Characterization of Lr46, a gene conferring partial resistance to wheat leaf rust

Components of resistance conferred by the Lr46 gene, reported as causing "slow rusting" resistance to leaf rust in wheat, were studied and compared with the effects of Lr34 and genes for quantitative resistance in cv. Akabozu. Lr34 is a gene that confers non-hypersensitive type of resistance. The effect of Lr46 resembles that of Lr34 and other wheats reported with partial resistance. At macroscopic level, Lr46 produced a longer latency period than observed on the susceptible recurrent parent Lalbahadur, and a reduction of the infection frequency not associated with hypersensitivity. Microscopically, Lr46 increased the percentage of early aborted infection units not associated with host cell necrosis and decreased the colony size. The effect of Lr46 is comparable to that of Lr34 in adult plant stage, but in seedling stage its effect is weaker than that of Lr34.     

AFLP and STS tagging of Lr19, a gene conferring resistance to leaf rust in wheat

Amplified fragment length polymorphism (AFLP) markers were used to enrich the map of the wheat chromosomal region containing the Thinopyrum-derived Lr19 leaf rust resistance gene. The region closest to Lr19 was targeted through the use of deletion and recombinant lines of the translocated segment. One of the AFLP bands thus identified was converted into a sequence-tagged-site (STS) marker. This assay generated a 130-bp PCR fragment in all Lr19-carrying lines tested, except for one deletion mutant, while non-carrier template failed to amplify any product. This sequence represents the first marker to map on the distal side of Lr19 on chromosome 7el₁. The conversion process of AFLP fragments to STS markers was technically difficult, mainly because of the presence of contaminating fragments. Various approaches were taken to reduce the frequency of false positives and to identify the correct clone. We were able to formulate a general verification strategy prior to clone sequencing. Various other factors causing problems with converting AFLP bands to an STS assays are also discussed. Received: 15 September 2000 / Accepted: 5 January 2001     

Number of genes controlling slow rusting resistance to leaf rust in five spring wheat cultivars

The number of genes controlling slow rusting resistance to leaf rust (Puccinia triticina) was estimated in five spring wheat (Triticum aestivum) cultivars using quantitative formulae. Parents and F₆ families were evaluated in replicated field trials under epidemics initiated by artificial inoculation. The F₆ families resulted from a diallel cross involving the fast-rusting cultivar Yecora 70 and five slow-rusting wheat cultivars: Sonoita 81, Tanager ‘S’, Galvez 87, Ures 81, and Moncho ‘S’. The area under the disease progress curve (AUDPC) was used to measure leaf rust severity over time. Results indicate that cultivar Sonoita 81 has three or four genes, Tanager ‘S’ has two or three genes, Galvez 87 has three genes, and both Ures 81 and Moncho ‘S’ have two genes for slow rusting resistance to leaf rust. Based on this result and previously reported moderate to high narrow-sense heritability estimates for slow rusting resistance in these materials, early-generation selection for slow leaf rusting would be effective.     

Molecular markers for wheat leaf rust resistance gene Lr41

Leaf rust, caused by Puccinia triticina Eriks., is an important foliar disease of common wheat (Triticum aestivum L.) worldwide. Pyramiding several major rust-resistance genes into one adapted cultivar is one strategy for obtaining more durable resistance. Molecular markers linked to these genes are essential tools for gene pyramiding. The rust-resistance gene Lr41 from T. tauschii has been introgressed into chromosome 2D of several wheat cultivars that are currently under commercial production. To discover molecular markers closely linked to Lr41, a set of near-isogenic lines (NILs) of the hard winter wheat cultivar Century were developed through backcrossing. A population of 95 BC₃F_2:6 NILs were evaluated for leaf rust resistance at both seedling and adult plant stages and analyzed with simple sequence repeat (SSR) markers using bulked segregant analysis. Four markers closely linked to Lr41 were identified on chromosome 2DS; the closest marker, Xbarc124, was about 1 cM from Lr41. Physical mapping using Chinese Spring nullitetrasomic and ditelosomic genetic stocks confirmed that markers linked to Lr41 were on chromosome arm 2DS. Marker analysis in a diverse set of wheat germplasm indicated that primers BARC124, GWM210, and GDM35 amplified polymorphic bands between most resistant and susceptible accessions and can be used for marker-assisted selection in breeding programs.     

Molecular mapping of leaf rust resistance gene Lr15 in hexaploid wheat

Leaf rust is a widespread and commonly occurring rust disease of wheat. Genetic resistance is the most economical method of reducing losses due to leaf rust. Lr15 has been shown to be present on wheat chromosome 2D and is reported to be a seedling resistance gene. However, tightly linked markers associated with Lr15 have not been reported to date. To identify molecular markers linked to Lr15, an F₂ mapping population of Thatcher × Thatcher-Lr15 was generated. Available wheat simple sequence repeat markers were utilized in parental screening and polymorphic markers were used to analyze the entire population of 221 plants. Phenotypic evaluations of the F₂-derived F₃ progenies with Puccinia triticina Eriks. pathotype 162A (93R15) confirmed the monogenic inheritance of Lr15. The linkage group representing chromosome 2DS was constructed at LOD 4.0 which revealed the closest flanking markers Xgwm4562 and Xgwm102 at a distance of 3.1 and 9.3 cM, respectively. Furthermore, utilization of these flanking markers in combination has successfully identified wheat lines with or without Lr15. These markers could potentially be useful in gene pyramiding with other genes to enhance rust resistance in wheat.     

