vitis to determine the existing polymorphism. For every microsatellite, about 8 homozygous cultivars (regarding the microsatellite genotype) were chosen for sequencing. A total of 45 different varieties of Vitis and 91 sequences were analysed. Sequence polymorphisms were detected for all the microsatellite flanking regions studied, including single nucleotide polymorphisms (SNPs), insertions and deletions. The number of identified changes varied considerably among the loci with a frequency of one polymorphism every 41 nucleotides, being VVMD5 the most polymorphic one. A number of SNPs were used to design SNP markers, which were scored by dideoxy single base primer extension and capillary electrophoresis methodology. These SNP markers were employed to genotype 21 cultivars of Vitis vinifera and 4 varieties of other Vitis species. The utility of the markers developed as well as their utility for varietal identification and pedigree studies is discussed, using a similar study carried out with the 10 microsatellites as a reference.     

Variation in recombination rates across Vitis species

Recombination rate data are presented for three populations of grape based on framework genetic linkage maps developed with simple-sequence repeat markers. These linkage maps were constructed from different Vitis species and represent three genetic backgrounds. The first population is pure Vitis vinifera, derived from a cross of the European cultivars Riesling and Cabernet Sauvignon. The second is an interspecific cross between two commercially used rootstock cultivars of different North American Vitis species parentage, Ramsey (Vitis champinii) and Riparia Gloire (Vitis riparia). The third population, D8909-15 (Vitis rupestris × (Vitis arizonica/Vitis girdiana form)) × F8909-17 (V. rupestris × (V. arizonica/Vitis candicans form)), is an F1 from two half-sibs. Genome-wide and chromosome-wide recombination rates varied across the three populations and among the six Vitis parents. Global recombination rates in the parents of the third F1 population, with a complex Vitis background, were significantly reduced. In the first and third populations, the recombination rate was significantly greater in the male parent. Specific genome locations with frequent heterogeneity in recombination were identified, suggesting that recombination rates are not equal across the Vitis genome. The identification of regions with suppressed or high recombination will aid grape breeders and geneticists who rely on recombination events to introgress disease resistance genes from the genomes of wild Vitis species, develop fine-scale genetic maps, and clone disease resistance genes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Characterization of new microsatellite loci from Vitis vinifera and their conservation in some Vitis species and hybrids

We present here characterization data for seven new microsatellite markers designed from new microsatellite loci isolated from a microsatellite‐enriched DNA library from Vitis vinifera. The observed heterozygosity varied from 0.73 up to 0.93 and the number of alleles per locus ranged from 12 to 26. This high polymorphism makes these new markers interesting for use in genotyping studies and completing the set of microsatellite markers already available for V. vinifera. Additionally these seven new markers appear to be conserved in four other Vitis species and 15 Vitis hybrids used as rootstocks for V. vinifera cultivation.     

Nuclear DNA content of Vitis vinifera cultivars and ploidy level analyses of somatic embryo-derived plants obtained from anther culture

Flow cytometry was employed to determine the ploidy level of Vitis vinifera L. somatic embryo-derived plants obtained from anther culture. Only one among the 41 analysed plants (2.4%) presented somaclonal variation (tetraploidy); the other plants were diploid. No significant differences (P≤0.05) were detected between diploid and parental field plants. No haploid or aneuploid plants were observed. The nuclear DNA content of nine V. vinifera cultivars was also estimated using flow cytometry. A non-significant variation was found among the cultivars, with DNA content ranging from 1.17 pg/2C (cv. ‘Tinta Barroca’ and ‘Viosinho’) to 1.26 pg/2C (cv. ‘Cabernet Sauvignon’). These results and previous studies on other Vitis species suggest that Vitis genome is stable with regard to nuclear DNA content.     

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.     

Retrotransposon-based molecular markers for grapevine species and cultivars identification

