Evidence of a secondary grapevine domestication centre detected by SSR analysis.

The origin of the grapevine was investigated with archaeobotanical, cultural and historical data. A primary domestication centre was located in the Near East region but there is no agreement on the existence or role of secondary domestication centres. In this work, PCR-based microsatellite analysis has been applied to study the origin of some Italian cultivated grapevines from in situ direct domestication of the wild autoctonous grapevine. Three different Italian locations in Grosseto, Cosenza and Nuoro were identified for this study, and domesticated grapevine as well as wild local accessions growing in these location, were analysed by SSR markers. Cluster analysis performed on Cosenza and Grosseto samples showed a high value of genetic distance between domesticated and wild accessions. On the contrary two cultivars (Bovale Murru and Bovale Muristellu) recovered in Nuoro (in the Sardinia island) were very close to some wild varieties. This suggests that the latter two cultivars may have originated from wild grapevines and consequently that in this location a secondary grapevine domestication event occurred. Six Lambrusco varieties were also included in this analysis as ancient putative ancestors of the cultivated grapevines. The molecular analysis excluded this hypothesis and suggest Lambrusco as an independent Vitis taxon.

Cloning and molecular analysis of structural genes involved in flavonoid and stilbene biosynthesis in grape (Vitis vinifera L.).

Genes involved in flavonoid and stilbene biosynthesis were isolated from grape (Vitis vinifera L.). Clones coding for phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), leucoanthocyanidin dioxygenase (LDOX) and UDP glucose:flavonoid 3-O-glucosyl transferase (UFGT), were isolated by screening a cDNA library, obtained from mRNA from seedlings grown in light for 48 h using snapdragon (Antirrhinum majus) and maize heterologous probes. A cDNA clone coding for stilbene synthase (StSy) was isolated by probing the library with a specific oligonucleotide. These clones were sequenced and when the putative products were compared to the published amino acid sequence for corresponding enzymes, the percentages of similarity ranged from 65% (UFGT) to 90% (CHS and PAL). The analysis of the genomic organization and expression of these genes in response to light shows that PAL and StSy genes belong to large multigene families, while the others are present in one to four copies per haploid genome. The steady-state level of mRNAs encoded by the flavonoid biosynthetic genes as determined in young seedlings is coordinately induced by light, except for PAL and StSy, which appear to be constitutively expressed.

Somatic embryogenesis from leaf- and petiole-derived callus of Vitis rupestris.

Somatic embryogenesis from leaf- and petiole-derived calli of Vitis rupestris was obtained with an efficiency of 3.2% and 4.2% of plated explants, respectively on two combinations of 6-benzyladenine and 2,4-dichlorophenoxyacetic acid (1/0.1 and 1/1 mgl(-1)) added to MS medium. Embryogenic callus, embryo subcultures and somatic embryogenesis from somatic embryos were obtained either in the presence of 1 mgl(-1) indole-3-acetic acid or 0.1 mgl(-1) indole-3-butyric acid added to MS or NN media. Within a 4-month culture, embryo germination occurred at a frequency of 13% of explanted embryos when chilling at 4°C was provided for two weeks and a combination of 6-benzyladenine (1 mgl(-1)) with indole-3-butyric acid (0.1 mgl(-1)) was added to NN medium supplemented with casein hydrolysate (250 mgl(-1)). A higher frequency (51%) was obtained in a longer culture time (9 months) when only indole-3-butyric acid was present in the medium and in absence of chilling.

Monitoring by two-dimensional electrophoresis somatic embryogenesis in leaf and petiole explants from Vitis.

A two-dimensional electrophoretic analysis of the total proteins was carried out in Vitis rupestris as model system in order to characterize the different developmental stages--from callus to plantlets--of somatic embryogenesis events in the grapevine. The patterns of callus, embryogenetic callus, somatic embryos and plantlets derived from leaf and petiole explants were compared. Each differentiation step was characterized by specific peptide spots.