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

Abstract

Background: Breeding 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.

Results: When 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.

Conclusions: The 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.

Figure 1

Scatter plots of fire blight severity. Scatter plot of percentage of the current season’s shoot length that became necrotic following inoculation with E. amylovora (% SLN) evaluated in A) 2006 and 2007 on 83 progeny of ‘Malling 9’ x ‘Robusta 5’-NZ, and B) 2005 and 2006 on 132 progeny of ‘Idared’ x ‘Robusta 5’-DE. The R² value is for the linear line of best fit, and not for the 1:1 line as shown in the figure. Dashed lines indicate boundary for more than 20% difference between years.

Figure 2

Distribution of fire blight severity in ‘Robusta 5’ populations from New Zealand and Germany. Histograms of fire blight mean % cumulative necrosis of current season’s shoot length over two seasons, presented as a percentage (top, A and B) and on the logit transformed scale (bottom, C and D), for the progeny of A/C: ‘Malling 9’ x ‘Robusta 5’ NZ and B/D: ‘Idared’ x ‘Robusta 5’ DE.

Figure 3

QTLs on Linkage Group (LG) 3 of ‘Robusta 5’ for fire blight resistance identified in the ‘Malling 9’ x ‘Robusta 5’ New Zealand (M9xR5-NZ) and ‘Idared’ x ‘Robusta 5’ Germany (IxR5-DE) populations. A)^* LOD score curves for Single QTL Composite interval mapping (CIM) in M9xR5-NZ following inoculation with Erwinia amylovora strain ICMP11176. B) LOD score curves for CIM in IxR5-DE following inoculation with E. amylovora strain ICMP11176 by the Multiple Imputation method using 128 imputed datasets and 1000 permutations to estimate LOD thresholds. C)^* LOD score curves for CIM in IxR5-DE following inoculation with E. amylovora strain Ea222_JKI. ^* In A and C markers outside a window length of 3 cM were used as co-factors; interval mapping method used was ‘Hayley-Knott’ regression; the 95% confidence limits estimated by 1.5-LOD drop-off method are shown at the top figure; and putative fire blight resistance genes NZsnEH034548 and NZsnEB140229 mapped to the same position (indicated by arrow) and were excluded from the analyses. The LOD thresholds at significance levels of 5% and 1% calculated from 1000 permutations were [1.8, 2.5], [1.6, 2.6] and [1.7, 2.3], respectively, for Figures 3A, 3B and 3C. Two alternative CIM methods (EM algorithm and Imputation) produced similar results.

Figure 4

‘Robusta 5’ QTLs for fire blight resistance identified in the ‘Ottawa3’ x ‘Robusta 5’ United States (O3xR5-US) population on Linkage Groups 3 and 7. ‘Robusta 5’ QTLs for fire blight resistance identified in the ‘Ottawa3’ x ‘Robusta 5’ United States (O3xR5-US) population on Linkage Groups 3 and 7. A) LOD score curves for Multiple QTL Mapping following inoculation with Erwinia amylovora strain Ea273 using MAPQTL 6 (Kyazma, Wageningen, NL) MQM mapping option. B) LOD score curves of Multiple QTL Mapping following inoculation with Erwinia amylovora strain E2002a using MAPQTL 6 (Kyazma, Wageningen, NL) MQM mapping option.

Figure 5

Relative transcript abundance of three peroxidase genes in ‘Geneva 41’ and ‘Malling 26’ apple rootstocks. Fold change in transcript abundance of three peroxidase genes in fire blight resistant ‘Geneva 41’ (light gray) and susceptible ‘Malling 26’ (dark gray) Malus rootstocks following inoculation with Erwinia amylovora. A: MxdPrx8, a class 3 peroxidase that co-located with a QTL for fire blight resistance on LG3 of ‘Robusta 5’-NZ; B: MxdPrx9, a class 3 peroxidase that mapped to Linkage Group 10; and C: a class 1 cytosolic ascorbate peroxidise. Transcript abundance was determined by RT-qPCR, is expressed as fold change in comparison to mock challenged tissue of the same genotype sampled at the same hpi and is represented on a log2 scale to equalize magnitude of induced and repressed gene expression; a fold change of 1 indicate no difference from mock-inoculated (reference) whereas values less than 1 indicate repression of gene expression. Transcript abundance was normalized to an elongation factor 1 internal control and fold change calculated by the 2^-ΔΔCt method; error bars are the ΔΔCt standard deviation calculated from the technical replicates [3] of all 3 biological replicates (total of 9) [103]. EST sequence used for PCR primer design is indicated in brackets (GenBank accession number).

Figure 6

Sequence and structure ofMalusleucine-rich repeat family receptor-like polypeptide. Predicted translation product of the MxdRLP1-1 allele cloned from cultivars ‘Malling 26’, ‘Malling 27’ and ‘Robusta 5’. A predicted signal peptide cleavage site is indicated by an inverted arrowhead, and the signal peptide is double underlined. Nine consensus (LXXLXXLXLXXNXXαGXαPXXαG, where α represents any hydrophobic amino acid substitution [105]) LRR elements are boxed; the tenth degenerate element is boxed with a dotted line. The two mutated alleles isolated from cultivar ‘G.41’ are shown in boxes above the corresponding sequence.

Figure 7

Alignment of monocot and dicot leucine-rich repeats (LRR) homologues of the alleleMxdRLP1. Only the regions where amino acid replacements were detected in the apple LRRs are shown in the alignment. Numbers of amino acids from the presumed translation start methionine are listed to the right of the sequence. Amino acid substitutions at indicated loci are shown color-coded to highlight conservation or variation. Rectangular boxes indicate the positions of consensus LRRs. A dashed rectangle indicates deviation from the LRR consensus sequence. AtLRR: Arabidopsis thaliana (GenBank:NP188718, locus tag AT5G20820) ribonuclease inhibitor-like (RIL) LRR subfamily; PoptreLRR: Populus tremula (GenBank:ACE97248) RIL LRR subfamily; PopdeltLRR: P. deltoides (GenBank:ABS18952) RIL LRR subfamily; PoptriLRR: P. trichocarpa (GenBank:ABK92966) LRR; Alfalfa: Medicago truncatula (GenBank:ACJ85058); VitisLRR: Vitis vinifera (GenBank:CAO21943) RIL LRR subfamily; CicerPGIP: Cicer arietinum (GenBank:CAD56505) polygalacturonase inhibitor-like protein (PILP); ZeaLRR: Zea mays (GenBank:ACF88180) RIL LRR subfamily; ZeaPGIP2: Z. mays (GenBank:NP001150670) PILP; RiceLRR: Oryza sativa (GenBank:NP001062185); MxdRLP1-1: allele (GenBank:XX000002) found in the fire blight susceptible Malus x domestica cultivars ‘Malling 26’, ‘Malling 27’ and fire blight resistant ‘Robusta 5’.