Molecular cloning and analysis of apple Emphasis

Authors: Frank Dunemann Regina Gläss Sabine Bartsch Mohamed Ali Saad Eldin Andreas Peil Vincent G M Bus

Publish Date: 2012/03/17

Volume: 8, Issue: 5, Pages: 1095-1109

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Abstract

Few complete genes belonging to the receptorlike 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 Markerassisted 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 markerbased screen of Rvi6 homologues supplemented by a polymerase chain reaction PCRbased fulllength cloning of HcrVf paralogs Known Rvi6 markers were analysed in a subset of accessions selected by a preceding SSRbased 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 subcluster consisting of 40 accessions including all M × floribunda two Malus × micromalus and two Malus baccata accessions was screened for Rvi6 markers CHVf1SSR and AL07SCAR and for the presence of HcrVf2 by using genespecific 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 567 scab isolate Vi158 To verify the assumed existence of the HcrVf2 gene in M × micromalus a PCRbased cloning method was used to clone fulllength HcrVf paralogs from this species and additionally from a scabsusceptible M baccata genotype also showing the Rvi6 markers The M × micromalus gene MAM31 was identified as an identical copy of HcrVf2 Another HcrVflike gene MAM6 newly cloned from M × micromalus showed 95 similarity to HcrVf2 MAM6 was chosen for the development of a genespecific 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

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