Identification of serine/threonine kinase and nucleotide-binding site-leucine-rich repeat (NBS-LRR) genes in the fire blight resistance quantitative trait locus of apple cultivar 'Evereste'

Abstract

Fire blight is the most destructive bacterial disease affecting apple (Malus×domestica) worldwide. So far, no resistance gene against fire blight has been characterized in apple, despite several resistance regions having been identified. A highly efficacious resistance quantitative trait locus (QTL) was localized on linkage group 12 (LG12) of the ornamental cultivar 'Evereste'. A marker previously reported to be closely linked to this resistance was used to perform a chromosome landing. A bacterial artificial chromosome (BAC) clone of 189 kb carrying the fire blight resistance QTL was isolated and sequenced. New microsatellite markers were developed, and the genomic region containing the resistance locus was limited to 78 kb. A cluster of eight genes with homologies to already known resistance gene structures to bacterial diseases was identified and the corresponding gene transcription was verified. From this cluster, two genes were recognized in silico as the two most probable fire blight resistance genes showing homology with the Pto/Prf complex in tomato.

Figure 1

Contig assembly with all bacterial artificial chromosome (BAC) clones originating from the resistant chromosome (black rectangles) and susceptible chromosome (grey rectangles) of apple cultivar ‘Evereste’, and genetic map of the end (bottom part) of the linkage group 12 of ‘Evereste’. Genetic distances are in centimorgan (cM). The positions of the markers developed from BAC end sequences and of flanking microsatellite markers are shown with arrows. The dashed boxes indicate the region of the contig in which the M45TA or 44RP probes, used to screen the BAC library and to confirm the overlap of the clones, hybridized. T7, T7 end; RP, RP end of the BAC inserts; ChFbE02‐07, microsatellite markers from ChFbE02 to ChFbE07.

Figure 2

Graphical output created using gff2ps software (Abril and Guigo, 2000) and edited using Designer 9.0 (Micrografx, Inc.) of all open reading frames (ORFs) predicted by fgenesh found on the 78‐kb resistant region of bacterial artificial chromosome (BAC) 44A20 (Salamov and Solovyev, 2000). Exons are illustrated with boxes; single genes and the last exon of a group are shown by thick arrows. The genes showing similarities to resistance genes known to induce resistance to bacterial disease are highlighted in black. At the top of the sequence, boxes were added indicating the positions of mapped microsatellite markers; triangles indicate the transcriptional starts and hexagons indicate the transcriptional ends of the interesting ORFs.

Figure 3

Alignment of the amino acids encoded in the ATP‐binding region and serine/threonine (Ser/Thr) kinase region of the seven putative kinases found by the ClustalW2 program (Larkin et al., 2007). Conserved amino acids of the Ser/Thr kinase and ATP‐binding signatures are shown in black boxes.

Figure 4

Alignment of the amino acids encoded by MdE‐EaK7 and MdE‐EaK7susc predicted by the ClustalW2 program (Larkin et al., 2007). Conserved amino acids are shown in black boxes; amino acids of the ATP‐binding region and Ser/Thr kinase domain are shown in bold.

Figure 5

Alignment of the amino acids encoded by MdE‐EaN and MdE‐EaNsusc predicted by the ClustalW2 program (Larkin et al., 2007). Conserved amino acids are shown in black boxes.

Figure 6

Amplification patterns of MdE‐EaKs and MdE‐EaN genes. +, bacterial artificial chromosome (BAC) 44A20 DNA used as template; −, negative control (double‐distilled H₂O); RT, cDNA used as template; gDNA, ‘Evereste’ genomic DNA used as template; L, 100‐bp DNA ladder (O'GeneRuler™ 100 bp Plus DNA Ladder, Fermentas).