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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.

ABSTRACT Leptosphaeria maculans causes blackleg of oilseed rape. Gene-for-gene interactions between race PG3 and Brassica napus cv. Quinta were related to interaction between the fungal avirulence (Avr) gene AvrLm1 and the corresponding resistance gene Rlm1. AvrLm1 isolates were aviru-lent on cvs. Doublol, Vivol, Columbus, and Capitol, and no recombinant phenotypes were observed in the progeny of two AvrLm1 x avrLm1 crosses, suggesting that all of these cultivars may possess Rlm1 or genes displaying the same recognition spectrum, or that a cluster of Avr genes is present at the Avrlm1 locus. In one cross, segregation distortion was observed at the AvrLm1 locus that could be explained by interaction between AvrLm1 and one unlinked deleterious gene, termed Del1. Incompatibility toward cvs. Jet Neuf and Darmor.bzh was governed by a single gene, unlinked to AvrLm1 or Del1. This avirulence gene was termed AvrLm4. Preliminary plant genetic analysis suggested the occurrence of a corresponding dominant resistance gene, termed Rlm4, present in the Quinta line analyzed and linked to Rlm1.

All Leptosphaeria maculans field isolates displayed a minichromosome (MC) clearly separated from the overall electrokaryotype following pulsed-field gel electrophoresis. MCs exhibited a length polymorphism ranging from 650 to 950 kb. Tetrad analyses revealed the parental inheritance of MC length polymorphism (50% of the tetrads) or else the generation of novel-sized MCs (27%), which suggested that recombination occurred between MCs. Nineteen percent of the tetrads displayed a lack of the MC band in the electrokaryotype for one or two of the four resulting genotypes. Crosses between isolates carrying or lacking MCs revealed non-Mendelian segregation and suggested that some isolates could display at least two copies of the MC. Only repeated sequences hybridising to all chromosomes were isolated from the MC. Finally, saprophytic or parasitic fitness was not modified when isolates apparently lacked the MC. All these data suggested that the L. maculans MC behaves like a 'B' chromosome.