No abstract

The generation and selection of novel fire blight resistant apple genotypes would greatly improve the management of this devastating disease, caused by Erwinia amylovora. Such resistant genotypes are currently developed by conventional breeding, but novel breeding technologies including cisgenesis could be an alternative approach. A cisgenic apple line C44.4.146 was regenerated using the cisgene FB_MR5 from wild apple Malus ×robusta 5 (Mr5), and the previously established method involving A. tumefaciens-mediated transformation of the fire blight susceptible cultivar ‘Gala Galaxy’ using the binary vector p9-Dao-FLPi. The line C44.4.146 was shown to carry only the cisgene FB_MR5, controlled by its native regulatory sequences and no transgenes were detected by PCR or Southern blot following heat induced recombinase-mediated elimination of the selectable markers. Although this line contains up to 452 bp of vector sequences, it still matches the original definition of cisgenesis. A single insertion of T-DNA into the genome of 'Gala Galaxy' in chromosome 16 was identified. Transcription of FB_MR5 in line C44.4.146 was similar to the transcription in classically bred descendants of Mr5. Three independent shoot inoculation experiments with a Mr5 avirulent strain of Erwinia amylovora were performed using scissors or syringe. Significantly lower disease symptoms were detected on shoots of the cisgenic line compared to those of untransformed 'Gala Galaxy'. Despite the fact that the pathogen can overcome this resistance by a single nucleotide mutation, this is, to our knowledge, the first prototype of a cisgenic apple with increased resistance to fire blight.

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.

Main conclusionThe approach presented here can be applied to reduce the time needed to introduce traits from wild apples into null segregant advanced selections by one-fourth.Interesting traits like resistances to pathogens are often found within the wild apple gene pool. However, the long juvenile phase of apple seedlings hampers the rapid introduction of these traits into new cultivars. The rapid crop cycle breeding approach used in this paper is based on the overexpression of the birch (Betula pendula) MADS4 transcription factor in apple. Using the early flowering line T1190 and ‘Evereste’ as source of the fire blight resistance (Fb_E locus), we successfully established 18 advanced selections of the fifth generation in the greenhouse within 7 years. Fifteen individuals showed the habitus expected of a regular apple seedling, while three showed very short internodes. The null segregants possessing a regular habitus maintained the high level of fire blight resistance typical for ‘Evereste’. Using SSR markers, we estimated the percentage of genetic drag from ‘Evereste’ still associated with Fb_E on linkage group 12 (LG12). Eight out of the 18 selections had only 4% of ‘Evereste’ genome left. Since genotypes carrying the apple scab resistance gene Rvi6 and the fire blight resistance QTL Fb_F7 were used as parents in the course of the experiments, these resistances were also identified in some of the null segregants. One seedling is particularly interesting as, beside Fb_E, it also carries Fb_F7 heterozygously and Rvi6 homozygously. If null segregants obtained using this method will be considered as not genetically modified in Europe, as is already the case in the USA, this genotype could be a very promising parent for breeding new fire blight and scab-resistant apple cultivars in European apple breeding programs.Electronic supplementary materialThe online version of this article (10.1007/s00425-018-2876-z) contains supplementary material, which is available to authorized users.

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.