Résumé
Clonal variation among cultivars of Vitis vinifera L. has been known formany years and its use achieved very significant gains for the wine industry. Nowadays, the basis of clonal phenotypic variation is barely known: hypothesis of genetic mutations or epigenetic modifications have nevertheless been considered. The objective of this study is to get a better insight of the genetic basis of clonal variation to develop a tool for clonal identification and selection. The current scientific context, access at the whole genome sequences of the individual PN40024 derived from Pinot noir and of a heterozygous clone of Pinot noir ENTAV-INRA® 115 and the development of the NGS (new generation sequencing) technologies, allows now exhaustive studies of the grapevine genome. Towards this aim, we used the 454 GX FLX Titanium technology on three official clones of Pinot noir ENTAV-INRA® 386, 583 and 777, selected for their different phenotypic traits. We firstly developed a nuclear DNA extraction method to limit the contamination from cytoplasmic DNA. We then realized one 454 run by clone and we obtained in mean 1 million reads of 350 bases by run. Our data sets from the 454 and the contigs from the whole sequence of Pinot noir ENTAV-INRA® 115 have been aligned on the reference genome, PN40024 with a compilation of different software: Mosaik-assembler, and RepeatMasker. Sixty-six percent of the reads obtained from each of the 454 runs have been aligned at a unique locus. Thanks to NGS technologies, we could compare a large portion of the genome with 3X coverage: i) in mean 8Mb between Pinot noir ENTAV-INRA® 115 and with each of the three others clones and ii) 0.5 Mb between the three clones themselves. The different sequence data obtained from these clones will be compared to identify the major molecular events which separate the clones analyzed. Preliminary results showed that SNPs are less important than insertions/deletions to be polymorphic between clones. We will validate this polymorphism and will develop method to perform identification of the clones.