An integrated SSR map of grapevine based on five mapping populations.

A grapevine (mainly Vitis vinifera L., 2n = 38) composite genetic map was constructed with CarthaGene using segregation data from five full-sib populations of 46, 95, 114, 139 and 153 individuals, to determine the relative position of a large set of molecular markers. This consensus map comprised 515 loci (502 SSRs and 13 other type PCR-based markers), amplified using 439 primer pairs (426 SSRs and 13 others) with 50.1% common markers shared by at least two crosses. Out of all loci, 257, 85, 74, 69 and 30 were mapped in 1, 2, 3, 4 and 5 individual mapping populations, respectively. Marker order was generally well conserved between maps of individual populations, with only a few significant differences in the recombination rate of marker pairs between two or more populations. The total length of the integrated map was 1,647 cM Kosambi covering 19 linkage groups, with a mean distance between neighbour loci of 3.3 cM. A framework-integrated map was also built, with marker order supported by a LOD of 2.0. It included 257 loci spanning 1,485 cM Kosambi with a mean inter-locus distance of 6.2 cM over 19 linkage groups. These integrated maps are the most comprehensive SSR-based maps available so far in grapevine and will serve either for choosing markers evenly scattered over the whole genome or for selecting markers that cover particular regions of interest. The framework map is also a useful starting point for the integration of the V. vinifera physical and genetic maps.

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).

A microsatellite marker based framework linkage map of Vitis vinifera L.

We have constructed a framework linkage map based on microsatellite markers for Vitis vinifera L., the European wine grape. The mapping population consisted of 153 progeny plants from a cross of Vitis vinifera cvs. Riesling x Cabernet Sauvignon. One hundred fifty-two microsatellite markers and one polymorphic EST marker have been mapped to 20 linkage groups (2 n=38). The map covers 1,728 cM with an average distance between markers of 11.0 cM. Estimates of genome size, expected genome coverage, and observed genome coverage were determined with 135-140 markers. Genome length estimates differed between paternal and maternal data sets. Observed approximate genome coverage was 65% versus an expected coverage of 90%. Meiotic recombination rates were not significantly different between maternal and paternal parents. This map has been adopted as a reference map for the International Grape Genome Program.

Microsatellite analysis of ancient alpine grape cultivars: pedigree reconstruction of Vitis vinifera L. 'Cornalin du Valais'.

Ancient and closely related grape cultivars from the Alps were analyzed with 50 microsatellite markers: 'Cornalin', 'Humagne Rouge' and 'Goron' from Valais (Switzerland); 'Cornalin', 'Petit Rouge' and 'Mayolet' from the Aosta Valley (Italy). Our results confirmed previous studies showing that the 'Cornalin' cultivars from Switzerland and Italy are distinct, and that 'Humagne Rouge' is identical to 'Cornalin' from the Aosta Valley. We propose the nomenclature 'Cornalin du Valais' and 'Cornalin d'Aoste' in order to prevent further confusion. At each locus, 'Goron', 'Petit Rouge', 'Mayolet' and 'Cornalin d'Aoste' all share at least one allele with 'Cornalin du Valais', strongly suggesting parent/offspring relationships. Alleles at 49 out of 50 microsatellite loci are consistent with 'Cornalin du Valais' being the progeny of 'Petit Rouge' and 'Mayolet'. The exception is a 10-base pair discrepancy at one locus, most likely the result of somatic mutation in one of the parents, since this parentage is supported by high likelihood ratios and historical data. We hypothesize that 'Cornalin du Valais' originated in the Aosta Valley through a natural cross and was then introduced into Valais centuries ago, probably via the Great St. Bernard Pass. Furthermore, 'Cornalin du Valais' is likely to be one of the parents of both 'Goron' and 'Cornalin d'Aoste', the respective second parents remaining unknown. This pedigree provides a convincing explanation for the allele-sharing patterns and is strongly supported by historical data. The present work is the first grapevine parentage study to deal with a multiple repeat unit discrepancy at a microsatellite locus. We suggest that the use of increasingly large numbers of loci in making parentage determinations leads to a corresponding increase in the probability of encountering a locus with intra-cultivar variability during the analysis. We therefore assume that a sole multiple repeat unit discrepancy is not sufficient to discard a parentage hypothesis.

