Generation of advanced fire blight-resistant apple (Malus × domestica) selections of the fifth generation within 7 years of applying the early flowering approach.

MAIN CONCLUSION: The 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.

Development of the First Cisgenic Apple with Increased Resistance to Fire Blight.

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.