Andreas Peil

Putative resistance gene markers associated with quantitative trait loci for fire blight resistance in Malus ‘Robusta 5’ accessions

BackgroundBreeding of fire blight resistant scions and rootstocks is a goal of several international apple breeding programs, as options are limited for management of this destructive disease caused by the bacterial pathogen Erwinia amylovora. A broad, large-effect quantitative trait locus (QTL) for fire blight resistance has been reported on linkage group 3 of Malus ‘Robusta 5’. In this study we identified markers derived from putative fire blight resistance genes associated with the QTL by integrating further genetic mapping studies with bioinformatics analysis of transcript profiling data and genome sequence databases.ResultsWhen several defined E.amylovora strains were used to inoculate three progenies from international breeding programs, all with ‘Robusta 5’ as a common parent, two distinct QTLs were detected on linkage group 3, where only one had previously been mapped. In the New Zealand ‘Malling 9’ X ‘Robusta 5’ population inoculated with E. amylovora ICMP11176, the proximal QTL co-located with SNP markers derived from a leucine-rich repeat, receptor-like protein ( MxdRLP1) and a closely linked class 3 peroxidase gene. While the QTL detected in the German ‘Idared’ X ‘Robusta 5’ population inoculated with E. amylovora strains Ea222_JKI or ICMP11176 was approximately 6 cM distal to this, directly below a SNP marker derived from a heat shock 90 family protein gene ( HSP90). In the US ‘Otawa3’ X ‘Robusta5’ population inoculated with E. amylovora strains Ea273 or E2002a, the position of the LOD score peak on linkage group 3 was dependent upon the pathogen strains used for inoculation. One of the five MxdRLP1 alleles identified in fire blight resistant and susceptible cultivars was genetically associated with resistance and used to develop a high resolution melting PCR marker. A resistance QTL detected on linkage group 7 of the US population co-located with another HSP90 gene-family member and a WRKY transcription factor previously associated with fire blight resistance. However, this QTL was not observed in the New Zealand or German populations.ConclusionsThe results suggest that the upper region of ‘Robusta 5’ linkage group 3 contains multiple genes contributing to fire blight resistance and that their contributions to resistance can vary depending upon pathogen virulence and other factors. Mapping markers derived from putative fire blight resistance genes has proved a useful aid in defining these QTLs and developing markers for marker-assisted breeding of fire blight resistance.

The MdTFL1 gene of apple (Malus × domestica Borkh.) reduces vegetative growth and generation time

TFL1 is known as a floral repressor in Arabidopsis thaliana (L.) Heynh. In apple there are two TFL1 homologs, MdTFL1-1 and MdTFL1-2. The MdTFL1-1 gene was silenced in transgenic clones expressing a hairpin gene construct of a 323 bp fragment of MdTFL1-1. The hairpin gene construct was transferred to three different apple genotypes. Of 22 transgenic clones, 21 showed a significant reduction in MdTFL1-1 mRNA expression. Precocious flowering was obtained for 20 clones, which flowered already during in vitro cultivation. Nineteen clones could successfully be transferred to the greenhouse where 18 of them flowered within a few weeks followed by the death or at least a strongly inhibited vegetative growth of the plant. Most of the transgenic flowers developed abnormally. Results obtained on greenhouse-grown plants of the transgenic clones and transgenic seedlings clearly demonstrated the major role of MdTFL1 genes in maintaining the vegetative growth as prerequisite for a perennial lifecycle. It was shown that MdTFL1 dsRNAi promotes a life history similar to annual plants. Preliminary results obtained from grafting experiments with non-transgenic scions grafted onto MdTFL1 dsRNAi transgenic rootstocks indicated that the flower-inducing signal obtained after silencing of MdTFL1 genes seems not to be graft-transmissible.

