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

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

Diplôme : Dr. d'UniversiteGrapevine (Vitis vinifera L.) varieties are composed by clones originated from a unique plant propagated by vegetative multiplication. This process theoretically ensure the transmission of genetic information from mother to daughter’s plants, but spontaneous mutations sometimes arise and can lead to the creation of new genotypes. Due to their ability to transpose, transposable elements are a source of mutation that causes genetic variability. To understand global mechanisms that participate to plant genome evolution during vegetative multiplication cycles, the main goal of this thesis is studying the structure and dynamics of grapevine retrotransposons. When this work started, only three grapevine retrotransposons were described. Therefore, the first part of this work is based on the characterization of ten new families, which are different from the elements previously described in grapevine or in other plant genomes. This ten new families represent near 1,700 copies, from full-length to degenerated, representing altogether about 1.2% of the genome. Some of these copies were integrated in the grapevine genome during the last two million years. In a second part, the transcription level of grapevine retrotransposons is evaluated by complementary approaches. The results show that their transcription level is regulated in different organs under normal growing conditions, each family showing a specific transcription profile. Moreover, the expression of many retrotransposons in leaves is induced by wounding and pathogen infection. The role of retrotransposons in grapevine genome evolution and in the genesis of intravarietal diversity has not been established, but induction of expression of several families under stress conditions allow us to suppose that this elements might have participated to genome adaptation in response to environmental challenges and grapevine culture by man. Moreover, the characterization of new transposable families contributes to the development of molecular tools allowing the identification and distinction of grapevine clones.Les variétés de vigne (Vitis vinifera L.) sont composées d’un ensemble de clones, tous issus de la même et unique plante propagée par multiplication végétative sur de longues périodes. Ce processus doit en principe assurer la transmission du patrimoine génétique de la plante mère à la plante fille, cependant des mutations spontanées peuvent apparaître et être, dans certains cas, à l’origine de nouveaux génotypes. Les éléments transposables, par la variabilité génétique que peut générer leur mobilité, sont une source potentielle de mutation. Dans un contexte général de compréhension des mécanismes biologiques qui participent à l’évolution des génomes au cours des cycles de multiplication végétative, l’objectif de la thèse est d’étudier la structure et la dynamique des rétrotransposons du génome de la vigne. Au début de la thèse, trois rétrotransposons du génome de la vigne étaient décrits. Une première partie de ce travail est consacrée à la caractérisation de dix nouvelles familles de rétrotransposons qui se distinguent des trois éléments déjà connus chez la vigne, ainsi que des rétrotransposons décrits chez d’autres plantes. L’ensemble de ces familles comptent environ 1700 copies, complètes et potentiellement actives ou partiellement dégénérées, dont certaines se sont insérées dans le génome de la vigne au cours des deux derniers millions d’années. Au total, ces familles représentent près de 1,2 % du génome. Dans une deuxième partie, l’étude du niveau de transcription de ces rétrotransposons par des approches complémentaires montre que la présence de leurs transcrits est régulée dans les organes de la vigne en conditions normales de croissance, chaque famille d’éléments présentant un profil d’expression particulier selon l’organe considéré. De plus, l’expression de plusieurs rétrotransposons est induite dans les feuilles en réponse à la blessure et à l’infection par le mildiou. Le rôle des rétrotransposons dans l’évolution du génome de la vigne et la genèse de diversité intra-variétale reste encore à déterminer. Cependant, l’induction de l’expression de différentes familles en condition de stress permet d’envisager que ces éléments pourraient avoir participé à l’adaptation du génome en réponse aux contraintes culturales et aux conditions environnementales. De plus, la caractérisation de nouvelles familles d’éléments, dont certaines sont actives et présentes en un grand nombre de copies, peut permettre le développement d’outils moléculaires adaptés à l’identification et à la distinction des clones de vigne