Simple sequence repeat analysis of a clonally propagated species: a tool for managing a grape germplasm collection.

The USDA germplasm repositories help to preserve the genetic variability of important crop species by collecting and maintaining representative cultivars and related germplasm. Simple sequence repeat markers with high allelic diversity were used to type 41 grapevines from 40 accessions. All vines were either seedless table grape cultivars or cultivars with names similar to table grape cultivars. The proportion of shared alleles was selected as the most appropriate statistical measure of genetic distance for this population. In conjunction with morphological traits, known synonyms were confirmed and a previously unknown synonym was discovered. An alleged synonym in the literature was disproved by the DNA data. The data were consistent with known parentage, where such data were available. Two mislabeled vines in the USDA collection were identified. UPGMA grouped the cultivars loosely into three groups: a group of nine mostly Middle Eastern cultivars, a group of 22 accessions mostly from Russia and Afghanistan that were morphologically similar to 'Thompson Seedless', and a third very loose group of 11 accessions consisting mostly of eastern European wine grape cultivars. The limitations and usefulness of this type of analysis are discussed.

Vitis vinifera must varietal authentication using microsatellite DNA analysis (SSR).

A microsatellite DNA-based method for Vitis vinifera grape must authentication is presented. Five of the most important port wine producing grape cultivars (Tinta Roriz, Tinto Cão, Touriga Francesa, Touriga Nacional, and Tinta Barroca) were typed at four microsatellite loci described by Bowers et al. (Genome 1996, 39, 628-633) and Thomas and Scott (Theor. Appl. Genet. 1993, 86, 985-990). The corresponding 5 varietal musts and 26 must mixtures that result from the combination of the five varieties were also typed at the four loci. There were no differences between the corresponding leaf and varietal must profiles. All must combinations showed the expected band profiles corresponding to the sum of the varietal band profile components. Among the 26 must mixtures, 8 could be discriminated using the four loci.

The parentage of a classic wine grape, Cabernet Sauvignon.

The world's great wines are produced from a relatively small number of classic European cultivars of Vitis vinifera L Most are thought to be centuries old and their origins have long been the subject of speculation. Among the most prominent of these cultivars is Cabernet Sauvignon, described as "the world's most renowned grape variety for the production of fine red wine". Although now grown in many countries, Cabernet Sauvignon derives its fame from its long association with the Bordeaux region of France, where it has been grown at least since the 17th century. We present microsatellite DNA evidence for the hypothesis that Cabernet Sauvignon is the progeny of two other Bordeaux cultivars, Cabernet franc and Sauvignon blanc. Likelihood ratios support this hypothesis to a very high degree of probability. A close relationship between Cabernet Sauvignon and Cabernet franc has been suspected but the genetic contribution of Sauvignon blanc, despite its similar name, is a surprise.

Isolation and characterization of new polymorphic simple sequence repeat loci in grape (Vitis vinifera L.).

Four new simple sequence repeat (SSR) loci (designated VVMD5, VVMD6, VVMD7, and VVMD8) were characterized in grape and analyzed by silver staining in 77 cultivars of Vitis vinifera. Amplification products ranged in size from 141 to 263 base pairs (bp). The number of alleles observed per locus ranged from 5 to 11 and the number of diploid genotypes per locus ranged from 13 to 27. At each locus at least 75% of the cultivars were heterozygous. Alleles differing in length by only 1 bp could be distinguished by silver staining, and size estimates were within 1 or 2 bp, depending on the locus, of those obtained by fluorescence detection at previously reported loci. Allele frequencies were generally similar in wine grapes and table grapes, with some exceptions. Some alleles were found only in one of the two groups of cultivars. All 77 cultivars were distinguished by the four loci with the exception of four wine grapes considered to be somatic variants of the same cultivar, 'Pinot noir', 'Pinot gris', 'Pinot blanc', and 'Meunier'; two table grapes that are known to be synonymous, 'Keshmesh' and 'Thompson Seedless'; and three table grapes, 'Dattier', 'Rhazaki Arhanon', and 'Markandi', the first two of which have been suggested to be synonymous. Although the high polymorphism at grape SSR loci suggests that very few loci would theoretically be needed to separate all cultivars, the economic and legal significance of grape variety identification requires the increased resolution that can be provided by a larger number of loci. The ease with which SSR markers and data can be shared internationally should encourage their broad use, which will in turn increase the power of these markers for both identification and genetic analysis of grape. Key words : grape, Vitis, microsatellite, simple sequence repeat, DNA typing, identification.