A candidate gene for fire blight resistance in Malus ×robusta 5 is coding for a CC–NBS–LRR

AbstractFire blight is the most important bacterial disease in apple (Malus ×  domestica) and pear (Pyrus communis) production. Today, the causal bacterium Erwinia amylovora is present in many apple- and pear-growing areas. We investigated the natural resistance of the wild apple Malus ×  robusta 5 against E. amylovora, previously mapped to linkage group 3. With a fine-mapping approach on a population of 2,133 individuals followed by phenotyping of the recombinants from the region of interest, we developed flanking markers useful for marker-assisted selection. Open reading frames were predicted on the sequence of a BAC spanning the resistance locus. One open reading frame coded for a protein belonging to the NBS–LRR family. The in silico investigation of the structure of the candidate resistance gene against fire blight of M. ×  robusta 5, FB_MR5, led us hypothesize the presence of a coiled-coil region followed by an NBS and an LRR-like structure with the consensus ‘LxxLx[IL]xxCxxLxxL’. The function of FB_MR5 was predicted in agreement with the decoy/guard model, that FB_MR5 monitors the transcribed RIN4_MR5, a homolog of RIN4 of Arabidopsis thaliana that could interact with the previously described effector AvrRpt2EA of E. amylovora.

Gene-for-gene relationship in the host–pathogen system Malus × robusta 5–Erwinia amylovora

Fire blight is a destructive bacterial disease caused by Erwinia amylovora affecting plants in the family Rosaceae, including apple. Host resistance to fire blight is present mainly in accessions of Malus spp. and is thought to be quantitative in this pathosystem. In this study we analyzed the importance of the E. amylovora effector avrRpt2(EA) , a homolog of Pseudomonas syringae avrRpt2, for resistance of Malus × robusta 5 (Mr5). The deletion mutant E. amylovora Ea1189ΔavrRpt2(EA) was able to overcome the fire blight resistance of Mr5. One single nucleotide polymorphism (SNP), resulting in an exchange of cysteine to serine in the encoded protein, was detected in avrRpt2(EA) of several Erwinia strains differing in virulence to Mr5. E. amylovora strains encoding serine (S-allele) were able to overcome resistance of Mr5, whereas strains encoding cysteine (C-allele) were not. Allele specificity was also observed in a coexpression assay with Arabidopsis thaliana RIN4 in Nicotiana benthamiana. A homolog of RIN4 has been detected and isolated in Mr5. These results suggest a system similar to the interaction of RPS2 from A. thaliana and AvrRpt2 from P. syringae with RIN4 as guard. Our data are suggestive of a gene-for-gene relationship for the host-pathogen system Mr5 and E. amylovora.

Differential Expression of Biphenyl Synthase Gene Family Members in Fire-Blight-Infected Apple ‘Holsteiner Cox’

Fire blight, caused by the bacterium Erwinia amylovora, is a devastating disease of apple (Malus × domestica). The phytoalexins of apple are biphenyls and dibenzofurans, whose carbon skeleton is formed by biphenyl synthase (BIS), a type III polyketide synthase. In the recently published genome sequence of apple ‘Golden Delicious’, nine BIS genes and four BIS gene fragments were detected. The nine genes fall into four subfamilies, referred to as MdBIS1 to MdBIS4. In a phylogenetic tree, the BIS amino acid sequences from apple and Sorbus aucuparia formed an individual cluster within the clade of the functionally diverse type III polyketide synthases. cDNAs encoding MdBIS1 to MdBIS4 were cloned from fire-blight-infected shoots of apple ‘Holsteiner Cox,’ heterologously expressed in Escherichia coli, and functionally analyzed. Benzoyl-coenzyme A and salicoyl-coenzyme A were the preferred starter substrates. In response to inoculation with E. amylovora, the BIS3 gene was expressed in stems of cv Holsteiner Cox, with highest transcript levels in the transition zone between necrotic and healthy tissues. The transition zone was the accumulation site of biphenyl and dibenzofuran phytoalexins. Leaves contained transcripts for BIS2 but failed to form immunodetectable amounts of BIS protein. In cell cultures of apple ‘Cox Orange,’ expression of the BIS1 to BIS3 genes was observed after the addition of an autoclaved E. amylovora suspension. Using immunofluorescence localization under a confocal laser-scanning microscope, the BIS3 protein in the transition zone of stems was detected in the parenchyma of the bark. Dot-shaped immunofluorescence was confined to the junctions between neighboring cortical parenchyma cells.

