Map-based cloning and functional analyses of the fire blight resistance candidate genes (FB_Mfu10) of Malus fusca Subtitle: Elucidating the fire blight resistance inherited from Malus fusca and functional analyses of candidate genes underlying the resistance QTL on linkage group 10

• Applicant: Dr. Ofere Francis Emeriewen, Ph.D.
• Subject Area: Plant Breeding and Plant Pathology
• Term: since 2019
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 433323507

The identification and use of fire blight resistance genes in breeding programmes is the silver bullet and the most eco-friendly strategy of tackling the threat posed by the very devastating and erratic fire blight disease of apples. This is especially true as most commercial cultivars are highly susceptible to the causal bacterium – Erwinia amylovora; and Germany forbids the use of antibiotics treatment. The wild apple Malus fusca (accession MAL0045) is highly resistant to fire blight, and it is one of only two Malus genotypes not broken down by any strain of the pathogen. A major fire blight resistance QTL was identified on linkage group 10 (LG10) of M. fusca (Mfu10) in an F1 population of 134 individuals. In the subsequent fine mapping of the locus in 1888 individuals, a single BAC clone (46H22) that harbours Mfu10 was identified and sequenced. Analyses of the BAC sequence led to the identification of a receptor-like protein kinase candidate gene. Inoculations of the original F1 population with several strains of E. amylovora differing in virulence led to the hypothesis that MAL0045 possesses other putative resistance factors e.g. masked QTLs that could not be mapped in the F1. Therefore, in the previous DFG project, we first proposed to clone and transfer the candidate gene into a susceptible cultivar to prove its function, and secondly perform genetic analyses and QTL mapping in F2 individuals to determine if homozygous resistance factors could be detected. We report here that our studies have led to the identification of two previously undetected significant QTLs on LG4 and LG15, and confirm that Mfu10 alone is not responsible for the resistance of MAL0045. Furthermore, we report the identification of two additional candidate genes, both serine/threonine kinase genes on the BAC sequence of 46H22 following Nanopore Minion resequencing and assembly into a single contig. Although transformation experiments with the first candidate gene has led to the generation of 10 transgenic lines overexpressing this candidate, we are yet to carryout functionality analyses on them due to their developmental stage. Moreover, overexpression experiments have already begun with the two additional candidate genes. It will therefore be impossible to prove the functionality of the three candidate genes within the timeframe of the current project, which runs until December 2022, hence it has become imperative to request for an extension by three years. In addition to the functionality of the candidate genes, the newly discovered QTLs will be verified by inoculation experiments with other E. amylovora strains, and these gene loci will be examined for neighboring candidate genes. This requires a high quality genome of MAL0045, which is an additional goal of the renewal phase. The aim is to elucidate the fire blight resistance of MAL0045 to all isolates and to use this knowledge to breed resistant apple cultivars.

• DFG Programme: Research Grants
• Co-Investigator: Dr. Andreas Peil