Financial support: The present work has been done in the framework of a research programme (1997-2001) developed in the area of viticulture and wine making funded by the Tuscan regional authority ARSIA (Agenzia Regionale per lo Sviluppo e l'Innovazione nel Settore Agricolo-Forestale).

Keywords: clone variation, polymerase chain reaction, sequence tagged sites, variety identification.

Abbreviations: PCR, polymerase chain reaction; AFLP, amplified fragment length polymorphism; Cy5, Cy^TM5 amidite (5'-cyamine-d[seq]) fluorochrome technology.

Grapevine diversity and biotechnology

Grapevine (Vitis vinifera L.) is an ancient fruit species and its domestication goes back to the 11^th century BC The long history of grapevine growth has determined a complex picture in which many biotypes or even cultivars are misidentified or called by different names in different areas. This often makes genetic identification difficult. Grapevines are propagated by cuttings and the resulting clones of a given population are genetically identical to each other (except for somatic mutations) and to the mother plant (the original seedling from which cultivars were derived).

Forensic technologies employ molecular markers, and this same technology is being used to solve problems in grapevine identification and parental analysis. Molecular markers have been used extensively in grapes due to the difficulty in distinguishing among similar groups of cultivars. DNA marker which are widely used nowadays include microsatellites and AFLP. As a result, there has been progress in understanding diversity within grapevine germplasm collections, relatedness of cultivars from different regions, and in the identification of multiple genotypes within supposedly homogeneous clonal cultivars. It seems evident that any scientific programme aimed at increasing plant quality or preserving the integrity of the genetic resources of this species must consider the problem of plant identification at variety or clone level. In order to evaluate variability within the "Sangiovese" variety and to confirm variety identification, genotype analysis, allele distribution and pedigree information were processed with a DNA-automated sequencer running AlleleLinks software on twenty-five grapevine accessions believed to belong to "Sangiovese" variety grown in different areas of Tuscany.

DNA typing revealed three cases of genetic dissimilarity compared to registered "Sangiovese" clones while the divergent accessions GA5, CH6 and LA4 were originally thought to be different clones of the same variety. They were different from "Sangiovese" at four microsatellite loci (VVS2, VVS4, VVMD7 and VVMD21). An innovative, non-radioactive modification of AFLP genome profiling confirmed the data obtained by microsatellite amplification test. Cy^TM5 fluorochrome labelled amplification products, were resolved on ReproGel™ High Resolution pre-made acrylamide-bisacrylamide solutions (8% w/v) and detected on a semi-automated DNA sequencer, the ALFexpress II DNA Analysis System by Amersham Pharmacia Biotech. Allele sizing, as well as genotyping and pre-linkage data on the studied population were performed by the use of AlleLinks software (Amersham Pharmacia Biotech).

Plant Material

Twenty-five grapevine accessions believed to belong to "Sangiovese" variety grown in different areas of Tuscany were analysed (Table 1). Plant material was kindly provided by Prof. G. Scalabrelli, University of Pisa and by Prof. M. Boselli, University of Florence. Several plants, including "Sangiovese" clones used as standards (SS-F9-A5-48, BB-S-11, VCR-10) were from germoplasm collection vineyards and are available commercially; BD1 and BD2 were from the Montalcino area, kindly provided by Prof. Pisani, University of Florence. The other accessions are grown in different parts of Tuscany, for example "Sangiovese" from the area of Scansano, Grosseto, (CH, LA, ROSSI3 and GA series), known for the DOC wine Morellino, and presumed clones of "Sangiovese" grown in the area of Val di Cornia (accessions labelled COR) under the 5-year (1997-2001) research breeding programme ("Miglioramento genetico delle produzioni vitivinicole e del materiale di propagazione" funded by ARSIA-Agenzia Regionale per lo Sviluppo e l'Innovazione nel Settore Agricolo forestale- the research promoting agency of the Tuscan Region).

Results

The allele sizes calculated by microsatellite amplification test and AFLP genotyping confirmed the genetic identity of most presumed clones of "Sangiovese". Only three individuals, GA5, CH6 and LA4 clearly differ from the rest of population. Previous traditional ampelometric studies on the three divergent accessions already suggested that they were not really "Sangiovese". Pre-linkage data and genotype report, for the probability evaluation of allelic configuration of each individual, is shown in Table 2. The data elaboration is based on the loci showing polymorphism in the Sangiovese-related family studied. Based on AFLP profiling, a phylogenetic diagram can be used to group the population (Figure 1) into four main clusters, which distinguish individuals at species level: A, B, and C correspond to G. biloba, A. thaliana and L. esculentum respectively, while D includes all the V. vinifera accessions analysed. It is noteworthy that the divergent accessions CH6 and LA4 form a cluster and GA5 is not far from them but separate from the other "Sangiovese" accessions. The present findings support the hypothesis that GA5, CH6 and LA4 differ from the "Sangiovese" standard and that they should be excluded from selection trials if accepting a strict definition of cultivar that poses the progeny as deriving from a single seedling (i.e. monoclonal origin).

Conclusions

Our aim was to demonstrate the utility of PCR-based DNA markers in Vitis vinifera for distinguishing accessions which diverge from a certain variety and for defining the phylogenetic relationship of unknown individuals of this species.

These results underline the importance of biotechnological testing, such as the PCR-based DNA tests together with traditional ampelography, in Vitis vinifera L. clone and variety selection programmes to avoid misnaming and erroneous identification and to evaluate genetic relatedness and variability within populations.

The use of multiple PCR-based markers helps to overcome some of the intrinsic limits of the microsatellite amplification test in Vitis vinifera populations, such as the partial or non-standard information on alleles size and frequencies. In addition, the use of multiple DNA tests is in keeping with the terms of the recent international Vitis genome project (http://www.genome.clemson.edu/projects/stc/grape/VV_SBa).

As already proposed elsewhere (Karp et al. 1997; Vignani et. al. 1999) the results presented in this paper clearly show that modern biotechnological tools, especially DNA tests based on easy-to-handle PCR-based techniques, can make significant contribution in crop breeding research. For V. vinifera in particular, molecular diagnostic tools can detect cases of genetic divergence and erroneous variety assignment early in the long process of traditional clonal selection. These cases could otherwise lead to misnaming and waste of human and financial resources.

KARP, A.; EDWARDS, K.J.; BRUFORD, M.; FUNK, S.; VOSMAN, B.; MORGANTE, M.; SEBERG, O.; KREMER, A.; BOURSOT, P.; ARCTANDER, P.; TAUTZ, D. and HEWITT, G.M. Molecular technologies for biodiversity evaluation: opportunities and challenges. Nature Biotechnology, July 1997, vol.15, no. 7, p.625-628.

VIGNANI, Rita; SCALI, Monica and CRESTI, Mauro. Grapevine biotechnology coming on to the scene. In: CRESTI, M.; CAI, G. and MOSCATELLI, A., eds. Fertilization in higher plants. Springer Verlag, Berlin, Heidelberg, New York, 1999, p. 413-425.

Note: EJB Electronic Journal of Biotechnology is not responsible if on-line references cited on manuscripts are not available any more after the date of publication.