In depth molecular characterization of T-DNA and Tos17 integration patterns in the Génoplante rice insertion collection : [P216]

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.