Breeding Elite Lines of Apple Carrying Pyramided Homozygous Resistance Genes Against Apple Scab and Resistance Against Powdery Mildew and Fire Blight

The development of high-quality cultivars, with durable disease resistance, is a major objective of apple breeding. The selection procedures of modern breeding programs no longer rely exclusively on phenotypic criteria but include marker-assisted breeding (MAB). Currently, molecular markers linked to several resistance genes and quantitative trait loci (QTLs) are available. In this study, we focused on markers available for resistance breeding against the major diseases scab (Venturia inaequalis), powdery mildew (Podosphaera leucotricha), and fire blight (Erwinia amylovora). One approach proposed to achieve durable resistance is the pyramiding of functionally different resistance genes against the same pathogen. This approach can be complemented with the incorporation of resistance genes against other pathogens. The resulting resistant apple cultivars would contribute considerably to low-input, sustainable, fruit production. Furthermore, apple cultivars can be developed carrying homozygous allele sets of specific resistance genes, and these genotypes can be used as parents for further crosses. Due to the ensured inheritance of the resistance genes to the progeny, MAB for these genes will become superfluous. In this study, we developed elite apple plants which are homozygous for three different scab resistance genes, Rvi6, Rvi2, and Rvi4. Furthermore, these apple selections tested positive for a resistance gene against powdery mildew (Pl1 or Pl2), and the FBF7 QTL from ‘Fiesta’ for enhanced fire blight resistance. Selected progeny plants were tested for their fire blight resistance after artificial shoot inoculation and evaluated for tree and fruit characteristics.

Improving resistance of different apple cultivars using the Rvi6 scab resistance gene in a cisgenic approach based on the Flp/FRT recombinase system

Cisgenic apple plants of two different cultivars were developed by transferring the Rvi6 scab resistance gene of Malus floribunda 821, using a new transformation vector based on the Flp/FRT recombinase system. Transformation experiments on seven different cultivars resulted in 22 transgenic lines for the cultivars ‘Brookfield Baigent’, ‘Mitchgla’, ‘Novajo’, and ‘Pinova’, whereby 16 lines thereof were resistant to Venturia inaequalis strain 104 (race 1). Analysis of the transgenic lines revealed Rvi6 mRNA expression levels comparable to several traditional bred Rvi6 containing cultivars and identified four transgenic lines, harboring a single T-DNA insertion, as suitable for the production of cisgenic lines. The T-DNA insertion site of these lines was determined, and lines were subject to induction of the recombinase system. Two cisgenic lines originating from the cultivars ‘Brookfield Baigent’ and ‘Pinova’ were obtained for which the exact excision of the recombinase cassette was confirmed by sequencing the previously determined T-DNA integration site. Further investigations revealed both cisgenic lines as fully resistant to V. inaequalis race 1. Rvi6 mRNA expression of the cisgenic lines and traditionally bred Rvi6 harboring cultivars was still comparable. The transformation vector developed is useable for the production of cisgenic apple plants to a certain extent.

‘Schmidt's Antonovka’ is identical to ‘Common Antonovka’, an apple cultivar widely used in Russia in breeding for biotic and abiotic stresses

Progenies of ‘Schmidts Antonovka’ (SA) have been widely used in Western breeding programs as a source of scab resistance. The identity of SA has remained obscure, especially due to the existence of a series of ‘Antonovka’ cultivars with different origins. In this paper we show Schmidts Antonovka to be identical to Анто́новка обыкновенный or ‘Common Antonovka’ (CA), an old Russian cultivar of unknown origin, by comparing simple sequence repeat (SSR) and SNP genotyping data from several first-generation descendants of SA from two European collections and a CA accession from the germplasm collection held at VNIISPK (The All-Russian Research Institute of Horticultural Breeding, Orel, Russia). The use of CA in Russian breeding programs is also briefly reviewed.

