Genome-Based Discovery of Polyketide-Derived Secondary Metabolism Pathways in the Barley Pathogen Ramularia collo-cygni.

Ramularia collo-cygni causes Ramularia leaf spot (RLS) disease of barley. The fungus develops asymptomatically within its host until late in the growing season, when necrotic lesions become visible on upper leaves. Fungal secondary metabolites (SM) have been proposed as important factors in RLS lesion formation but the biosynthetic pathways involved remain largely unknown. Mining the R. collo-cygni genome revealed the presence of 10 polyketide synthases (PKS), 10 nonribosomal peptide synthetases (NRPS), and 3 hybrid PKS-NRPS (HPS) identified within clusters of genes with predicted functions associated with secondary metabolism. SM core genes along with their predicted transcriptional regulators exhibited transcriptional coexpression during infection of barley plants. Moreover, their expression peaked during early stages of host colonization and preceded or overlapped with the appearance of disease symptoms, suggesting that SM may manipulate the host to promote colonization or protect R. collo-cygni from competing organisms. Accordingly, R. collo-cygni inhibited the growth of several fungi in vitro, indicating that it synthesized and excreted antifungal agents. Taken together, these findings demonstrate that the R. collo-cygni genome contains the genetic architecture to synthesize a wide range of SM and suggests that coexpression of PKS and HPS is associated with competitive colonization of the host and early symptom development.

Development and use of microsatellite markers to study diversity, reproduction and population genetic structure of the cereal pathogen Ramularia collo-cygni.

Ramularia collo-cygni (Rcc) is a major pathogen of barley that causes economically serious yield losses. Disease epidemics during the growing season are mainly propagated by asexual air-borne spores of Rcc, but it is thought that Rcc undergoes sexual reproduction during its life cycle and may also disperse by means of sexual ascospores. To obtain population genetic information from which to infer the extent of sexual reproduction and local genotype dispersal in Rcc, and by implication the pathogen's ability to adapt to fungicides and resistant cultivars, we developed ten polymorphic microsatellite markers, for which primers are presented. We used these markers to analyse the population genetic structure of this cereal pathogen in two geographically distant populations from the Czech Republic (n=30) and the United Kingdom (n=60) that had been sampled in a spatially explicit manner. Genetic diversity at the microsatellite loci was substantial, Ht=0.392 and Ht=0.411 in the Czech and UK populations respectively, and the populations were moderately differentiated at these loci (Θ=0.111, P<0.01). In both populations the multilocus genotypic diversity was very high (one clonal pair per population, resulting in >96% unique genotypes in each of the populations) and there was a lack of linkage disequilibrium among loci, strongly suggesting that sexual reproduction is an important component of the life cycle of Rcc. In an analysis of spatial genetic structure, kinship coefficients in all distance classes were very low (-0.0533 to 0.0142 in the Czech and -0.0268 to 0.0042 in the Scottish population) and non-significant (P>0.05) indicating lack of subpopulation structuring at the field scale and implying extensive dissemination of spores. These results suggest that Rcc possesses a high evolutionary potential for developing resistance to fungicides and overcoming host resistance genes, and argue for the development of an integrated disease management system that does not rely solely on fungicide applications.

Inbreeding depression in self-incompatible North-American Arabidopsis lyrata: disentangling genomic and S-locus-specific genetic load.

Newly formed selfing lineages may express recessive genetic load and suffer inbreeding depression. This can have a genome-wide genetic basis, or be due to loci linked to genes under balancing selection. Understanding the genetic architecture of inbreeding depression is important in the context of the maintenance of self-incompatibility and understanding the evolutionary dynamics of S-alleles. We addressed this using North-American subspecies of Arabidopsis lyrata. This species is normally self-incompatible and outcrossing, but some populations have undergone a transition to selfing. The goals of this study were to: (1) quantify the strength of inbreeding depression in North-American populations of A. lyrata; and (2) disentangle the relative contribution of S-linked genetic load compared with overall inbreeding depression. We enforced selfing in self-incompatible plants with known S-locus genotype by treatment with CO(2), and compared the performance of selfed vs outcrossed progeny. We found significant inbreeding depression for germination rate (δ=0.33), survival rate to 4 weeks (δ=0.45) and early growth (δ=0.07), but not for flowering rate. For two out of four S-alleles in our design, we detected significant S-linked load reflected by an under-representation of S-locus homozygotes in selfed progeny. The presence or absence of S-linked load could not be explained by the dominance level of S-alleles. Instead, the random nature of the mutation process may explain differences in the recessive deleterious load among lineages.

The effect of mating system on growth of Arabidopsis lyrata in response to inoculation with the biotrophic parasite Albugo candida.

The effects of variation in host reproductive systems on response to pathogens are not well understood. We inoculated individuals from outcrossing and inbreeding populations of North American Arabidopsis lyrata with Albugo candida (white blister rust) to test the effect of mating system and heterozygosity on disease response. We observed three host infection phenotypes, classified as fully resistant, partially resistant and fully susceptible. Overall, inbreeding populations had more susceptible and fewer partially resistant individuals than outcrossing populations, but the highest proportion of resistant individuals was found in two of the inbreeding populations. Mating system did not affect relative growth rate of inoculated plants, but there were strong effects of population and infection phenotype. We conclude that mating system per se does not determine the resistance of natural A. lyrata populations to infection by Albugo, but that the increased variability in responses among inbreeding populations may be due to reduced effective population size.

Multiple losses of self-incompatibility in North-American Arabidopsis lyrata?: phylogeographic context and population genetic consequences.

Arabidopsis lyrata is mostly outcrossing due to a sporophytic self-incompatibility (SI) system but around the Great Lakes of North America some populations have experienced a loss of SI. We researched the loss of SI in a phylogeographic context. We used cpDNA and microsatellite markers to test if populations of North-American A. lyrata around the Great Lakes have experienced different (recent) histories, and linked this with individually established selfing phenotype and population level realized outcrossing rates calculated based on variation in progeny arrays at multi-locus microsatellite markers. We found three chloroplast haplotypes, in two of which the loss of self-incompatibility had occurred independently. Shifts to high rates of inbreeding were most apparent in one of these lineages but individuals showing loss of SI occurred in all three. Self-compatible individuals usually showed a reduction of observed heterozygosity (H(O)) compared to outcrossing individuals. In the lineage that included the populations with the highest levels of inbreeding, this reduction was more substantial. This may indicate that the loss of SI in this lineage did not occur as recently as in the other lineage. The geographic distribution of the haplotypes suggested that there had been at least two independent colonization routes to the north of the Great Lakes following the last glaciation. This is consistent with postglacial migration patterns that have been suggested for other organisms with limited dispersal, such as reptiles and amphibians.