Characterization of ten novel Ty1/copia-like retrotransposon families of the grapevine genome.

BACKGROUND: Retrotransposons make a significant contribution to the size, organization and genetic diversity of their host genomes. To characterize retrotransposon families in the grapevine genome (the fourth crop plant genome sequenced) we have combined two approaches: a PCR-based method for the isolation of RnaseH-LTR sequences with a computer-based sequence similarity search in the whole-genome sequence of PN40024. RESULTS: Supported by a phylogenic analysis, ten novel Ty1/copia families were distinguished in this study. To select a canonical reference element sequence from amongst the various insertions in the genome belonging to each retroelement family, the following screening criteria were adopted to identify the element sequence with: (1) perfect 5 bp-duplication of target sites, (2) the highest level of identity between 5' and 3'-LTR within a single insertion sequence, and (3) longest, un-interrupted coding capacity within the gag-pol ORF. One to eight copies encoding a single putatively functional gag-pol polyprotein were found for three families, indicating that these families could be still autonomous and active. For the others, no autonomous copies were identified. However, a subset of copies within the presumably non-autonomous families had perfect identity between their 5' and 3' LTRs, indicating a recent insertion event. A phylogenic study based on the sequence alignment of the region located between reverse transcriptase domains I and VII distinguished these 10 families from other plant retrotransposons. Including the previously characterized Ty1/copia-like grapevine retrotransposons Tvv1 and Vine 1 and the Ty3/gypsy-like Gret1 in this assessment, a total of 1709 copies were identified for the 13 retrotransposon families, representing 1.24% of the sequenced genome. The copy number per family ranged from 91-212 copies. We performed insertion site profiling for 8 out of the 13 retrotransposon families and confirmed multiple insertions of these elements across the Vitis genus. Insertional polymorphism analysis and dating of full-length copies based on their LTR divergence demonstrated that each family has a particular amplification history, with 71% of the identified copies being inserted within the last 2 million years. CONCLUSION: The strategy we used efficiently delivered new Ty1/copia-like retrotransposon sequences, increasing the total number of characterized grapevine retrotrotransposons from 3 to 13. We provide insights into the representation and dynamics of the 13 families in the genome. Our data demonstrated that each family has a particular amplification pattern, with 7 families having copies recently inserted within the last 0.2 million year. Among those 7 families with recent insertions, three retain the capacity for activity in the grape genome today.

Evaluation of tolerance to Pierce's disease and Botrytis in transgenic plants of Vitis vinifera L. expressing the pear PGIP gene.

SUMMARY Polygalacturonase-inhibiting proteins (PGIPs) are plant cell-wall proteins that specifically inhibit fungal endo-polygalacturonases (PGs) that contribute to the aggressive decomposition of susceptible plant tissues. The inhibition of fungal PGs by PGIPs suggests that PGIPs have a role in plant tolerance to fungal infections and this has been observed in transgenic plants expressing PGIPs. Xylella fastidiosa, the causal agent of Pierce's disease (PD) in grapevines, has genes that encode cell-wall-degrading enzymes, including a putative PG. Therefore, we hypothesized that PGIP expression could confer tolerance against this bacterium as well as against the fungal pathogen Botrytis cinerea. To test this hypothesis, Vitis vinifera cvs. 'Thompson Seedless' and 'Chardonnay' were transformed to express pear fruit PGIP-encoding gene (pPGIP) under the control of the CaMV 35S promoter. Substantial pear PGIP (pPGIP) activity was found in crude extracts from leaves and in xylem exudate of transgenic lines obtained from independent transformation events, but not in untransformed controls. pPGIP activity was detected in xylem exudate of untransformed scions grafted on to transgenic rootstocks expressing pPGIP. Leaves of transgenic plants infected with B. cinerea had reduced rates of lesion expansion. The development of PD was delayed in some transgenic lines with increased pPGIP activity. PD-tolerant transgenic lines had reduced leaf scorching, lower Xylella titres and better re-growth after pruning than the untransformed controls.

Genetic structure and differentiation in cultivated grape, Vitis vinifera L.

222 cultivated (Vitis vinifera) and 22 wild (V. vinifera ssp. sylvestris) grape accessions were analysed for genetic diversity and differentiation at eight microsatellite loci. A total of 94 alleles were detected, with extensive polymorphism among the accessions. Multivariate relationships among accessions revealed 16 genetic groups structured into three clusters, supporting the classical eco-geographic grouping of grape cultivars: occidentalis, pontica and orientalis. French cultivars appeared to be distinct and showed close affinity to the wild progenitor, ssp. sylvestris from south-western France (Pyrenees) and Tunisia, probably reflecting the origin and domestication history of many of the old wine cultivars from France. There was appreciable level of differentiation between table and wine grape cultivars, and the Muscat types were somewhat distinct within the wine grapes. Contingency chi2 analysis indicated significant heterogeneity in allele frequencies among groups at all loci. The observed heterozygosities for different groups ranged from 0.625 to 0.9 with an overall average of 0.771. Genetic relationships among groups suggested hierarchical differentiation within cultivated grape. The gene diversity analysis indicated narrow divergence among groups and that most variation was found within groups (approximately 85%). Partitioning of diversity suggested that the remaining variation is somewhat structured hierarchically at different levels of differentiation. The overall organization of genetic diversity suggests that the germplasm of cultivated grape represents a single complex gene pool and that its structure is determined by strong artificial selection and a vegetative mode of reproduction.