Molecular cloning and analysis of apple HcrVf resistance gene paralogs in a collection of related Malus species
Few complete genes belonging to the receptor-like protein class of plant resistance (R) genes (called HcrVf genes in Malus) have been cloned from apple cultivars. To date, the HcrVf2 gene from the Rvi6 locus of ‘Florina’, a derivative of Malus × floribunda 821, is the only cloned apple scab R gene with a proven function. The breakdown of the Rvi6 scab resistance in several apple growing regions has forced the search for new resistance sources for R gene pyramiding through traditional and biotechnological breeding. Marker-assisted breeding is aimed at the selection of the desired R gene combinations but might be extended for monitoring putative risks of resistance breakdown in potential scab R gene donors. Here we report on a marker-based screen of Rvi6 homologues supplemented by a polymerase chain reaction (PCR)-based full-length cloning of HcrVf paralogs. Known Rvi6 markers were analysed in a sub-set of accessions selected by a preceding SSR-based genetic relationship analysis from a large Malus species germplasm collection, which has been evaluated for scab resistance in an unsprayed orchard for a period of 3 years. The Rvi6 breakdown in several M. × floribunda accessions was confirmed, and several other Malus species putatively related to M. × floribunda were also infected by scab. The selected sub-cluster consisting of 40 accessions, including all M. × floribunda, two Malus × micromalus and two Malus baccata accessions, was screened for Rvi6 markers CH-Vf1-SSR and AL07-SCAR and for the presence of HcrVf2 by using gene-specific primers. The two M. × micromalus accessions, which proved to be identical genotypes, were found to be closely related to M. × floribunda. They also displayed the Rvi6 markers and could be infected by race (5,6,7) scab isolate Vi158. To verify the assumed existence of the HcrVf2 gene in M. × micromalus, a PCR-based cloning method was used to clone full-length HcrVf paralogs from this species and additionally from a scab-susceptible M. baccata genotype also showing the Rvi6 markers. The M. × micromalus gene MAM31 was identified as an identical copy of HcrVf2. Another HcrVf-like gene (MAM6) newly cloned from M. × micromalus showed 95 % similarity to HcrVf2. MAM6 was chosen for the development of a gene-specific PCR marker, which was analysed in the selected apple group and additionally mapped in an apple progeny derived from a cross with M. × micromalus. The cloning method described in this paper might be used in future to mine for more HcrVf gene variants to develop highly specific markers for R gene deployment in traditional breeding and to use cloned genes for gene transfer and functional studies.