Chromosome replacement and deletion lead to clonal polymorphism of berry color in grapevine.

Clonal polymorphism mainly results from somatic mutations that occur naturally during plant growth. In grapevine, arrays of clones have been selected within varieties as a valuable source of diversity, among them clones showing berry color polymorphism. To identify mutations responsible for this color polymorphism, we studied a collection of 33 clones of Pinot noir, Pinot gris, and Pinot blanc. Haplotypes of the L2 cell layer of nine clones were resolved by genotyping self-progenies with molecular markers along a 10.07 Mb region of chromosome 2, including the color locus. We demonstrated that at least six haplotypes could account for the loss of anthocyanin biosynthesis. Four of them resulted from the replacement of sections of the 'colored' haplotype, sized from 31 kb to 4.4 Mb, by the homologous sections of the 'white' haplotype mutated at the color locus. This transfer of information between the two homologous sequences resulted in the partial homozygosity of chromosome 2, associated in one case with a large deletion of 108 kb-long. Moreover, we showed that, in most cases, somatic mutations do not affect the whole plant; instead, they affect only one cell layer, leading to periclinal chimeras associating two genotypes. Analysis of bud sports of Pinot gris support the hypothesis that cell layer rearrangements in the chimera lead to the homogenization of the genotype in the whole plant. Our findings shed new light on the way molecular and cellular mechanisms shape the grapevine genotypes during vegetative propagation, and enable us to propose a scheme of evolutionary mechanism of the Pinot clones.

The Tvv1 retrotransposon family is conserved between plant genomes separated by over 100 million years.

KEY MESSAGE: Combining several different approaches, we have examined the structure, variability, and distribution of Tvv1 retrotransposons. Tvv1 is an unusual example of a low-copy retrotransposon metapopulation dispersed unevenly among very distant species and is promising for the development of molecular markers. Retrotransposons are ubiquitous throughout the genomes of the vascular plants, but individual retrotransposon families tend to be confined to the level of plant genus or at most family. This restricts the general applicability of a family as molecular markers. Here, we characterize a new plant retrotransposon named Tvv1_Sdem, a member of the Copia superfamily of LTR retrotransposons, from the genome of the wild potato Solanum demissum. Comparative analyses based on structure and sequence showed a high level of similarity of Tvv1_Sdem with Tvv1-VB, a retrotransposon previously described in the grapevine genome Vitis vinifera. Extending the analysis to other species by in silico and in vitro approaches revealed the presence of Tvv1 family members in potato, tomato, and poplar genomes, and led to the identification of full-length copies of Tvv1 in these species. We were also able to identify polymorphism in UTL sequences between Tvv1_Sdem copies from wild and cultivated potatoes that are useful as molecular markers. Combining different approaches, our results suggest that the Tvv1 family of retrotransposons has a monophyletic origin and has been maintained in both the rosids and the asterids, the major clades of dicotyledonous plants, since their divergence about 100 MYA. To our knowledge, Tvv1 represents an unusual plant retrotransposon metapopulation comprising highly similar members disjointedly dispersed among very distant species. The twin features of Tvv1 presence in evolutionarily distant genomes and the diversity of its UTL region in each species make it useful as a source of robust molecular markers for diversity studies and breeding.

An extensive study of the genetic diversity within seven French wine grape variety collections.

The process of vegetative propagation used to multiply grapevine varieties produces, in most cases, clones genetically identical to the parental plant. Nevertheless, spontaneous somatic mutations can occur in the regenerative cells that give rise to the clones, leading to consider varieties as populations of clones that conform to a panel of phenotypic traits. Using two sets of nuclear microsatellite markers, the present work aimed at evaluating and comparing the intravarietal genetic diversity within seven wine grape varieties: Cabernet franc, Cabernet Sauvignon, Chenin blanc, Grolleau, Pinot noir, Riesling, Savagnin, comprising a total number of 344 accessions of certified clones and introductions preserved in French repositories. Ten accessions resulted in being either self-progeny, possible offspring of the expected variety or misclassified varieties. Out of the 334 remaining accessions, 83 displayed genotypes different from the varietal reference, i.e., the microsatellite profile shared by the larger number of accessions. They showed a similarity value ranging from 0.923 to 0.992, and thus were considered as polymorphic monozygotic clones. The fraction of polymorphic clones ranged from 2 to 75% depending on the variety and the set of markers, the widest clonal diversity being observed within the Savagnin. Among the 83 polymorphic clones, 29 had unique genotype making them distinguishable; others were classified in 21 groups sharing the same genotype. All microsatellite markers were not equally efficient to show diversity within clone collections and a standard set of five microsatellite markers (VMC3a9, VMC5g7, VVS2, VVMD30, and VVMD 32) relevant to reveal clonal polymorphism is proposed.

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.

Retention of the virus-derived sequences in the nuclear genome of grapevine as a potential pathway to virus resistance.

