Keller B

Krattinger SG

Lagudah, E

Spielmeyer W

English

Sydney eScholarship

 1Fine-mapping of the durable leaf rust resistance gene Lr34 using sequence information from Brachypodium and Aegilops tauschii  Krattinger SG1, Lagudah ES2, Spielmeyer W2 and Keller B1 1 Institute of Plant Biology, University of Zurich, Switzerland 2 CSIRO Plant Industy, Canberra, Australia  THE DURABLE LEAF RUST RESISTANCE GENE LR34 One of the most relevant genes in wheat disease resistance breeding is Lr34. This locus has contributed towards durable resistance to leaf rust, stripe rust, stem rust and powdery mildew, making Lr34 a unique resource for breeding and a model for understanding the molecular basis of partial horizontal resistance. Lr34 has been used in breeding programs since many decades and has not been overcome. A detailed QTL study mapped this gene to the short arm of chromosome 7D (Schnurbusch et al. 2004). The gene explained up to 42% of the phenotypic variance observed in the field. Lr34 is associated with the morphological marker ‘Leaf Tip Necrosis’ (Ltn1). Ltn1 can be used as a morphological marker to assist breeding for Lr34 (Figure1), but is limited by variable expression under different environmental conditions and genetic background.   Fig.1: A wheat plant carrying the durable resistance gene Lr34 and exhibiting the morphological marker ‘Leaf Tip Necrosis’. INCREASING MARKER DENSITY AROUND LR34 Previous work identified the two SSR markers gwm1220 (distal) and SWM10 (proximal) as the closest flanking markers for Lr34 (Lagudah et al. 2006, Bossolini et al. 2006). To increase marker density around Lr34, we used molecular information from the orthologous region in Brachypodium sylvaticum and rice. Brachypodium was recently proposed as a new model organism for temperate grasses and it showed closer homology to wheat than rice (Bossolini et al. 2007). In Brachypodium there are four coding sequences spanning the homologous Lr34 target interval between the two flanking markers, namely a serine kinase, a pectate lyase, a hypothetical protein and a GTP binding protein. Wheat expressed sequence tags (wESTs) were identified by homology with these Brachypodium genes. These wESTs were then used to screen a fingerprinted bacterial artificial chromosome (BAC) library from Aegilops tauschii (Jan Dvorak, UC Davis), which is the donor of the wheat D-genome. Three BAC contigs of Ae. tauschii consisting of 124 BAC clones and spanning about 2.1 Mb of the Lr34 target interval were identified by hybridization screening. They contained all the orthologous genes of Brachypodium as well as the two flanking markers gwm1220 and SWM10. We therefore concluded that a significant portion of the Lr34 target interval was covered by these contigs. Thirteen BAC clones were selected for low-pass sequencing, resulting in a total of 790 kb of sequence information. These sequences were mined for simple sequence repeats (SSR), since these elements are known to be very powerful tools for genetic mapping and marker assisted selection even in genomes with a low degree of polymorphism. In addition locus-specific sequence-tagged sites (STS) were developed and their sequences were analysed for single nucleotide polymorphism (SNPs) and insertion / deletion (InDel) markers on the parents of the mapping populations. A third strategy was to develop RFLP markers based on low-copy probes. Using these strategies we were able to develop ten new molecular markers in the region of interest. All of them mapped to the expected position according to the homology with Brachypodium. Hence, Brachypodium sylvaticum proved to be a good model organism to derive locus-specific molecular markers in wheat. While the targeted gene region from Brachypodium was also identified in rice, the latter species carried a large inverted segment relative to the orthologous regions in Ae. tauschii, the D genome of hexaploid wheat and Brachypodium (Bossolini et al 2007, Spielmeyer et al 2008). Using sequence information from the diploid D-genome progenitor Ae. tauschii turned out to be a successful strategy, since most PCR-based probes were highly transferable to hexaploid wheat cultivars. Of more significance were the additional markers that were obtained from Ae. tauschii but absent from the corresponding region in Brachypodium; these additional markers mapped closer to, as well as at the Lr34 locus.   