POSITIONAL CLONING OF THE APPLE SCAB RESISTANCE GENE VF

Abstract

The apple scab disease resistance gene Vf, originating from Malus floribunda 821, is an important gene locus for the development of apple scab disease resistant cultivars. A total of 11 tightly linked sequence-characterized amplified region (SCAR) markers have been tagged to the Vf locus by using the amplified fragment length polymorphism (AFLP) technique followed by successful conversion of AFLP markers into SCAR markers. These 11 SCAR markers along with three previously identified SCAR markers were used to screen two bacterial artificial chromosome (BAC) libraries constructed from M. floribunda 821 and the apple cultivar 'GoldRush' (also containing Vf), respectively. As a result, a BAC contig, consisting of five overlapping BAC clones, was constructed spanning a genetic distance of ∼290 kb of the Vf region. Four receptor-like resistance gene homologues were unveiled in the Vf region after searching for encoding sequences from overlapping BAC clones. These four Vf gene homologues were designated as Vfal, Vfa2, Vfa3, and Vfa4, and co-segregated with the Vf locus in a segregant apple mapping population. The first three homologues, Vfal, Vfa2, and Vfa3, were expressed in young leaves, while the fourth homologue, Vfa4, was expressed in young leaves and strongly expressed in mature leaves. Four cDNA clones corresponding to these four homologues were recovered by using rapid cDNA end amplification (RACE) method. Sequence comparisons between cDNA and genomic DNA were conducted. Deduced amino acid sequences revealed that each of the Vf gene homologues contained both an extracellular leucine-rich repeat (LRR) domain and a transmembrane (TM) domain.