Map-based isolation of the leaf rust disease resistance gene Lr10 from the hexaploid wheat (Triticum aestivum L.) genome

Abstract

More than 50 leaf rust resistance (Lr) genes against the fungal pathogen Puccinia triticina have been identified in the wheat gene pool, and a large number of them have been extensively used in breeding. Of the 50 Lr genes, all are known only from their phenotype and/or map position except for Lr21, which was cloned recently. For many years, the problems of molecular work in the large (1.6 x 10(10) bp), highly repetitive (80%), and hexaploid bread wheat (Triticum aestivum L.) genome have hampered map-based cloning. Here, we report the isolation of the Lr gene Lr10 from hexaploid wheat by using a combination of subgenome map-based cloning and haplotype studies in the genus Triticum. Lr10 is a single-copy gene on chromosome 1AS. It encodes a CC-NBS-LRR type of protein with an N-terminal domain, which is under diversifying selection. When overexpressed in transgenic wheat plants, Lr10 confers enhanced resistance to leaf rust. Lr10 has similarities to RPM1 in Arabidopsis thaliana and to resistance gene analogs in rice and barley, but is not closely related to other wheat Lr genes based on Southern analysis. We conclude that map-based cloning of genes of agronomic importance in hexaploid wheat is now feasible, opening perspectives for molecular bread wheat improvement trough transgenic strategies and diagnostic allele detection.

Fig. 1.

Two haplotypes defined by the presence or absence of two candidates for the Lr10 resistance gene on chromosome 1AS are present in the hexaploid wheat gene pool. (A) Schematic representation of the H1 and H2 haplotypes at the Lr10 locus. The candidate genes rga1 and rga2 are present on chromosome 1AS in lines with the H1 haplotype (e.g., ThatcherLr10), but not in lines with the H2 haplotype (e.g., Frisal). (B) The H2 haplotype is predominant and is very conserved in the wheat gene pool. Southern hybridization with HindIII (Upper)- and DraI(Lower)-digested genomic DNA isolated from a subset of 56 hexaploid European wheat breeding lines. Hybridizations were performed with rga1 (Upper) and the LRR domain of rga2 (Lower) as probes. Lines with an H1 haplotype are underlined. The fragments corresponding to T10rga2-1A on chromosome 1AS and T10rga2-1D on chromosome 1DS (25) are indicated with arrowheads.

Fig. 2.

Amino acid sequence of the Lr10 gene. The CC, NBS, spacer, and LRR domains are indicated. Amino acids belonging to characteristic motives in each domain are bold. In the NBS domain they are in the following order: P-loop, RNBS-A, kinase2, RNBS-B, RNBS-C, GLPL, RNBS-D, and MHDV. The four domains that have been used in the K_a/K_s analysis are separated from each other by an empty line. The spacer sequence is indicated in italics and the aliphatic (a) residues in the consensus (xxaxaxx) region of the LRR domain are boxed in yellow. The amino acid residues that are modified in the three EMS mutant genes are highlighted with red boxes with the number of the mutant above them.

Fig. 3.

Transgenic wheat seedlings overexpressing T10rga1 show enhanced resistance to leaf rust. (A) Southern hybridization of HindIII-digested genomic DNA extracted from Bobwhite SH 98 56 (S56), ThatcherLr10 (Th10), and 17 transgenic T1 plants of the T17 family (T17A_x) with T10rga1 as a probe. The arrowhead indicates the 5.6-kb fragment, which is expected from a HindIII digest of the pUbi_T10rga1 construct. (B) Northern blot of total RNA extracted from the same seedlings hybridized with T10rga1 as a probe. The same blot was hybridized with the housekeeping GAPDH gene as a control. The relative intensity of the hybridization signals in the transgenics vs. wild-type plants was estimated with the Cyclone gene array system (Perkin–Elmer, Boston). Transgenic plants showing chlorotic hypersensitive resistance reaction (i.e., T17A_2) are blue, those with a necrotic phenotype (i.e., T17A_3) are red, while susceptible T1 plants are black. (C) Phenotypes of transgenic plants overexpressing Lr10 compared with the resistant ThacherLr10 and the susceptible Bobwhite SH 98 56, 10 days after artificial infection with the leaf rust isolate TCB/TD AvrLr10.

Fig. 4.

NJ phylogenetic tree of LR10, LR21, and products of RGAs from different grass species. Except for Td_RGA1 (unpublished results) and the rice homologs on chromosome 8, which were annotated from a BAC sequence (GenBank accession no. AP005158) in this work, the accession numbers of the proteins are given next to the gene names. Genes that did not have names in the database were named after the species of origin (OS, rice; Sb, sorghum) and the homology defined by the authors in the database annotation. The chromosomal location of the genes is indicated on the branch of the phylogenetic tree.