The genetics and mechanism of avoidance of rust infection in Hordeum chilense

Hordeum chilense is a perennial species occurring in Chile and Argentina. This wild barley species shows a very wide range of variation of morphological and agronomic characters and crosses easily with other members of the Triticeae tribe.H. chilense is one of the five wild barley species in which avoidance of rust fungi as been reported. The avoidance mechanism to rust fungi is characterised by stomata overgrowth by the fungal germ tube, with no appressorium formation or penetration of the stomata, resulting in early failure of the infection process. This avoidance is an interesting mechanism of defence against rust infection, especially when it could be transferred to cultivated cereal species.In this thesis the genetic basis of this avoidance mechanism and associations of avoidance with other characters in H. chilense were established.We tested the hypothesis that the avoidance character occurs in a certain morphologically and molecularly distinct ecotype of H. chilense . A H. chilense collection of 88 accessions was characterised for morphological and agronomic traits, level of avoidance of Puccinia hordei , habitat of origin and AFLP fingerprint. Cluster analysis using both morphological/agronomic and AFLP fingerprint data suggested three distinct clusters of accessions. High avoidance was typical to the accessions of one of these three clusters. The accessions in the cluster with the higher levels of avoidance had been collected in humid habitats. This putative subspecific taxon was further characterised by shorter and wider spikes, more erect culms, a shorter uppermost internode until flag leaf and a greater amount of stomata density on the abaxial leaf side. We conclude that H. chilense consists of at least three rather well defined morphologically and genetically distinct subspecific taxa, one of which has a very high level of avoidance of barley leaf rust.We studied the effect of the cuticular wax layer on the orientation of germ tube growth and on appressorium differentiation of P. hordei . Several orientation parameters and appressorium differentiation of P. hordei germ tubes were measured on H. chilense leaves with and without the wax layer.Removal of the cuticular wax layer did not result in poor and also not in better germ tube orientation. Evidence was obtained that epidermal cell junctions rather than the wax crystals provided the landmarks to guide germ tubes along the transverse axis of the leaf. On high avoidance accessions the removal of the wax layer allowed appressoria to develop over stomata that would otherwise be overgrown. This suggests that the overgrowth of stomata on H. chilense leaves by P. hordei germ tubes is mainly due to the wax covering of the stomatal apparatus.A molecular map of the wild barley H. chilense would greatly facilitate to map and efficiently transfer agronomic traits from H. chilense to cereal genomes. As a first step towards a map construction, we evaluated AFLP markers in H. chilense using diploid wheat ( Triticum monococcum ) and cultivated barley ( H. vulgare ) as references. H. chilense showed a higher percentage of polymorphisms than diploid wheat. It was remarkable that, based on AFLP markers generated by 12 Eco RI/ Mse I primer combinations, the cultivated barley was more similar to diploid wheat than to H. chilense . Even more surprisingly, the genetic distance between the interfertile H. chilense accessions (H1 and H7) was almost as large as the genetic distance between the non-crossable cultivated barley and diploid wheat. Eco RI/ Mse I AFLP fingerprints revealed more polymorphism than Pst I/ Mse I AFLP fingerprints for all species tested and were chosen for generating the first AFLP linkage map of H. chilense .The mapping population consisted of 100 F 2 plants derived from a cross between two genetically distinct H. chilense accessions that were contrasting for the level of avoidance, H1 and H7. The constructed map contained 443 AFLP markers, on nine long and ten shorter linkage groups, covering a genetic distance of 714 cM. Nine of these linkage groups were assigned to H. chilense chromosomes using a set of H. chilense in T. aestivum addition and substitution lines. Strong clustering of AFLP markers was observed at putative centromeric regions. A skeletal map with a uniform distribution of markers was extracted from this linkage map. This skeletal map was applied to detect and map QTLs underlying avoidance and stoma density on the abaxial leaf epidermis.Three QTLs were detected for avoidance and three other QTLs for stoma density. Both traits segregated independently in the F 2 .Avoidance on H. chilense is effective to barley leaf rust, wheat leaf rust and rye leaf rust. As H. chilense shows good crossability with several cultivated cereals avoidance of leaf rusts may be introgressed into these cultivated cereals, especially wheat. Addition and substitution lines of H. chilense in Chinese Spring wheat cv. are already available or being developed as a first step towards transfer of the genes governing the avoidance trait.</p

Orientation of Germ Tubes of Puccinia hordei on the Hordeum chilense Leaf Surface

The directional growth of urediospores germ tubes along the transverse axis of a cereal's leaf is considered to be a response to stimuli from the plant surface. In order to find out if the germ tube growth is directed towards stomata, and if the cuticular wax layer plays a role in this orientated growth, several orientation parameters of Puccinia hordei Otth germ tube were measured on Hordeum chilense Roem. and Schult, leaves with and without the wax layer. Orientated growth of the germ tubes seems to start only upon contact with the epidermal cell junctions. The lateral growing of the germ tube over the first epidermal cell junction that it meets is longer than the subsequent lateral growings. These lateral growings, especially the first one, may help the germ tube to grow along the transverse axis of the leaf. No evidence was found of attraction of the germ tube by stomata. Removal of the cuticular wax layer did not result in poor germ tube orientation. This suggests that the leaf wax layer has no role on the orientation of the germ tube

Orientation of Germ Tubes of Puccinia hordei on the Hordeum chilense Leaf Surface

