Identification and characterization of DNA markers associated with a locus conferring virulence on barley in the plant pathogenic fungus Cochliobolus sativus

Abstract.

Cochliobolus sativus is a plant pathogenic fungus that causes spot blotch on barley and wheat. Virulence of a pathotype-2 isolate (ND90Pr) on barley cultivar Bowman was previously determined to be controlled by a single locus. To identify DNA markers associated with this virulence locus, amplified fragment length polymorphism (AFLP) analysis was conducted on 104 progeny isolates derived from a cross between isolates ND90Pr (exhibiting high virulence on Bowman) and ND93-1 (exhibiting low virulence on Bowman). Among 115 AFLP markers identified, 14 were linked to the virulence locus VHv1 in isolate ND90Pr, six of which co-segregated with VHv1. Two (E-AG/M-CA-207 and E-AG/M-CG-121) of the six co-segregating AFLP markers were cloned and used to probe genomic DNAs from the fungal parents and progeny. Both markers hybridized only with DNAs from ND90Pr and the virulent progeny. These two cloned markers were also used as probes to survey field isolates of C. sativus collected from different regions of the world and again only hybridized to DNAs from isolates that had the same virulence phenotype as ND90Pr. The results of this study indicate that E-AG/M-CA-207 and E-AG/M-CG-121 are closely linked to VHv1 and are unique to isolates carrying the virulence locus. Development of a linkage group, coupled with the identification of closely linked molecular markers, will facilitate the cloning of the virulence gene VHv1 in C. sativus by map-based cloning.