Genetic diversity and mechanisms of resistance to Aphanomyces euteiches in legumes. – Immunit-Ae

Aphanomyces euteiches, a root oomycete of legumes is the main limiting biotic factor for French and European pea production. To accelerate genetic improvement of resistance in pea to A. euteiches we propose to use genetic and genomic resources developed in the model legume M. truncatula. This includes notably the recent discovery of a major locus of resistance to A. euteiches in this plant. This locus was identified in two different sources of resistance with several isolates of this parasite. In the A17 sequenced line,this locus, named prAe1 conferred a partial recessive resistance and was narrowed to a 135 kb genomic region which does not contain any classical NBS-LRR resistance gene. In the DZA45.5 line, the AER1 gene, located in the same genomic region is responsible for a complete dominant resistance and act epistatically with other loci to confer resistance against several A. euteiches isolates.
From this results, the first objective of this proposal is to identify sequence(s) located in the prAe1/AER1 sequences that are involved in the resistance to A. euteiches. Following the sequencing of this locus in DZA45.5 and in the susceptible F83005.5 line, data comparison with A17 will lead to fine mapping studies to identify the gene(s) (or sequences) involved in resistance. These sequence(s) will then be functionally validated in complementation studies performed in transformed M. truncatula roots, in available mutants and/or in transgenic M. sativa plants. . To analyze prAe1-related molecular mechanisms, near-isogenic lines (NIL) at this locus will be analyzed following A. euteiches inoculation with Affymetrix chips in microarray experiments. The transcriptome of divided pericycle cells, which are only observed in the resistant NIL, wil also be analyzed by using RNAs from these cells that will be microdissected by laser capture microdissection.
The second objective of this proposal is to mine the diversity of loci involved in resistance to several strains of A. euteiches in different resistance sources of a M. truncatula collection. We will take advantage of a privileged access to data generated by an ongoing NSF Hapmap re-sequencing genome project in M. truncatula. Two approaches will be used : i)- a genome-wide association mapping analysis in a core-collection of 192 M. truncatula lines, which genomes are currently under re-sequencing and, ii)- a QTL mapping approach in three available joint family populations, which could use whole genome sequence data of the founder lines for developing a nested association mapping study for increasing resolution of QTL detection. Results from the two approaches will be compared.
The third goal of this project is to assess conservation of genes, loci and mechanisms involved in resistance to A. euteiches between M. truncatula and main leguminous crops, especially pea. To reach it, we will take advantage of the high amount of knowledge and data previously acquired, particularly in pea (markers, sequences, QTL). First, synteny of Aphanomyces resistance loci identified in M. truncatula and pea will be studied by generating bridge markers in target regions between the two genomes. Second, orthologous genes to candidate genes at the main Aphanomyces resistance loci detected in M. truncatula will be searched in different leguminous crops, especially pea. Markers at these orthologous genes will be developed. In pea, association between resistance and allelic diversity at best candidate genes (i.e. in syntenic regions and/or colocalizing with QTL) will be validated in a pea core-collection. Functional role in resistance of a few validated candidate genes will be analyzed with TILLING mutants. Finally, cytological studies will be undertaken to compare resistance mechanisms observed in M. truncatula and several other crop legumes.