2 HIGH RESOLUTION GENETIC MAP  Three high-resolution back-cross populations were used to determine the mapping positions of the newly derived molecular markers. Phentoypic analysis was done independently on all the populations over several years. Lr34 showed close linkage to markers csLVMS, csLVE17, SWM10 and SWSNP3. The two microsatellite markers csLVMS and SWM10 showed the same allele in the three independent sources of Lr34 ‘Frontanta’, ‘Chinese Spring’ and ‘Forno’ as well as in many additional cultivars containing Lr34 (Lagudah et al. 2006, Bossolini et al. 2006). Hence, these are highly useful markers to assist selection for Lr34 in breeding programs worldwide. Genetic mapping defined a 0.15 cM interval that is flanked by the two new markers csLVE17 and SWSNP3. Five markers showed co-segregation with Lr34. Figure 2 summarizes the mapping data based on a consensus map from three fine-mapping populations.  Xgwm12200.00XSWSNP30.67Lr340.79XcsLVE170.82XcsLVMS0.85XSWM100.88  Fig.2: High resolution genetic consensus map of the Lr34 region based on three fine-mapping populations. The map displays the position of the two newly developed flanking markers csLVE17 and SWSNP3 as well as the earlier described markers SWM10, csLVMS and gwm1220.  FROM GENETIC TO PHYSICAL INFORMATION  The two newly derived markers csLVE17 and SWSNP3 define a 0.15 cM target interval for Lr34. This interval was spanned physically by a BAC contig from Ae. tauschii. To get corresponding sequence information from hexaploid wheat, BAC libraries from the wheat cultivars ‘Chinese Spring’ (INRA, Toulouse) and ‘Glenlea’ (Sylvie Cloutier, Agriculture and Agri-Food, Canada) were screened using locus-specific PCR and RFLP probes derived from the Ae. tauschii contig. Four ‘Chinese Spring’ BAC clones spanning the whole target interval were identified and fully sequenced. These sequencing data revealed the presence of a gene-rich region surrounded by repeat-rich sequences. This finding corroborates the hypothesis that the wheat genome is organized in gene-rich islands surrounded by large stretches of repetitive sequences. The gene-rich region consists of ten candidate genes. None of these coding sequences has a homolog in Brachypodium sylvaticum or Brachypodium distachyon. Probably several independent insertion events led to the complexity of this gene-rich island. We propose that one of these coding sequences is responsible for the durable resistance conferred by Lr34.  REFERENCES  Bossolini E, Krattinger SG and Keller B (2006) Development of simple sequence repeat markers specific for the Lr34 resistance region of wheat. Theor. Appl. Genet. 113: 1049-1062. Bossolini E, Wicker T, Knobel PA and Keller B (2007) Comparison of orthologous loci from small grass genomes Brachypodium and rice: implications for wheat genomics and grass genome annotation. Plant Journal 49: 704-717. Lagudah ES, McFadden H, Singh RP, Huerta-Espino J, Bariana HS and Spielmeyer W (2006) Molecular genetic characterization of the Lr34/Yr18 slow rusting resistance gene region in wheat. Theor. Appl. Genet. 114: 21-30. Schnurbusch T, Bossolini E, Messmer M and Keller B (2004) Tagging and validation of a major QTL for leaf rust resistance and leaf tip necrosis in the winter wheat cultivar ‘Forno’. Phytopathology 94: 1036-1041. Spielmeyer W, Singh RP, McFadden H, Wellings CR, Huerta-Espino J, Kong X, Appels R, Lagudah ES (2008) Fine scale genetic and physical mapping using interstitial deletion mutants of Lr34/Yr18: A disease resistance locus effective against multiple pathogens in wheat. Theor. Appl. Genet. 116: 481-490. 

Fine-mapping of the durable leaf rust resistance gene Lr34 using sequence information from Brachypodium and Aegiliops tauschii

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Fine-mapping of the durable leaf rust resistance gene Lr34 using sequence information from Brachypodium and Aegiliops tauschii

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