Publications des agents du Cirad

Cirad

High throughput T-DNA insertion mutagenesis in rice : A first step towards in silico reverse genetics

Sallaud C., Gay C., Larmande P., Bes M., Piffanelli P., Piegu B., Droc G., Regad F., Bourgeois E., Meynard D., Perin C., Sabau X., Ghesquière A., Glaszmann J.C., Delseny M., Guiderdoni E.. 2004. Plant Journal, 39 (3) : p. 450-464.

DOI: 10.1111/j.1365-313X.2004.02145.x

A library of 29 482 T-DNA enhancer trap lines has been generated in rice cv. Nipponbare. The regions flanking the T-DNA left border from the first 12 707 primary transformants were systematically isolated by adapter anchor PCR and sequenced. A survey of the 7480 genomic sequences larger than 30 bp (average length 250 bp), representing 56.4% of the total readable sequences and matching the rice bacterial artificial chromosome/ phage artificial chromosome (BAC/PAC) sequences assembled in pseudomolecules allowed the assigning of 6645 (88.8%) T-DNA insertion sites to at least one position in the rice genome of cv. Nipponbare. T-DNA insertions appear to be rather randomly distributed over the 12 rice chromosomes, with a slightly higher insertion frequency in chromosomes 1, 2, 3 and 6. The distribution of 723 independent T-DNA insertions along the chromosome 1 pseudomolecule did not differ significantly from that of the predicted coding sequences in exhibiting a lower insertion density around the centromere region and a higher density in the subtelomeric regions where the gene density is higher. Further establishment of density graphs of T-DNA inserts along the recently released 12 rice pseudomolecules confirmed this non-uniform chromosome distribution. T-DNA appeared less prone to hot spots and cold spots of integration when compared with those revealed by a concurrent assignment of the Tos17 retrotransposon flanking sequences deposited in the National Center for Biotechnology Information (NCBI). T-DNA inserts rarely integrated into repetitive sequences. Based on the predicted gene annotation of chromosome 1, preferential insertion within the first 250 bp from the putative ATG start codon has been observed. Using 4 kb of sequences surrounding the insertion points, 62% of the sequences showed significant similarity to gene encoding known proteins (E-value <1.00 e-05). To illustrate the in silico reverse genetic approach, identification of 83 T-DNA insertions within gen

Mots-clés : oryza; génie génétique; gène; adn; mutation; mutagène; méthode d'amélioration génétique

Documents associés

Article (a-revue à facteur d'impact)

Agents Cirad, auteurs de cette publication :