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

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Hypothesis</p> <p>Analyses of two nuclear genomes of grapevine revealed multiple events of horizontal gene transfer from pararetroviruses. The ~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.</p> <p>Conclusion</p> <p>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.</p> <p>Reviewers</p> <p>This article was reviewed by Arcady R. Mushegian, I. King Jordan, and Eugene V. Koonin.</p

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

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.Peer reviewe

Analyse structurale et transcriptionnelle des rétrotransposons du génome de la vigne, Vitis vinifera L.

STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

La diversité génétique intra-variétale chez la vigne (Caractérisation et origines)

Les variétés de vigne (Vitis vinifera L.), ou cépages, sont composées d'un ensemble de clones présentant des caractéristiques ampélographiques homogènes avec des différences mineures. Cette diversité clonale peut avoir pour origine, d'une part, par différents évènements de reproduction sexuée et, d'autre part, une accumulation de mutations somatiques durant les nombreux cycles de propagation végétative.Ce mémoire de thèse présente une étude de la diversité génétique intra-variétale réalisée sur un échantillon de 477 clones du groupe variétal des pinots, provenant de cinq régions viticoles et appartenant à six variétés. Cette analyse, réalisée à 50 loci microsatellites, a montré l'existence de deux groupes de clones présentant des niveaux de similarité génétique distincts. Dans le premier groupe composé de 474 clones, les niveaux de similarité génétique sont très élevés, compris entre 0.97 et 1.00. Un génotype majoritaire est partagé par 45 % des clones appartenant à différentes variétés. Les clones variants de ce génotype se différencient par une variation sur un, deux, voire trois loci. Vingt marqueurs polymorphes ont permis d'identifier 35 génotypes différents. Dans 70 % de ces cas, la variabilité est révélée par des génotypes tri-alléliques et, plus rarement, tétra-alléliques. Ces différents résultats indiquent que les clones de ce premier groupe sont très probablement issus d'un même zygote ancestral (origine monozygotique). Dans le deuxième groupe, trois clones de pinot noir présentent des niveaux de similarité génétique comparable à ceux observés au niveau inter-variétal (0.74 à 0.92). Deux d'entre eux sont très probablement issus de croisements entre un pinot et un apparenté, le troisième de l'autofécondation d'un pinot noir, donc de zygotes différents de celui dont sont issus les clones du premier groupe (origine polyzygotique).Afin de comprendre l'origine des variations tri-alléliques chez une espèce diploïde, nous avons, dans un premier temps, confirmé par séquençage l'appartenance à un même locus des trois allèles observés. Le clone agréé de pinot gris PG52, tri-allélique à deux loci, a ensuite été utilisé pour analyser (i) le mode de transmission des allèles dans une descendance d'autofécondation, (ii) la composition allélique de différents organes de la plante et (iii) la ségrégation de ces allèles dans des plantes issues d'embryogénèse somatique. Nous avons ainsi montré que la présence simultanée de trois allèles résultait d'une structure tissulaire chimérique, dans laquelle les deux couches cellulaires du méristème apical étaient génétiquement différentes. Une telle structure ne peut être maintenue que par multiplication végétative.L'origine monozygotique de la majorité des pinots et l'état chimérique des clones variants nous ont permis de retracer une phylogénie possible des différents évènements mutationnels ayant conduit à la diversité actuelle. Ces résultats représentent les premières bases pour l'identification et la protection des obtentions clonales, le chimérisme apportant de nouvelles perspectives pour l'amélioration de la vigne.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

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

BackgroundRetrotransposons 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.ResultsSupported 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.ConclusionThe 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

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

Biochemical and Immunological Characterization of Nitrate Reductase Deficient nia Mutants of Nicotiana plumbaginifolia

Sixty-five Nicotiana plumbaginifolia mutants affected in the nitrate reductase structural gene (nia mutants) have been analyzed and classified. The properties evaluated were: (a) enzyme-linked immunosorbent assay (two-site ELISA) using a monoclonal antibody as coating reagent and (b) presence of partial catalytic activities, namely nitrate reduction with artificial electron donors (reduced methyl viologen, reduced flavin mononucleotide, or reduced bromphenol blue), and cytochrome c (Cyt c) reduction with NADH. Four classes have been defined: 40 mutants fall within class 1 which includes all mutants that have no protein detectable in ELISA and no partial activities; mutants of classes 2 and 3 exhibit an ELISA-detectable nitrate reductase protein and lack either Cyt c reductase activity (class 2: fourteen mutants) or the terminal nitrate reductase activities (class 3: eight mutants) of the enzyme. Three mutants (class 4) are negative in the ELISA test, lack Cyt c reductase activity, and lack or have a very low level of reduced methyl viologen or reduced flavin mononucleotide-nitrate reductase activities; however, they retain the reduced bromphenol blue nitrate reductase activity. Variations in the degrees of terminal nitrate reductase activities among the mutants indicated that the flavin mononucleotide and methyl viologen-dependent activities were linked while the bromphenol blue-dependent activity was independent of the other two. The putative positions of the lesions in the mutant proteins and the nature of structural domains of nitrate reductase involved in each partial activity are discussed

Assessment of two highly polymorphic microsatellite loci in 103 accessions of <em>Vitis</em> species

We assessed the discriminative power of two nuclear microsatellite (nSSR, nuclear simple sequence repeated) loci, ssrVvUCH12 and ssrVvUCH29, which had been suspected to be highly polymorphic and to display specific alleles in the frame of a previous characterization work. As more genotyping of more plant material at a higher number of nSSR loci was needed for an extensive comparison, one hundred and three accessions of Vitis vinifera, other Vitis species, and 3 related genera were submitted to microsatellite profiling at fourteen nuclear nSSR loci, including ssrVvUCH12 and vsrVvUCH29. The loci ssrVvUCH12 and ssrVvUCH29 loci displayed each 30 alleles found in 103 accessions while the most polymorphic loci among the 12 other nSSR loci tested, were ssrVrZAG93 and VMc8a7, which displayed 31 and 23 alleles respectively. These 2 last loci had however the disadvantage to display a three banded pattern in some individuals, probably due to chimerism. Most interestingly average similarity found in this pool of 103 accessions was also lower with the combination ssrVvUCH12-ssrVvUCH29 than with the combination ssrVrZAG-VMC8a7, while observed heterozygocity was higher with the combination ssrVvUCH12-ssrVvUCH29. Comparisons were made easily visible under the form of UPGMA phenograms for each combination of loci and plant sets and the most resolutive combination was always the combination ssrVvUCH12- ssrVvUCH29. At last, numerous specific alleles were found for rootstocks and Vitis species at loci ssrVvUCH12 and ssrVvUCH29, while the other combination did not show many specific alleles. These features allow to think that this 2-loci combination could be considered for managing collections of Vitis species in addition of previously evaluated highly polymorphic microsatellite markers. It could also be useful as a quality control tool for rootstock material in nurseries or trade