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Molecular cloning and analysis of apple HcrVf resistance gene paralogs in a collection of related Malus species

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Abstract

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.

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Authors and Affiliations

  1. Institute for Breeding Research on Horticultural and Fruit Crops, Julius Kühn Institute (JKI), Federal Research Centre for Cultivated Plants, Erwin-Baur-Straße 27, 06484, Quedlinburg, Germany

    Frank Dunemann

  2. Institute for Breeding Research on Horticultural and Fruit Crops, Julius Kühn Institute (JKI), Federal Research Centre for Cultivated Plants, Pillnitzer Platz 3a, 01326, Dresden, Germany

    Regina Gläss, Sabine Bartsch & Andreas Peil

  3. Plant Pathology Department, Zagazig University, Faculty of Agriculture, 44511, Zagazig, Egypt

    Mohamed Ali Saad Eldin

  4. The New Zealand Institute for Plant & Food Research Limited, Hawke’s Bay Research Centre, Private Bag 1401, Havelock North, 4157, New Zealand

    Vincent G. M. Bus

Authors
  1. Frank Dunemann
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  2. Regina Gläss
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  3. Sabine Bartsch
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  4. Mohamed Ali Saad Eldin
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  5. Andreas Peil
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  6. Vincent G. M. Bus
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Correspondence to Frank Dunemann.

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Communicated by E. Dirlewanger

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Figure 1

Multiple amino acid sequence alignment of six HcrVf members identified by full-length cloning in this study (MAM/MAB paralogs) and “reference” genes HcrVf2 (Vinatzer et al. 2001) and HcrVf4 (Broggini et al. 2009). Sequences were compared by ClustalW alignment (Lasergene). Sequence regions used for MAM6 primer design are in bold letters and underlined. (PDF 182 kb)

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Dunemann, F., Gläss, R., Bartsch, S. et al. Molecular cloning and analysis of apple HcrVf resistance gene paralogs in a collection of related Malus species. Tree Genetics & Genomes 8, 1095–1109 (2012). https://doi.org/10.1007/s11295-012-0489-4

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