Piffanelli Pietro, Mieulet Delphine, Bes Martine, Lanau Nadège, Rouvière Claire, Droc Gaëtan, Meynard Donaldo, Bourgeois Emmanuelle, Sallaud Christophe, Guiderdoni Emmanuel.
2005. In depth molecular characterization of T-DNA and Tos17 integration patterns in the Génoplante rice insertion collection : [P216].
In : 5th International Rice Genetics Symposium (A platform for exploring developments in rice genetics and their applications) and 3rd International Symposium of Rice Functional Genomics. Abstract. IRRI
Résumé : In the framework of the Genoplante project, we have produced a library of rice (cv. Nipponbare) lines containing T-DNA and novel insertions of Tos 17 retrotransposon. Using a subset of 400 primary transformants harbouring the pC-4978 T-DNA construct, we attempted to establish a relationship between T-DNA organization, walk-PCR patterns of flanking regions and nature of the FSTs. Thorough Southern blot analyses revealed integration of an average of 2.2 T-DNA copies per plant, associated with an often-complex T-DNA organisation resulting from sequence rearrangements. Moreover, we observed that backbone vector sequences are often integrated in transformants harbouring multiple T-DNA copies. A highly efficient digestion-ligation walk-PCR protocol to amplify genomic sequences flanking both right and left T-DNA borders was developed for five restriction enzymes. Our study revealed that the analysis by walk-PCR with three enzymes enabled to amplify at least one Flanking Sequence Tag (FST) from 90% of the primary transformants. Comparative analysis of FST sequences obtained from both right and left TDNA borders revealed that in approximately 25% of cases novel FST sequences were obtained. In the same subset of 400 lines we estimated that on average insertion of 3.2 new copies of TOS17 had occurred per primary transformant. A novel highly efficient protocol of selective amplification of newly inserted copies of Tos17 retrotransposon was developed and enabled us to recover at least one sequence flanking a newly-transposed Tos17 copy in 70% the lines.Walk-PCR based protocols for both T-DNA and TOS17 enabled to obtain on average 70% of monoband and 30% of multiband products for every restriction enzyme we used. The development of a pipeline of bioanalysis of the sequences derived from multiband products, enabled us to maximize the efficiency of our walk-PCR based technology. The results of this pilot study will serve as a guideline to efficiently analyse the entire rice collection and help us to understand the mechanisms of integration of both T-DNA and TOS17 in the rice genome.
Mots-clés Agrovoc : Oryza
Classification Agris : F30 - Génétique et amélioration des plantes
Auteurs et affiliations
- Piffanelli Pietro, CIRAD-AMIS-UMR PIA (FRA)
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Mieulet Delphine
ORCID: 0000-0001-6220-0372
- Bes Martine, CIRAD-AMIS-UMR PIA (FRA)
- Lanau Nadège
- Rouvière Claire
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Droc Gaëtan, CIRAD-AMIS-BIOTROP (FRA) ORCID: 0000-0003-1849-1269
- Meynard Donaldo, CIRAD-AMIS-UMR PIA (FRA)
- Bourgeois Emmanuelle
- Sallaud Christophe
- Guiderdoni Emmanuel, CIRAD-AMIS-UMR PIA (FRA)
Autres liens de la publication
Source : Cirad - Agritrop (https://agritrop.cirad.fr/550230/)
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