Molecular mapping of resistance to Leptosphaeria maculans in Australian cultivars of Brassica napus

Abstract

Doubled haploid (DH) lines together with a cotyledon bioassay were employed for the molecular analysis of resistance to the blackleg fungus Leptosphaeria maculans in the Australian Brassica napus cultivars Shiralee and Maluka. We used bulked segregant analysis to identify 13 RAPD and two RFLP markers linked to the resistance phenotype and mapped these markers in the segregating DH population. Our data suggest the presence of a single major locus controlling resistance in the cultivar Shiralee, confirming our previous results obtained from Mendelian genetic analyses. In addition, preliminary mapping data for the cultivar Maluka also support a single locus model for resistance and indicate that the resistance genes from 'Shiralee' and 'Maluka' are either linked or possibly identical. The molecular markers identified in this study should be a useful tool for breeding blackleg resistant varieties using marker-assisted selection, and are the essential first step towards the map-based cloning of this resistance gene.

Complexities of Chromosome Landing in a Highly Duplicated Genome: Toward Map-Based Cloning of a Gene Controlling Blackleg Resistance in Brassica napus

“…As expected, in both populations the resistance phenotype mapped as a single dominant locus to linkage group N7 (Mayerhofer et al 1997;Rimmer et al 1999). Further mapping in each population was targeted to the resistance gene regions by assessing only the phenotype of lines with recombination events between the markers flanking the resistance loci, with the most extensive mapping carried out in the Shiralee population.…”

“…Genomewide analyses of the organization of NBS-LRR genes in Arabidopsis suggest that tandem duplications distribute and separate this class of resistance genes in the genome and contribute to the diversification of gene families (Arabidopsis Genome Initiative 2000; reviewed in Leister 2004). Linkage group N7 (alternative nomenclature LG6) has already been shown to be a valuable source of blackleg resistance loci, harboring at least five different loci (Ferreira et al 1995;Mayerhofer et al 1997;Rimmer et al 1999). It seems likely that the tandem duplication could harbor a homolog of LmR1, which although nonfunctional in Shiralee, might reveal the identity of one of the additional blackleg loci already assigned to N7.…”

“…Greenhouse screening for blackleg resistance: The cotyledon resistance response to blackleg was determined for the BC1 populations as described in Mayerhofer et al (1997). The resistance phenotype of each backcross line was determined using 16 self progeny from each BC1.…”

“…DNA isolations, Southern hybridizations, PCR reactions, and sequencing: Genomic DNA preparations from fresh or lyophilized leaf tissue, Southern analyses, and RAPD reactions were carried out as described in Sharpe et al (1995) and Mayerhofer et al (1997). PCR reactions for sequencecharacterized amplified region (SCAR) markers were performed in a Gene Amp 9700 thermocycler (Perkin-Elmer, Norwalk, CT) in 25-ml reaction volumes containing 10 mm Tris-HCl (pH 8.0); 50 mm KCl; 0.01% gelatin; 2.5 mm MgCl; 0.4 mm each of dATP, dCTP, dGTP, and dTTP; 0.2 mm each of primer; 0.5 units/reaction Taq DNA polymerase (PerkinElmer); and 25 ng of template DNA.…”

“…Single dominant genes were identified in the cultivars Major, RB87-62, Shiralee, and Maluka (Ferreira et al 1995;Mayerhofer et al 1997;Rimmer et al 1999) and mapped to the same linkage group LG6, which has been shown subsequently to be equivalent to the A genome linkage group N7 (Ferreira et al 1995;Parkin et al 1995). Interestingly, a single major locus controlling field disease resistance in the cultivar Cresor has also been mapped to the same region of N7 (Dion et al 1995).…”