The directional growth of urediospores germ tubes along the transverse axis of a cereal's leaf is considered to be a response to stimuli from the plant surface. In order to find out if the germ tube growth is directed towards stomata, and if the cuticular wax layer plays a role in this orientated growth, several orientation parameters of Puccinia hordei Otth germ tube were measured on Hordeum chilense Roem. and Schult, leaves with and without the wax layer. Orientated growth of the germ tubes seems to start only upon contact with the epidermal cell junctions. The lateral growing of the germ tube over the first epidermal cell junction that it meets is longer than the subsequent lateral growings. These lateral growings, especially the first one, may help the germ tube to grow along the transverse axis of the leaf. No evidence was found of attraction of the germ tube by stomata. Removal of the cuticular wax layer did not result in poor germ tube orientation. This suggests that the leaf wax layer has no role on the orientation of the germ tube

Morphology and AFLP markers suggest three Hordeum chilense ecotypes that differ in avoidance to rust fungi

In Hordeum chilense Roem. & Schult., a high variation in the level of avoidance to infection of barley leaf rust (Puccinia hordei Otth) occurs. Probably resulting from the properties of the stomata, the rust germ tube overgrows stomata, and the infection process fails in an early stage. In the present study we tested the hypothesis that the avoidance character occurs in certain morphologically and molecularly distinct ecotypes of H. chilense. Eighty-eight H. chilense accessions were inoculated with P. hordei to assess the level of avoidance. The accessions were described for 30 morphological characters and three AFLP primer combinations. Cluster analysis using both morphological and AFLP fingerprint data suggested three distinct clusters of accessions. One of the clusters had a particularly high level of avoidance. This putative subspecific taxon was characterized by shorter and wider spikes, more erect culms, a greater number of stomata per square centimetre on the abaxial leaf side, and a shorter uppermost internode until flag leaf. All accessions clustered in this subspecific taxon were collected from humid habitats. We conclude that H. chilense consists of at least three rather well defined, morphologically and genetically distinct subspecific taxa, one of which has a very high level of avoidance to barley leaf rust

QTL mapping provides evidence for lack of association of the avoidance of leaf rust in Hordeum chilense with stomata density

In cereals, rust fungi are among the most harmful pathogens. Breeders usually rely on short-lived hypersensitivity resistance. As an alternative, "avoidance" may be a more durable defence mechanism to protect plants to rust fungi. In Hordeum chilense avoidance is based on extensive wax covering of stomata, which interferes with the induction of appressorium formation by the rust fungi. High avoidance levels are associated with a higher stoma density on the abaxial leaf epidermis. The avoidance level was assessed as the percentage of germ tube/stoma encounters that did not result in appressorium differentiation by Puccinia hordei, the barley leaf rust fungus. One hundred F-2 individuals from the cross between two H. chilense accessions with contrasting levels of avoidance showed a continuous distribution for avoidance of the rust fungus and for stoma density, indicating quantitative inheritance of the traits. No significant correlation was found between avoidance and stoma density in the segregating F2 population. In order to map quantitative trait loci (QTLs) for both traits, an improved molecular marker linkage map was constructed, based on the F2 population. The resulting linkage map spanned 620 cM and featured a total of 437 AFLP markers, thirteen RFLPs, four SCARs, nine SSRs, one STS and two seed storage protein markers. It consisted of seven long and two shorter linkage groups, and was estimated to cover 81% of the H. chilense genome. Restricted multiple interval mapping identified two QTLs for avoidance and three QTLs for stoma density in the abaxial leaf surface. The QTLs for avoidance were mapped on chromosome 3 and 5; those for stoma density on chromosomes 1, 3 and 7. Only the two QTLs regions located on chromosome 3 (one for avoidance and the other for stoma density) overlapped. The wild barley H. chilense has a high crossability with other members of the Triticeae tribe. The knowledge on the location of the QTLs responsible for the avoidance trait is a prerequisite to transfer this favourable agronomic trait from H. chilense to cultivated cereal genomes

Breeding annual legumes for sustainable agricultures must target for new and more complex variety ideotypes

International audienceAlthough yield and total biomass produced by annual legumes remain major objectives for breeders, environment-friendly, resource use efficient including symbiotic performance, resilient production in the context of climate change, adaptation to sustainable cropping systems (reducing leaching and glasshouse gas emissions), adaptation to diverse uses (seeds for feeds foods, non-food, forage or green manures), and finally new ecological services such as pollinator protection, imply the development of innovative genotypes, definition of new ideotypes and acceptance of their commercialisation. Taken as a whole, this means more complex and integrated objectives for breeders. Several illustrations will be given of breeding such complex traits for different annual legume species. Genetic diversity for root development and for ability to establish efficient symbioses with rhizobia and mycorrhiza can contribute to better resource management (N, P, water). Shoot architectures and phenologies can contribute to yield and biotic protection (weeds, disease, parasitic insects). Long winter cycles or short cycles, tolerance to biotic or abiotic stresses, are key features for the introduction of annual legumes in low input cropping systems. Adaptation to intercropping requires adapted genotypes. Improved health and nutritional value for humans are key objectives for developing new markets. Modifying product composition often requires the development of specific varieties and sometimes the need to break negative genetic correlations with yield. A holistic approach in legume breeding is important for defining objectives with farmers, processors and consumers. The varietal structures may be more complex, combining genotypes, plant species and associated symbionts. New tools to build, evaluate and register them are important