Transgenic apple plants overexpressing the chalcone 3-hydroxylase gene of Cosmos sulphureus show increased levels of 3-hydroxyphloridzin and reduced susceptibility to apple scab and fire blight.

AbstractMain conclusionOverexpression of chalcone-3-hydroxylase provokes increased accumulation of 3-hydroxyphloridzin inMalus. Decreased flavonoid concentrations but unchanged flavonoid class composition were observed. The increased 3-hydroxyphlorizin contents correlate well with reduced susceptibility to fire blight and scab. The involvement of dihydrochalcones in the apple defence mechanism against pathogens is discussed but unknown biosynthetic steps in their formation hamper studies on their physiological relevance. The formation of 3-hydroxyphloretin is one of the gaps in the pathway. Polyphenol oxidases and cytochrome P450 dependent enzymes could be involved. Hydroxylation of phloretin in position 3 has high similarity to the B-ring hydroxylation of flavonoids catalysed by the well-known flavonoid 3′-hydroxylase (F3′H). Using recombinant F3′H and chalcone 3-hydroxylase (CH3H) from Cosmos sulphureus we show that F3′H and CH3H accept phloretin to some extent but higher conversion rates are obtained with CH3H. To test whether CH3H catalyzes the hydroxylation of dihydrochalcones in planta and if this could be of physiological relevance, we created transgenic apple trees harbouring CH3H from C. sulphureus. The three transgenic lines obtained showed lower polyphenol concentrations but no shift between the main polyphenol classes dihydrochalcones, flavonols, hydroxycinnamic acids and flavan 3-ols. Increase of 3-hydroxyphloridzin within the dihydrochalcones and of epicatechin/catechin within soluble flavan 3-ols were observed. Decreased activity of dihydroflavonol 4-reductase and chalcone synthase/chalcone isomerase could partially explain the lower polyphenol concentrations. In comparison to the parent line, the transgenic CH3H-lines showed a lower disease susceptibility to fire blight and apple scab that correlated with the increased 3-hydroxyphlorizin contents.

Phenotypic and genetic analysis of the German Malus Germplasm Collection in terms of type 1 and type 2 red-fleshed apples.

Red fruit flesh is a desirable trait in apple breeding, because red-fleshed apples are a novelty and therefore considered to be more attractive to consumers and contain more health beneficial compounds. The red fruit flesh coloration is based on an increased level of cyanidin 3-galactoside, an anthocyanin whose biosynthesis is regulated by the MYB-type transcription factors MdMYB10 or MdMYB110a, respectively. A repeated segment in the MdMYB10 promoter allele R6 results in a gain-of-function mutation visible as red pigmentation of fruit skin and flesh and all vegetative tissues. Red-fleshed apple genotypes containing this R6 allele belong to the type 1 red-fleshed apple, which is known to be linked to some negative traits like astringent taste and internal flesh browning disorder. In type 2 red-fleshed apples the fruit flesh coloration is not inevitably linked with skin and leaf color. This red-fleshed apple phenotype, which is a result of increased expression of MdMYB110a, seems to be more useful for breeding, but it can be found rather seldom. In the present study 357 Malus accessions of the German Malus Germplasm Collection were evaluated for red fruit flesh coloration and the presence of the MdMYB10 R1 (not mutated) and R6 promoter alleles. Among them a total of 40 accessions were identified which contain the R6 allele. 37 accessions showed a red coloration of the fruit flesh. All these accessions belong to type 1 red-fleshed apple. No type 2 red-fleshed apple could be found. Three accessions with R6 allele had non-red-fleshed apples. 312 other non-red-fleshed accessions contained only the R1 allele. Five non-red-fleshed accessions contained a new promoter allele with an unexpected size of ~1 kbp. Sequencing of this allele detected the insertion of a non-autonomous apple transposon.