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

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.

A Molecular Marker Identification Study for Allow Early Selection in Determination Resistant Apple Varieties and Genotypes against to fire blight (Erwinia amylovora)

“…Moreover, it should be known that; the resistance to fire blight and the degree of sensitivity changed even in year-to-year climate conditions (Koski and Jacobi 2018). According to the information in Table1, Kasel-37 and Galaxy Gala apple varieties are expressed in moderate resistance while Gala apple variety is very sensitive and sensitive (Zwet 1996;Warner 1992;Hampson and Kemp 2003;Yılmaz and Aysan 2009;Kost et al 2015;Beckerman 2018;Koski and Jacobi 2018). In a gene transfer study by Kellerhals et al (2014), has been mentioned to transfer the resistance gene from 'Malus x robusta' wild apple plant to 'Gala' apple variety, which is susceptible to fire blight.…”

“…In a gene transfer study by Kellerhals et al (2014), has been mentioned to transfer the resistance gene from 'Malus x robusta' wild apple plant to 'Gala' apple variety, which is susceptible to fire blight. In a study on a gene transfer (Kost et al 2015), it has stated that Galaxy Gala apple cultivar was a sensitive species against Erwinia amylovora by Norelli et al (2003) and (Turchek 2004). Gala and Royal Gala varieties are expressed as apple varieties obtained by Kidd's Orange Red x Golden Delicious hybridization (Kiddle 2006).…”

“…Table 1. Some literature information about apple varieties used in the study and their resistance or sensitivity against to fire blight Varieties Resistance/Sensitivity Gala Very Sensitive (Zwet 1996) Very Sensitive (Warner 1992) Moderately Resistant (Vanneste 2000) Very Sensitive (Hampson and Kemp 2003) Sensitive (Yılmaz and Aysan 2009) Sensitive (Kellerhals et al 2014) Sensitive ( Sensitive (Koski and Jacobi 2018) Sensitive (Beckerman, 2018) Resistant (Sehic et al 2009) Royal Gala Sensitive (Koski and Jacobi 2018) Galaxy Gala Moderately Resistant (Akgül et al 2005;Özçağıran et al 2004) Sensitive (Norelli et al 2003;Turchek 2004) Sensitive (Kost et al 2015) 2.3. DNA isolation and determination of DNA concentration and purity The DNA isolation protocol was carried out in the following steps: 1000 µl of DNA extraction buffer (containing 0.2% of β-mercaptoethanol and 0.2 g of PVP) was added to on 0.3 g plant material in 2 ml tube which is lysed by liquid nitrogen.…”

Chromosome-scale de novo diploid assembly of the apple cultivar ‘Gala Galaxy’

“…Genomic DNA was isolated from field-grown apple leaves of ‘Gala Galaxy’ and processed for library construction for long- and short-reads sequencing as described in the supplementary methods. Additional long-reads libraries were also generated from four genotypes of the ‘Gala’ sport mutants group; ‘Gala’ original, ‘Gala Royal’, ‘Gala Schnico Red’ and the cisgenic apple line C44.4.146 (Kost, et al 2015). The long-reads libraries from ‘Gala Galaxy’ and the additional genotypes were sequenced using PacBio RSII and Sequel instruments, respectively.…”

New Biotechnological Tools for the Genetic Improvement of Major Woody Fruit Species

“…Fruit breeders are developing solutions for the various diseases affecting apples, including fire blight disease caused by Erwinia amylovora . Kost et al ( 2015 ) recently developed a cisgenic apple line C44.4.146 from a fire blight susceptible cultivar “Gala Galaxy” using the cisgene FB_MR5 from wild apple Malus × robusta 5 ( Mr5 ), which confers resistance to fire blight (Peil et al, 2007 ). After elimination of the selectable markers through heat-induced recombinase, both PCR and Southern blot analysis did not detect any transgenes.…”