Although genetic and genomic studies have progressed to a very advanced level in apple, the application of this acquired knowledge for markerassisted breeding (MAB) remains limited mainly to pyramiding monogenetically inherited resistances against apple scab, powdery mildew and fire blight. Crucial contributing reasons are the uncertainty in map position of some genes and the lack of tightly linked markers suitable for high-throughput analysis (HTA) that reduces the costs of MAB. Single-nucleotide polymorphism (SNP) markers have the potential to resolve these major issues. Here we present the refined map positions of the apple scab resistance genes Rvi2, Rvi4 and Rvi11, and the systematic search for SNPs associated with apple scab (Rvi2, Rvi4, Rvi6, Rvi11, Rvi15), powdery mildew (Pl2) and fire blight (FB_E and FB_MR5) resistances. With the aid of the 'Golden Delicious' sequence, several SNPs linked to each of the eight resistances were identified in the genomic regions around the resistance loci previously delimited by simple sequence repeat markers. The specificity of the alleles in coupling with the resistances was determined by screening eight apple genotypes, six of them being founding clones of modern apple cultivars. These SNPs can now be used to develop SNP-based HTA assays for MAB.
Monilinia fructicola is a quarantine fungal pathogen in Europe, but many major stone fruit growing countries in Europe have reported its presence recently. In Switzerland, the fungus was first found in a single apricot orchard in 2008. This study confirms the presence of M. fructicola in nine out of 22 commercial orchards in Canton Valais, Switzerland. Five simple sequence repeat markers (SSRs) were developed for M. fructicola and samples from Switzerland, Spain, Italy, France and the United States were analysed and compared in order to assess the genetic diversity of the pathogen, identify the origin of the disease, and verify if the fungus reproduces sexually in Europe. In the 119 European samples analysed, 12 different haplotypes were found, indicating a relatively high genetic diversity of the pathogen considering that the first report in Europe was 10 years ago. Three haplotypes found in Europe were identical to those found in the American samples (two from the east coast and one from the west coast). Population structure analysis suggests that the European population is derived from at least two ‘invasion’ events probably originating from the US (one from the east coast, the other from the west coast). Preliminary evidence of sexual reproduction of M. fructicola in Europe is reported.
The causal agent of brown rot on stone and pome fruits, Monilinia fructicola (G. Wint.), is a quarantine pathogen in Europe. It has been detected in Austria (later eradicated), Spain, the Czech Republic, Italy, Germany, and Switzerland (1). In the United States and other countries, M. fructicola isolates were reported to show resistance to different classes of fungicides, including methyl benzimidazole carbamates (MBC) (2). Lichou et al. (2) reported the presence of isolates resistant to the MBC carbendazim in France, but the mechanisms inducing MBC resistance in these isolates were not studied. Ma et al. (3) in California, and more recently, Zhu et al. (4) in South Carolina, demonstrated that the molecular mechanisms accounting for low and high levels of resistance to MBC fungicides in M. fructicola isolates were the mutations H6Y and E198A, respectively, in the β-tubulin gene. Four M. fructicola isolates each from Italy, France, Spain, and Switzerland (16 isolates total), all having an unknown level of MBC resistance, were selected. In each isolate, the section of the β-tubulin gene containing the two potentially mutant codons was PCR-amplified with the primers TubA and TubR1 (3) and the amplicons were sequenced directly. Sequence analysis revealed the amino acid histidine (H) at codon 6 in all the isolates, which would not predict MBC resistance, while alanine (A) at codon 198 (the mutation predictive of a high level of MBC resistance) was found in all isolates from Spain and Switzerland and in three isolates each from France and Italy. A representative sequence of the four identical partial β-tubulin gene sequences from the Swiss isolates was submitted to GenBank under the Accession No. HQ709265. All isolates were tested in a potato dextrose agar (PDA) petri dish assay for resistance to the MBC fungicide thiophanate-methyl (Nippon Soda Co., Ltd., Tokyo, Japan) at the discriminatory dose of 50 μg/ml (4). All isolates with the E198A mutation were able to grow on the media, while the two isolates without the E198A mutation were not able to grow. The result indicated that most isolates had a high level of resistance to the MBC fungicide. To our knowledge, this is the first report of the presence of the E198A mutation conferring resistance to MBC fungicides in European isolates of M. fructicola. As the mutation appears to be widely distributed, we anticipate that MBC fungicides may be ineffective at controlling brown rot in countries with occurrence of M. fructicola. References: (1) M. Hilber-Bodmer et al. Plant Dis. 94:643, 2010. (2) J. Lichou et al. Phytoma 547:22, 2002. (3) Z. H. Ma et al. Appl. Environ. Microbiol. 69:7145, 2003. (4) F. X. Zhu et al. Plant Dis. 94:1511, 2010.
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