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

Diplôme : Dr. d'UniversiteGrapevine (Vitis vinifera L.) varieties are composed by clones originated from a unique plant propagated by vegetative multiplication. This process theoretically ensure the transmission of genetic information from mother to daughter’s plants, but spontaneous mutations sometimes arise and can lead to the creation of new genotypes. Due to their ability to transpose, transposable elements are a source of mutation that causes genetic variability. To understand global mechanisms that participate to plant genome evolution during vegetative multiplication cycles, the main goal of this thesis is studying the structure and dynamics of grapevine retrotransposons. When this work started, only three grapevine retrotransposons were described. Therefore, the first part of this work is based on the characterization of ten new families, which are different from the elements previously described in grapevine or in other plant genomes. This ten new families represent near 1,700 copies, from full-length to degenerated, representing altogether about 1.2% of the genome. Some of these copies were integrated in the grapevine genome during the last two million years. In a second part, the transcription level of grapevine retrotransposons is evaluated by complementary approaches. The results show that their transcription level is regulated in different organs under normal growing conditions, each family showing a specific transcription profile. Moreover, the expression of many retrotransposons in leaves is induced by wounding and pathogen infection. The role of retrotransposons in grapevine genome evolution and in the genesis of intravarietal diversity has not been established, but induction of expression of several families under stress conditions allow us to suppose that this elements might have participated to genome adaptation in response to environmental challenges and grapevine culture by man. Moreover, the characterization of new transposable families contributes to the development of molecular tools allowing the identification and distinction of grapevine clones.Les variétés de vigne (Vitis vinifera L.) sont composées d’un ensemble de clones, tous issus de la même et unique plante propagée par multiplication végétative sur de longues périodes. Ce processus doit en principe assurer la transmission du patrimoine génétique de la plante mère à la plante fille, cependant des mutations spontanées peuvent apparaître et être, dans certains cas, à l’origine de nouveaux génotypes. Les éléments transposables, par la variabilité génétique que peut générer leur mobilité, sont une source potentielle de mutation. Dans un contexte général de compréhension des mécanismes biologiques qui participent à l’évolution des génomes au cours des cycles de multiplication végétative, l’objectif de la thèse est d’étudier la structure et la dynamique des rétrotransposons du génome de la vigne. Au début de la thèse, trois rétrotransposons du génome de la vigne étaient décrits. Une première partie de ce travail est consacrée à la caractérisation de dix nouvelles familles de rétrotransposons qui se distinguent des trois éléments déjà connus chez la vigne, ainsi que des rétrotransposons décrits chez d’autres plantes. L’ensemble de ces familles comptent environ 1700 copies, complètes et potentiellement actives ou partiellement dégénérées, dont certaines se sont insérées dans le génome de la vigne au cours des deux derniers millions d’années. Au total, ces familles représentent près de 1,2 % du génome. Dans une deuxième partie, l’étude du niveau de transcription de ces rétrotransposons par des approches complémentaires montre que la présence de leurs transcrits est régulée dans les organes de la vigne en conditions normales de croissance, chaque famille d’éléments présentant un profil d’expression particulier selon l’organe considéré. De plus, l’expression de plusieurs rétrotransposons est induite dans les feuilles en réponse à la blessure et à l’infection par le mildiou. Le rôle des rétrotransposons dans l’évolution du génome de la vigne et la genèse de diversité intra-variétale reste encore à déterminer. Cependant, l’induction de l’expression de différentes familles en condition de stress permet d’envisager que ces éléments pourraient avoir participé à l’adaptation du génome en réponse aux contraintes culturales et aux conditions environnementales. De plus, la caractérisation de nouvelles familles d’éléments, dont certaines sont actives et présentes en un grand nombre de copies, peut permettre le développement d’outils moléculaires adaptés à l’identification et à la distinction des clones de vigne

