A. Vanden Broeck

Identification of AFLP molecular markers for resistance against Melampsora larici-populina in Populus

We have identified AFLP markers tightly linked to the locus conferring resistance to the leaf rust Melampsora larici-populina in Populus. The study was carried out using a hybrid progeny derived from an inter-specific, controlled cross between a resistant Populus deltoides female and a susceptible P. nigra male. The segregation ratio of resistant to susceptible plants suggested that a single, dominant locus defined this resistance. This locus, which we have designated Melampsora resistance (Mer), confers resistance against E1, E2, and E3, three different races of Melampsora larici-populina. In order to identify molecular markers linked to the Mer locus we decided to combine two different techniques: (1) the high-density marker technology, AFLP, which allows the analysis of thousands of markers in a relatively short time, and (2) the Bulked Segregant Analysis (BSA), a method which facilitates the identification of markers that are tightly linked to the locus of interest. We analyzed approximately 11,500 selectively amplified DNA fragments using 144 primer combinations and identified three markers tightly linked to the Mer locus. The markers can be useful in current breeding programs and are the basis for future cloning of the resistance gene.

Ex-situ conservation of Black poplar in Europe: genetic diversity in nine gene bank collections and their value for nature development

Populus nigra L. is a pioneer tree species of riparian ecosystems that is threatened with extinction because of the loss of its natural habitat. To evaluate the existing genetic diversity of P. nigra within ex-situ collections, we analyzed 675 P. nigra L. accessions from nine European gene banks with three amplified fragment length polymorphism (AFLP) and five microsatellite [or simple sequence repeat (SSR)] primer combinations, and 11 isozyme systems. With isozyme analysis, hybrids could be detected, and only 3% were found in the gene bank collection. AFLP and SSR analyses revealed effectively that 26% of the accessions were duplicated and that the level of clonal duplication varied from 0% in the French gene bank collection up to 78% in the Belgian gene bank collection. SSR analysis was preferred because AFLP was technically more demanding and more prone to scoring errors. To assess the genetic diversity, we grouped material from the gene banks according to topography of the location from which the accessions were originally collected (river system or regions separated by mountains). Genetic diversity was expressed in terms of the following parameters: percentage of polymorphic loci, observed and effective number of alleles, and Nei’s expected heterozygosity or gene diversity (for AFLP). Genetic diversity varied from region to region and depended, to some extent, on the marker system used. The most unique alleles were identified in the Danube region (Austria), the Rhône region (France), Italy, the Rijn region (The Netherlands), and the Ebro region (Spain). In general, the diversity was largest in the material collected from the regions in Southern Europe. Dendrograms and principal component analysis resulted in a clustering according to topography. Material from the same river systems, but from different countries, clustered together. The genetic differentiation among the regions (Fst/Gst) was moderate.

Natural hybridisation between Populus nigra L. and P. x canadensis Moench. Hybrid offspring competes for niches along the Rhine river in the Netherlands

Black poplar (Populus nigra L.) is a major species for European riparian forests but its abundance has decreased over the decades due to human influences. For restoration of floodplain woodlands, the remaining black poplar stands may act as source population. A potential problem is that P. nigra and Populus deltoides have contributed to many interspecific hybrids, which have been planted in large numbers. As these Populus x canadensis clones have the possibility to intercross with wild P. nigra trees, their offspring could establish themselves along European rivers. In this study, we have sampled 44 poplar seedlings and young trees that occurred spontaneously along the Rhine river and its tributaries in the Netherlands. Along these rivers, only a few native P. nigra L. populations exist in combination with many planted cultivated P. x canadensis trees. By comparison to reference material from P. nigra, P. deltoides and P. x canadensis, species-specific AFLP bands and microsatellite alleles indicated that nearly half of the sampled trees were not pure P. nigra but progeny of natural hybridisation that had colonised the Rhine river banks. The posterior probability method as implemented in NewHybrids using microsatellite data was the superior method in establishing the most likely parentage. The results of this study indicate that offspring of hybrid cultivated poplars compete for the same ecological niche as native black poplars.

Temperature‐related natural selection in a wind‐pollinated tree across regional and continental scales

Adaptive genetic variation is a key factor in evolutionary biology, but the detection of signatures of natural selection remains challenging in nonmodel organisms. We used a genome scan approach to detect signals of natural selection in the Black alder (Alnus glutinosa), a widespread wind‐pollinated tree. Gene flow through pollen dispersal is believed to be high in this species, and we therefore expected to find a clear response to natural selection. In combination with two different landscape genetic approaches, we determined which environmental variables were most associated with the inferred selection. This analysis was performed on a regional scale (northern Belgium) and on a continental scale (Europe). Because climate‐related differences are much more pronounced at the continental scale, we expected to find more selection‐sensitive genetic markers across Europe than across northern Belgium. At both spatial scales, a substantial number of genetic loci were considered outliers, with respect to neutral expectations, and were therefore identified as selective. Based on results from our combined approach, four putative selective loci (or 2.5%) were recovered with high statistical support. Although these loci seemed to be associated with different environmental variables, they were mainly temperature‐related. Our study demonstrates that the use of complementary methods in landscape genetics allows the discovery of selective loci which otherwise might stay hidden. In combination with a genome scan, the selective loci can be verified and the nature of the selection pressure can be identified.

THE USE OF BULKED SEGREGANT ANALYSIS TO IDENTIFY AFLP"" MOLECULAR MARKERS CLOSELY LINKED TO MELAMPSORA LARICI-POPULINA RESISTANCE IN POPULUS

We have identified three AFLP markers tightly linked to the locus conferring resistance to the leaf rust Melampsora larici-populina in Populus by Bulked Segregant Analysis. The study was carried out using a hybrid progeny derived from an inter-specific, controlled cross between a resistant Populus deltoides female and a susceptible P. nigra male. The segregation ratio of resistant to susceptible plants suggested that a single, dominant locus (Mer) defined the resistance to Melampsora.