Insertional polymorphisms of two copia-like (Vine-1, Tvv1) and one gypsy-like (Gret1) retrotransposon found in the grapevine genome were studied in 29 Vitis genotypes (Vitis arizonica, Vitis cinerea, Vitis labrusca, Vitis rupestis, Vitis rotundifolia, Vitis vinifera subsp. sylvestris and 23 V. vinifera subsp. sativa) using inter-retrotransposon amplified polymorphism (IRAP), retrotransposon-microsatellite amplified polymorphism (REMAP) and sequence-specific amplified polymorphism (SSAP) techniques. IRAP, REMAP and SSAP polymorphisms were compared with amplified fragment length polymorphism (AFLP), Inter-single sequence repeats (ISSR) and SSR polymorphisms by evaluating the information content, the number of loci simultaneously analysed per experiment, the effectiveness of the analyses in assessing the relationship between accessions and the number of loci needed to obtain a coefficient of variation of 10%. The UPGMA dendrograms of each molecular marker system were compared and the Mantel matrix correspondence test was applied. Furthermore, the corresponding insertion ages of the transposable elements were estimated for each retrotransposon subfamily analysed. The presence of Gret1, Tvv1 and Vine-1 retrotransposons in all analysed genotypes suggests that copia-like and gypsy-like retrotransposons are widespread in Vitis genus. The results indicate that these retrotransposons were active before Vitis speciation and contributed to Vitis genus evolution. IRAP, REMAP and SSAP markers allow the discrimination of Vitis species and V. vinifera subsp. sativa cultivars with certainty as has been shown with AFLP, ISSR and SSR analyses, but phylogenetic trees obtained by retrotransposon-based molecular markers polymorphisms show some significant differences in the allocation of the analysed accessions compare to those obtained by ISSR, AFLP and SSR molecular markers. The phylogenetic tree resulting from REMAP polymorphism appeared the most representative of the effective relationship between all analysed accessions.     

Candidate genes within a 143 kb region of the flower sex locus in <Emphasis Type="Italic">Vitis</Emphasis>

Wild Vitis species are dioecious plants, while the cultivated counterpart, Vitis vinifera subspec. vinifera, generally shows hermaphroditic flowers. In Vitis the genetic determinants of flower sex have previously been mapped to a region on chromosome 2. In a combined strategy of map-based cloning and the use of the publicly available grapevine reference genome sequence, the structure of the grapevine flower sex locus has been elucidated with the subsequent identification of candidate genes which might be involved in the development of the different flower sex types. In a fine mapping approach, the sex locus in grapevine was narrowed down using a population derived from a cross of a genotype with a Vitis vinifera background (‘Schiava Grossa’ × ‘Riesling’) with the male rootstock cv. ‘Börner’ (V. riparia × V. cinerea). A physical map of 143 kb was established from BAC clones spanning the 0.5 cM region defined by the closest flanking recombination break points. Sequencing and gene annotation of the entire region revealed several candidate genes with a potential impact on flower sex formation. One of the presumed candidate genes, an adenine phosphoribosyltransferase, was analysed in more detail. The results led to the development of a marker for the presence or absence of the female alleles, while the male and hermaphroditic alleles are still to be differentiated. The impact of other candidate genes is discussed, especially with regard to plant hormone actions. The markers developed will permit the selection of female breeding lines which do not require laborious emasculation thus considerably simplifying grapevine breeding. The genetic finger prints displayed that our cultivated grapevines frequently carry a female allele while homozygous hermaphrodites are rare.     

Genetic variation and biogeography of the disjunct Vitis subg. Vitis (Vitaceae)

Aim Vitis subg. Vitis provides an example of a plant disjunction occurring in the Northern Hemisphere. It shows broad morphological variation but is assumed to be a species complex with limited genetic differentiation. Based on a comprehensive sampling of taxa and polymorphism in both chloroplast and nuclear DNA, we assessed genetic variation within this subgenus. Our aims were to clarify the relationships among species and to examine their historical biogeography. Location Asia, Europe, North America. Methods We analysed a total of 30 species and putative hybrids from subgenus Vitis and examined the infra‐specific variation in some species. Polymorphism in chloroplast DNA was assessed in trnL and trnH–psbA–trnK sequences (c. 2170 bp) and in 15 microsatellite loci. We also obtained nuclear data for size variation at 24 microsatellite loci. Phylogenetic inference was performed with Bayesian analyses. A maximum parsimony network was constructed to depict the evolutionary relationships among haplotypes, and microsatellite data were also subjected to hierarchical clustering analysis using the Ward distance. In addition, we assessed size homoplasy by sequencing both chloroplast and nuclear microsatellite loci. Results Chloroplast polymorphisms resolved subgenus Vitis as a monophyletic group with limited genetic variation. The ancestral haplotypes were found in Eurasia. American taxa all harboured derived haplotypes. Most of them formed a monophyletic group that did not include Vitis californica. The four main haplotypes in Vitis vinifera corresponded to two different origins. Nuclear microsatellites indicated that genetic variation was especially large in North America. Asian species exhibited a lower level of nuclear divergence and the European V. vinifera corresponded to a differentiated nuclear lineage. Main conclusions We obtained some evidence that subgenus Vitis has an Asian origin and then dispersed to Europe and North America. Geographic separation was followed by diversification, presumably during the Pleistocene, resulting in phylogeographic patterns similar to other biota. In contrast to chloroplast DNA, nuclear DNA shows a larger than expected genetic variation. Our molecular data also highlight the need to re‐examine certain aspects of the current subgeneric classification.     

