Analysis of Molecular Markers Genetically Linked to the Leptosphaeria maculans Avirulence Gene AvrLm1 in Field Populations Indicates a Highly Conserved Event Leading to Virulence on Rlm1 Genotypes

Abstract

Map-based cloning of the avirulence gene AvrLm1 of Leptosphaeria maculans was initiated utilizing a genetic map of the fungus and a BAC library constructed from an AvrLm1 isolate. Seven polymorphic DNA markers closely linked to AvrLm1 were identified. Of these, two were shown to border the locus on its 5' end and were present, with size polymorphism, in both the virulent and the avirulent isolates. In contrast, three markers, J19-1.1, J53-1.3 (in coupling phase with avirulence), and Vir1 (in repulsion phase with avirulence), cosegregated with AvrLm1 in 312 progeny from five in vitro crosses. J19-1.1 and J53-1.3 were never amplified in the virulent parents or progeny, whereas Vir1 was never amplified in the avirulent parents or progeny. J19-1.1 and J53-1.3 were shown to be separated by 40 kb within a 184-kb BAC contig. In addition, the 1.6-cM genetic distance between J53-1.3 and the nearest recombinant marker corresponded to a 121-kb physical distance. When analyzing a European Union-wide collection of 192 isolates, J53-1.3, J19-1.1, and Vir1 were found to be closely associated with the AvrLm1 locus. The results of polymerase chain reaction amplification with primers for the three markers were in accordance with the interaction phenotype for 92.2% (J53-1.3), 90.6% (J19-1.1), and 88.0% (Vir1) of the isolates. In addition, genome organization of the AvrLm1 region was highly conserved in field isolates, because 89.1% of the avirulent isolates and 79.0% of the virulent isolates showed the same association of markers as that of the parents of in vitro crosses. The large-scale analysis of field isolates with markers originating from the genetic map therefore confirms (i) the physical proximity between the markers and the target locus and (ii) that AvrLm1 is located in (or close to) a recombination-deficient genome region. As a consequence, map-based markers provided us with high-quality markers for an overview of the occurrence of race "AvrLm1" at the field scale. These data were used to propose hypotheses on evolution towards virulence in field isolates.

Frequency of Avirulence Alleles in Field Populations of Leptosphaeria maculans in Europe

“…Undoubtedly, such specific resistance will be of only short-term value unless combined with quantitative (polygenic) resistance or used in combination with other effective resistance genes. Numerous studies show virulence is achieved or lost through a set of small, highly conserved genetic events (Joosten et al, 1994;Bryan et al, 2000;Attard et al, 2002). Therefore, knowledge of race structure of a pathogen population before and after the introduction of a new resistance gene is essential for the good management of resistance to maintain its durability over several seasons.…”

Genetic Variability and Distribution of Mating Type Alleles in Field Populations of Leptosphaeria maculans from France

“…The suppression of recombination observed in some genetic maps may be related to nonhomologous sequences present in this region in the avirulent and virulent parental isolates used (L. Gout, unpublished data). The nonhomologous genome organization of the AvrLm1 region in avirulent and virulent isolates has been conserved in field populations from Western Europe based on the lack of apparent recombination between markers spanning this region (5). In the Le Rheu and Oucques populations, the presence of avirulent and virulent isolates could have reduced recombination near AvrLm1 and have led to the linkage disequilibrium between MinLm1 and AvrLm1 via "hitchhiking" to AvrLm1, which would be selected for when cultivars carrying Rlm1 were planted.…”

“…To date, there have been no studies of the genetic structure of L. maculans field populations in France and other countries of Western Europe. In these regions, previous work has focused mainly on field population race structure and has used virulence markers to assess genetic diversity in the fungus (5,9,22,35).…”

Fitness Cost Associated with Loss of the AvrLm4 Avirulence Function in Leptosphaeria maculans (Phoma Stem Canker of Oilseed Rape)

“…Two sister isolates of opposite mating-type, v23.1.2 (avrLm4, MAT1-1) and v23.1.3 (AvrLm4, MAT1-2), were crossed in vitro and the F 1 progeny isolates were characterised at the AvrLm4 locus by inoculation of cotyledons of cv Jet Neuf (carrying Rlm4) and cv Westar (susceptible with no resistance genes) Attard et al, 2002). The mating types of progeny isolates were determined by PCR (Cozijnsen and Howlett, 2003).…”