BACKGROUND: Previous studies have revealed a wide-spread occurence of the partial and complete genomes of the reverse-transcribing pararetroviruses in the nuclear genomes of herbaceous plants. Although the absence of the virus-encoded integrases attests to the random and incidental incorporation of the viral sequences, their presence could have functional implications for the virus-host interactions. HYPOTHESIS: Analyses of two nuclear genomes of grapevine revealed multiple events of horizontal gene transfer from pararetroviruses. The approximately 200-800 bp inserts that corresponded to partial ORFs encoding reverse transcriptase apparently derived from unknown or extinct caulimoviruses and tungroviruses, were found in 11 grapevine chromosomes. In contrast to the previous reports, no reliable cases of the inserts derived from the positive-strand RNA viruses were found. Because grapevine is known to be infected by the diverse positive-strand RNA viruses, but not pararetroviruses, we hypothesize that pararetroviral inserts have conferred host resistance to these viruses. Furthermore, we propose that such resistance involves RNA interference-related mechanisms acting via small RNA-mediated methylation of pararetroviral DNAs and/or via degradation of the viral mRNAs. CONCLUSION: The pararetroviral sequences in plant genomes may be maintained due to the benefits of virus resistance to this class of viruses conferred by their presence. Such resistance could be particularly significant for the woody plants that must withstand years- to centuries-long virus assault. Experimental research into the RNA interference pathways involving the integrated pararetroviral inserts is required to test this hypothesis. REVIEWERS: This article was reviewed by Arcady R. Mushegian, I. King Jordan, and Eugene V. Koonin.

Structural variability of Tvv1 grapevine retrotransposons can be caused by illegitimate recombination.

Structural variability of Tvv1, a grapevine retrotransposon Ty1 copia-like family, was investigated within the grape genome and the canonical sequence of Tvv1 determined. Then, two remarkable elements, Tvv1-Delta3001 and Tvv1-Delta3640, which had suffered large deletions 3,001 bp and 3,460 bp in length of their coding sequences were compared to the canonical copy. In both deleted elements, the deletion breakpoint was characterized by a stretch 13 bp-long in Tvv1-Delta3001 and 11 bp-long in Tvv1-Delta3640 found duplicated in the canonical copy at each bound of the deleted regions. Tvv1-Delta3001 and Tvv1-Delta3460 were both shown to be unique copies fixed at a single locus in the grapevine genome. Their presence was very variable in a set of 58 varieties and wild vines. These elements have most likely been dispersed through natural intermixing after their initial insertion whose chronology was estimated. The model that we propose to explain the structure of Tvv1-Delta3001 and Tvv1-Delta3640, implies illegitimate recombination involving template switching between two RNA molecules co-packaged in the VLP prior to the integration of the deleted daughter copy into the host genome.

Untranslated leader region polymorphism of Tvv1, a retrotransposon family, is a novel marker useful for analyzing genetic diversity and relatedness in the genus Vitis.

Grapevine retrotransposons belonging to the Tvv1 family share a single, highly conserved open reading frame but differ by their untranslated leader (UTL) region, which is highly variable in size. Amplification of the UTL region of Tvv1 elements from 94 Vitaceae accessions reveals that each of them shows a unique pattern of UTL-derived bands, which is inherited in progenies but conserved between clones vegetatively propagated. The overall organization of genetic diversity of the Vitaceae at the inter and intraspecific level and relatedness among accessions described by UTL-derived bands was compared to those obtained using 15 microsatellite loci. Both fingerprinting methods show a similar grouping of Vitis vinifera accessions but UTL-based fingerprinting more accurately isolates the muscadine grapes from the American and Asian Vitis. Finally, sequence analysis of seven UTL regions determines that their size variation is essentially caused by large deletions/insertions within the internal region, whereas flanking regions are more conserved. UTL-based fingerprinting could be considered as a novel marker system specific of the genus Vitis; moreover, as this multiband genotype is stable between clones it is suitable to be used as a "DNA barcode" for Vitis identification.

Genetic diversity and geographical dispersal in grapevine clones revealed by microsatellite markers.

Intravarietal genetic diversification associated with geographical dispersal of a vegetatively propagated species was studied using grapevine Vitis vinifera L. 'Cabernet Sauvignon' as a model. Fifty-nine clonal samples obtained from 7 countries (France, Chile, Spain, Australia, Hungary, USA, and Italy) were analyzed using 84 microsatellite markers. Eighteen polymorphic microsatellite loci (21.4%) were detected, finding 22 different genotypes in the population analyzed with a genetic similarity of over 97%. The presence of chimeric clones was evidenced at locus VMC5g7 by means of a segregation analysis of descendants by self-pollination of a triallelic Chilean clone and by somatic embryogenesis analysis, showing a mutation in L2 cell layer. Only 2 clones (obtained from France and Australia) presented the ancestral genotype, and the most divergent genotype was exhibited by another French clone, which had accumulated 5 somatic mutations. The 2 largest populations considered (from France and Chile) showed a clear divergency in the polymorphisms detected. These antecedents enabled the tracing of geographical dispersal with a phylogenetic hypothesis supporting France as the center of origin of diversification of Cabernet Sauvignon. The results obtained could help to explain diversification processes in other grapevine cultivars. The possibility that this kind of genetic variability occurs in other vegetatively propagated species is discussed, focusing on possible fingerprinting applications.