DNA typing of grapevines: A universal methodology and database for describing cultivars and evaluating genetic relatedness

With established ampelographic techniques for grapevine identification it is often difficult to achieve a satisfactory, objective result. We have developed a DNA typing system using sequence-tagged microsatellite site markers as a means of differentiating cultivars of grapevine. A semi-automated analysis procedure was linked to an electronic database and found to be an objective and reliable system for cultivar identification using this simple marker type. The accumulated DNA typing data from over eighty cultivars demonstrated that cultivars that are difficult to differentiate phenotypically using ampelographic techniques can be distinguished by DNA typing. Parentage analysis uncovered errors in parent assignment of cultivar identification in specific cases. The electronic database has a conservative format to take into account the occurrence of null alleles and the possibility of missed alleles. Computer-assisted comparisons of cultivars in the database can be performed and various approaches for estimating the match probability that two unrelated cultivars have the same genotype simply due to chance are discused. We suggest that further development of the database through international co-operation using standardised sequence-tagged site markers offers the possibility of achieving a universal grapevine identification system.     

Multiple loss-of-function 5-O-glucosyltransferase alleles revealed in Vitis vinifera,but not in other Vitis species

Key message

Wild and loss-of-function alleles of the 5 - O - glucosyltransferase gene responsible for synthesis of diglucoside anthocyanins in Vitis were characterized. The information aids marker development for tracking this gene in grape breeding.

Abstract

Anthocyanins in red grapes are present in two glycosylation states: monoglucoside (3-O-glucoside) and diglucoside (3, 5-di-O-glucoside). While monoglucoside anthocyanins are present in all pigmented grapes, diglucoside anthocyanins are rarely found in the cultivated grape species Vitis vinifera. Biochemically 3-O-glucoside anthocyanins can be converted into 3,5-di-O-glucoside anthocyanins by a 5-O-glucosyltransferase. In this study, we surveyed allelic variation of the 5-O-glucosyltransferase gene (5GT) in 70 V. vinifera ssp. vinifera cultivars, 52 V. vinifera ssp. sylvestris accessions, 23 Vitis hybrid grapes, and 22 accessions of seven other Vitis species. Eighteen 5GT alleles with apparent loss-of-function mutations, including seven premature stop codon mutations and six frameshift indel mutations, were discovered in V. vinifera, but not in the other Vitis species. A total of 36 5GT alleles without apparent loss-of-function mutations (W-type) were identified. These W-type alleles were predominantly present in wild Vitis species, although a few of them were also found in some V. vinifera accessions. We further evaluated some of these 5GT alleles in producing diglucoside anthocyanins by analyzing the content of diglucoside anthocyanins in a set of representative V. vinifera cultivars. Through haplotype network analysis we revealed that V. vinifera ssp. vinifera and its wild progenitor V. vinifera ssp. sylvestris shared many loss-of-function 5GT alleles and extensive divergence of the 5GT alleles was evident within V. vinifera. This work advances our understanding of the genetic diversity of 5GT and provides a molecular basis for future marker-assisted selection for improving this important wine quality trait.     

Progress in grapevine breeding

Summary The European, or bunch grape, Vitis vinifera, is widely grown because of its high fruit quality and its capacity to grow in a wide range of climatic conditions. However, they are susceptible to fungal diseases and insect pests, especially when grown in cool, wet climates. The aim of a number of grapevine breeding programs throughout the world is to develop new varieties resistant to diseases using complex hybrids between European and American species of Vitis. Within these breeding programs it is essential to maintain heterozygosity and desirable hybrids are multiplied by asexual propagation. New approaches to grapevine improvement include the use of protoplast fusion to overcome sexual barriers, however the routine regeneration of plantlets from protoplasts and calluses is difficult. In vitro rescue of ovules from varieties with stenospermocarpic seeds shows considerable promise for breeding new seedless grapes. Eventually the use of plant transformation techniques to insert specific pieces of DNA coding for desirable genetic characteristics will provide opportunities for equipping well known grape cultivars with new characteristics.     

Construction and characterization of BAC libraries from major grapevine cultivars

Genome projects were initiated on grapevine (Vitis vinifera L., 2n=38, genome size 475 Mb) through the successful construction of four bacterial artificial chromosome (BAC) libraries from three major cultivars, Cabernet Sauvignon (Cabernet S), Syrah and two different clones of Pinot Noir (Pinot N). Depending on the library, the genome coverage represented 4.5–14.8 genome equivalents with clones having a mean insert size of 93–158 kb. BAC pools suitable for PCR screening were constructed for two of these BAC libraries [Cabernet S and Pinot N clone (cl) 115] and subsequently used to confirm the genome coverage of both libraries by PCR anchoring of 74 genetic markers sampled from the 19 linkage groups. For ten of these markers, two bands on separate BAC pools were differentiated that could correspond either to different alleles or to a duplication of the locus being studied. Finally, a preliminary assessment of the correspondence between genetic and physical distances was made through the anchoring of all the markers mapped along linkage group 1 of the V. vinifera genetic map. A pair of markers, 2.1 cM apart, anchored the same BAC clones, which allowed us to estimate that 1 cM corresponded in this particular region to a maximum length of 130 kb.     