Specific transcription profiles of 11 grapevine LTR-retrotransposon families

National audienceGrapevine clones belonging to a same variety originate from a unique seedling through vegetative propagation. Thus, it is expected that clones are identical to their mother plant and consequently also among them. However, some clones show variations due to somatic mutations. Our work aims to understand the nature of these mutations, with a special focusing on the role of retrotransposons. The choice of retrotransposons is justified because transcription of these elements, which are able to insert new copies at different positions around the host genome, is often induced by biotic and abiotic stresses. Using as query a canonical element for each retrotransposon family identified in the grapevine genome, we searched EST sequences released in the grapevine TIGR database. Most of the EST showing identity with query sequences were mined from a cDNA library obtained from leaves under abiotic stress. Then, the level of expression of 11 grapevine retrotransposons was evaluated by RT-PCR in plant organs at different stages of vegetative and reproductive development. Our results show that each retrotransposon family has a specific transcription pattern during development, from silence to near constitutive expression. The expression was observed mainly in growing tissues such as flower buds, apical meristems and somatic embryos. Changes in the transcription level were also evaluated in leaves in response to wounding or downy mildew infection, two stresses naturally undergoes by grapevine plants during its long productive growth. Depending on the retrotransposon family, we have observed an increase in the level of transcription in leaves, 6 or 12 hours after the stress application. Thus, our results suggest that retrotransposons of the grapevine genome are activated by environmental challenges

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

STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Tvv1-SD, un nouveau rétrotransposon du génome de S.demissum, proche de la famille Tvv1 de la vigne

National audienceThe Tvv1 retrotransposon family has been previously described in the grapevine genome (Pelsy and Merdinoglu 2002). Searches on databases, using the gag domain of Tvv1 as query, revealed the presence of a transposable element in a BAC clone of Solanum demissum, a wild potato variety. The sequence of this element, 4821 bp-long, shares all the classic features of LTR-retrotransposons. An internal sequence is flanked by two LTRs (sized 180 bp), and contains an ORF showing, in this order, gag, protease, integrase, reverse transcriptase and RnaseH domains, which characterize Ty1-copia-like retrotransposons. Sequence analysis of the complete S.demissum element showed that most domains present a high level of similarity to the Tvv1 family. Based on this observation, this new element was named Tvv1-SD. Our results suggest that the Tvv1 family of V.vinifera and Tvv1-SD of S.demissum may have a common origin despite their presence in very distant species

Are grapevine retrotransposons expressed during development and activated by abiotic stress ?

National audienceGrapevine varieties are composed of clones that derived by vegetation propagation from a same and unique seedling. It is expected that all clones are identical to their mother plant, however some can show variations in their morphological and agronomical traits due to somatic mutations. Retrotransposons, that are genetic entities able to insert new copies at different positions around the host genome, are known to be activated by biotic or abiotic stress. Grapevine is a perennial plant strongly stressed during a long productive growth –by grafting, pruning, pathogen attack, UV…- therefore retrotransposons may be able to play a role in the differentiation of clones belonging to the same variety. Three retrotransposons from the grapevine genome are described : GRET-1, Tvv1 and Vine-1. The main goal of this work consisted in evaluating by RT-PCR the level of expression of these three retrotransposons during development and under abiotic stress. To target each retrotransposon, two different specific domains were amplified, RT and gag domains. During shoot development, expression was only detected in roots and growing tissues such as flower buds, apical meristems and somatic embryos. Induction of retrotransposons transcription was evaluated under two abiotic stress. Depending of the retrotransposon family, a differential increase of expression was detected in UV exposed or wounded leaves, in general 6 or 12 hours after stress application. This result is the first evidence that grapevine retrotransposons are specifically expressed in undifferentiated tissues. Moreover, their expression is induced by stress conditions undergone by grapevine during its long productive growth and vegetative propagation. If the level of transposition is proportional to the level of transcription, we could believe that retrotransposons participate in clone differentiation

Régulation de l'activité des éléments transposables

National audienceLes éléments transposables sont des éléments génétiques autonomes capables de se multiplier dans le génome. Ils sont en général maintenus à l’état dormant, mais certains stress peuvent induire leur transposition, c’est-à-dire leur déplacement vers un nouveau site. Leur activité est considérée comme un facteur évolutif qui participe à l’adaptation des organismes aux variations de l’environnemen