In-depth molecular and phenotypic characterization in a rice insertion line library facilitates gene identification through reverse and forward genetics approaches.

We report here the molecular and phenotypic features of a library of 31,562 insertion lines generated in the model japonica cultivar Nipponbare of rice (Oryza sativa L.), called Oryza Tag Line (OTL). Sixteen thousand eight hundred and fourteen T-DNA and 12,410 Tos17 discrete insertion sites have been characterized in these lines. We estimate that 8686 predicted gene intervals--i.e. one-fourth to one-fifth of the estimated rice nontransposable element gene complement--are interrupted by sequence-indexed T-DNA (6563 genes) and/or Tos17 (2755 genes) inserts. Six hundred and forty-three genes are interrupted by both T-DNA and Tos17 inserts. High quality of the sequence indexation of the T2 seed samples was ascertained by several approaches. Field evaluation under agronomic conditions of 27,832 OTL has revealed that 18.2% exhibit at least one morphophysiological alteration in the T1 progeny plants. Screening 10,000 lines for altered response to inoculation by the fungal pathogen Magnaporthe oryzae allowed to observe 71 lines (0.7%) developing spontaneous lesions simulating disease mutants and 43 lines (0.4%) exhibiting an enhanced disease resistance or susceptibility. We show here that at least 3.5% (four of 114) of these alterations are tagged by the mutagens. The presence of allelic series of sequence-indexed mutations in a gene among OTL that exhibit a convergent phenotype clearly increases the chance of establishing a linkage between alterations and inserts. This convergence approach is illustrated by the identification of the rice ortholog of AtPHO2, the disruption of which causes a lesion-mimic phenotype owing to an over-accumulation of phosphate, in nine lines bearing allelic insertions.

Building a mutant resource for the study of disease resistance in rice reveals the pivotal role of several genes involved in defence.

In Arabidopsis, gene expression studies and analysis of knock-out (KO) mutants have been instrumental in building an integrated view of disease resistance pathways. Such an integrated view is missing in rice where shared tools, including genes and mutants, must be assembled. This work provides a tool kit consisting of informative genes for the molecular characterization of the interaction of rice with the major fungal pathogen Magnaporthe oryzae. It also provides for a set of eight KO mutants, all in the same genotypic background, in genes involved in key steps of the rice disease resistance pathway. This study demonstrates the involvement of three genes, OsWRKY28, rTGA2.1 and NH1, in the establishment of full basal resistance to rice blast. The transcription factor OsWRKY28 acts as a negative regulator of basal resistance, like the orthologous barley gene. Finally, the up-regulation of the negative regulator OsWRKY28 and the down-regulation of PR gene expression early during M. oryzae infection suggest that the fungus possesses infection mechanisms that enable it to block host defences.

A cytomolecular approach to assess the potential of gene transfer from a crop (Triticum turgidum L.) to a wild relative (Aegilops geniculata Roth.).

When a crop hybridizes with a wild relative, the potential for stable transmission to the wild of any crop gene is directly related to the frequency of crop-wild homoeologous pairing for the chromosomal region where it is located within the crop genome. Pairing pattern at metaphase I (MI) has been examined in durum wheat x Aegilops geniculata interspecific hybrids (2n=4x=ABUgMg) by means of a genomic in-situ hybridization procedure that resulted in simultaneous discrimination of A, B and wild genomes. The level of MI pairing in the hybrids varied greatly depending on the crop genotype. However, their pattern of homoeologous association was very similar, with a frequency of wheat-wild association close to 60% in all genotype combinations. A-wild represented 80-85% of wheat-wild associations which supports that, on average, A genome sequences are much more likely to be transferred to this wild relative following interspecific hybridization and backcrossing. Combination of genomic DNA probes and the ribosomal pTa71 probe has allowed to determine the MI pairing behaviour of the major NOR-bearing chromosomes in these hybrids (1 B, 6B, 1 Ug and 5 Ug), in addition to wheat chromosome 4A which could be identified with the sole use of genomic probes. The MI pairing pattern of the wild chromosome arms individually examined has confirmed a higher chance of gene escape from the wheat A genome. However, a wide variation regarding the amount of wheat-wild MI pairing among the specific wheat chromosome regions under analysis suggests that the study should be extended to other homoeologous groups.