Cryptic diversity of <Emphasis Type="Italic">Plasmopara viticola</Emphasis> (Oomycota,Peronosporaceae) in North America

Plasmopara viticola is the causal agent of grapevine downy mildew and is among the most important diseases in viticulture. It originates from North America, where it coevolved with wild Vitis species. Beginning in the 1870s it turned into a global epidemic that has been causing severe yield losses. It is generally believed that a single species is causing downy mildew on a large variety of economically important cultivars. Here we report, based on one nuclear and two mitochondrial markers, that isolates from vineyards in the United States fall into three highly distinct phylogenetic lineages. One of these contains European strains and affects Vitis vinifera cultivars, while the other two lineages affect also other species of Vitis. The divergence between these lineages is high, and, judging from the genetic variation in other Plasmopara lineages, might reflect distinct species. Due to the potentially significant implications for quarantine regulations and resistance breeding, detailed studies will be necessary to clarify whether these genetically distinct lineages occur outside of North America or are still confined there.     

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.     

A high quality draft consensus sequence of the genome of a heterozygous grapevine variety

Background

Worldwide, grapes and their derived products have a large market. The cultivated grape species Vitis vinifera has potential to become a model for fruit trees genetics. Like many plant species, it is highly heterozygous, which is an additional challenge to modern whole genome shotgun sequencing. In this paper a high quality draft genome sequence of a cultivated clone of V. vinifera Pinot Noir is presented.

Principal Findings

We estimate the genome size of V. vinifera to be 504.6 Mb. Genomic sequences corresponding to 477.1 Mb were assembled in 2,093 metacontigs and 435.1 Mb were anchored to the 19 linkage groups (LGs). The number of predicted genes is 29,585, of which 96.1% were assigned to LGs. This assembly of the grape genome provides candidate genes implicated in traits relevant to grapevine cultivation, such as those influencing wine quality, via secondary metabolites, and those connected with the extreme susceptibility of grape to pathogens. Single nucleotide polymorphism (SNP) distribution was consistent with a diffuse haplotype structure across the genome. Of around 2,000,000 SNPs, 1,751,176 were mapped to chromosomes and one or more of them were identified in 86.7% of anchored genes. The relative age of grape duplicated genes was estimated and this made possible to reveal a relatively recent Vitis-specific large scale duplication event concerning at least 10 chromosomes (duplication not reported before).

Conclusions

Sanger shotgun sequencing and highly efficient sequencing by synthesis (SBS), together with dedicated assembly programs, resolved a complex heterozygous genome. A consensus sequence of the genome and a set of mapped marker loci were generated. Homologous chromosomes of Pinot Noir differ by 11.2% of their DNA (hemizygous DNA plus chromosomal gaps). SNP markers are offered as a tool with the potential of introducing a new era in the molecular breeding of grape.     

Conservation of microsatellite loci within the genus Vitis

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. Received: 24 October 1999 / Accepted: 11 November 1999     

Construction of a reference linkage map of Vitis amurensis and genetic mapping of Rpv8, a locus conferring resistance to grapevine downy mildew

Downy mildew, caused by the oomycete Plasmopara viticola, is one of the major threats to grapevine. All traditional cultivars of grapevine (Vitis vinifera) are susceptible to downy mildew, the control of which requires regular application of fungicides. In contrast, many sources of resistance to P. viticola have been described in the Vitis wild species, among which is V. amurensis Rupr. (Vitaceae), a species originating from East Asia. A genetic linkage map of V. amurensis, based on 122 simple sequence repeat and 6 resistance gene analogue markers, was established using S1 progeny. This map covers 975?cM on 19 linkage groups, which represent 82% of the physical coverage of the V. vinifera reference genetic map. To measure the general level of resistance, the sporulation of P. viticola and the necrosis produced in response to infection, five quantitative and semi-quantitative parameters were scored 6?days post-inoculation on the S1 progeny. A quantitative trait locus (QTL) analysis allowed us to identify on linkage group 14 a major QTL controlling the resistance to downy mildew found in V. amurensis, which explained up to 86.3% of the total phenotypic variance. This QTL was named ??Resistance to Plasmopara viticola 8?? (Rpv8).     

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