Kanamycin sensitivity of cultured tissues of Vitis.

The kanamycin sensitivity of callus growth and adventitious shoot and root formation was studied in several cultivars of Vitis vinifera L. and in V. rupestris Scheele cv. St. George to investigate the suitability of kanamycin resistance as a selectable marker for grape transformation. Kanamycin concentrations ranged from 0 to 30 mg/l. Carbenicillin was added to the medium in all experiments at concentrations of 500 or 250 mg/l, as normally used in cocultivation experiments with Agrobacterium. Callus formation, root initiation, and adventitious shoot formation were completely inhibited by 20, 10, and 7 mg/l kanamycin, respectively; suggesting that these are the minimum concentrations that should be necessary to select transformed plants. Carbenicillin produced inhibitory effects that sometimes resembled those of growth regulators. The high kanamycin sensitivity of adventitious shoot formation in grape exceeds that reported for any other plant species and is likely to hinder the recovery of transformed plants.

Strategies for the selection and characterization of aluminum-resistant variants from cell cultures of Nicotiana plumbaginifolia.

The development of strategies for selecting and characterizing aluminum-resistant variants from Nicotiana plumbaginifolia Viv. cell cultures is described. Plated cells, smeared callus, in-vitro-grown shoots, and seedlings of wild-type N. plumbaginifolia all showed similar responses to Al, with total growth inhibition at or above 600 µM Al. The strict control of both cell density and aggregate size is important in selection experiments for total inhibition of the growth of wild-type cells. Two approaches for the selection of Al-resistant variants were used. In a direct method, cells were plated onto medium containing 600 µM Al which inhibited growth and chlorophyll synthesis in wildtype cells. A double selection strategy based on both cell growth and greening was used to isolate 29 Al-resistant variants. In the other approach, a rescue method, suspensions were cultured for 10 d in medium containing 600 µM Al, then plated onto standard medium for recovery of survivors. Using this strategy, 217 Al-resistant variants were selected. After six to twelve weeks of growth in the absence of Al, each variant was cloned and reselected from single cells. Al resistance was retained in 31% and 51% of the variants selected by the direct and rescue strategies, respectively. Seedling segregation data are presented for the progeny (selfed and backcrossed) of plants regenerated from one of the variants and are consistent with those expected for a single dominant mutation.

Large scale selection of aluminum-resistant mutants from plant cell culture: expression and inheritance in seedlings.

A large number of aluminum-resistant variants, selected from non-mutagenized homozygous diploid cell cultures of Nicotiana plumbaginifolia Viv., are characterized. Of 115 variants cloned and reselected from single cells, 67 retained Al resistance in callus cultures after 6-9 months of growth in the absence of Al. There was no association between Al resistance and callus growth in the absence of Al, suggesting that the Al-resistant phenotype is not detrimental in the absence of Al challenge and that Al resistance is not the result of increased vigor. Plants regenerated from initially resistant callus lines that subsequently lost their resistance failed, with one exception, to transmit resistance to their seedling progeny. Fertile plants were regenerated from 40 of the 67 variants that retained stable Al resistance in callus culture. All 40 transmitted Al resistance to their seedling progeny (selfed and backcrossed) in segregation ratios expected for a single dominant mutation. The selfed progeny of many variants also segregated for recessive lethal mutations which were attributed to independent mutations that occurred during cell culture.