An N-band marker for gene Lr18 for resistance to leaf rust in wheat

The leaf rust resistance gene, Lr18, of common wheat cultivars has been derived from Triticum timopheevi and is located on chromosome arm 5BL. Chromosome banding (N-banding) analyses revealed that in the wheat cultivars carrying Lr18 that were examined, which had been bred in 6 different countries, chromosome arm 5BL possessed a specific terminal band not carried by their susceptible parental cultivars. It was suggested that this terminal N-band was introduced from T. timopheevi together with Lr18. N-banding analysis of a T. timopheevi strain showed that one of two timopheevi chromosomes had provided Japanese wheat lines containing Lr18 with the terminal band.     

Lr70, a new gene for leaf rust resistance mapped in common wheat accession KU3198

Key message

KU3198 is a common wheat accession that carries one novel leaf rust resistance (Lr) gene, Lr70 , and another Lr gene which is either novel, Lr52 or an allele of Lr52.

Abstract

Leaf rust, caused by Puccinia triticina Eriks. (Pt), is a broadly distributed and economically important disease of wheat. Deploying cultivars carrying effective leaf rust resistance (Lr) genes is a desirable method of disease control. KU3198 is a common wheat (Triticum aestivum L.) accession from the Kyoto collection that was highly resistant to Pt in Canada. An F₂ population from the cross HY644/KU3198 showed segregation for two dominant Lr genes when tested with Pt race MBDS which was virulent on HY644. Multiple bulk segregant analysis (MBSA) was employed to find putative chromosome locations of these Lr genes using SSR markers that provided coverage of the genome. MBSA predicted that the Lr genes were located on chromosomes 5B and 5D. A doubled haploid population was generated from the cross of JBT05-714 (HY644*3/KU3198), a line carrying one of the Lr genes from KU3198, to Thatcher. This population segregated for a single Lr gene conferring resistance to Pt race MBDS, which was mapped to the terminal region of the short arm of chromosome 5B with SSR markers and given the temporary designation LrK1. One F₃ family derived from the HY644/KU3198 F₂ population that segregated only for the second Lr gene from KU3198 was identified. This family was treated as an F₂-equivalent population and used for mapping the Lr gene, which was located to the terminal region of chromosome 5DS. As no other Lr gene has been mapped to 5DS, this gene is novel and has been designated as Lr70.     

Development of PCR markers for wheat leaf rust resistance gene Lr47

The leaf rust resistance gene Lr47 confers resistance to a wide spectrum of leaf rust strains. This gene was recently transferred from chromosome 7S of Triticum speltoides to chromosome 7A of hexaploid wheat Triticum aestivum. To facilitate the transfer of Lr47 to commercial varieties, the completely linked restriction fragment length polymorphism (RFLP) locus Xabc465 was converted into a PCR-based marker. Barley clone ABC465 is orthologous to the type-I wheat sucrose synthase gene and primers were designed for the conserved regions between the two sequences. These conserved primers were used to amplify, clone and sequence different alleles from T. speltoides and T. aestivum. This sequence information was used to identify the T. speltoides sequence, detect allele-specific mutations, and design specific primers. Cosegregation of the PCR product of these primers and the T. speltoides chromosome segment was confirmed in four backcross-populations. To complement this dominant marker, a cleavage amplified polymorphic sequence (CAPS) was developed for the 7A allele of Xabc465. This CAPS marker is useful to select homozygous Lr47 plants from F₂ or backcross-F₂ segregating populations, and in combination with the T- speltoides specific primers is expected to facilitate the deployment of Lr47 in new bread wheat varieties. Received: 11 October 1999 / Accepted: 30 December 1999     

Molecular genetic characterization of the Lr34/Yr18 slow rusting resistance gene region in wheat

Wheat expressed sequence tags (wESTs) were identified in a genomic interval predicted to span the Lr34/Yr18 slow rusting region on chromosome 7DS and that corresponded to genes located in the syntenic region of rice chromosome 6 (between 2.02 and 2.38 Mb). A subset of the wESTs was also used to identify corresponding bacterial artificial chromosome (BAC) clones from the diploid D genome of wheat (Aegilops tauschii). Conservation and deviation of micro-colinearity within blocks of genes were found in the D genome BACs relative to the orthologous sequences in rice. Extensive RFLP analysis using the wEST derived clones as probes on a panel of wheat genetic stocks with or without Lr34/Yr18 revealed monomorphic patterns as the norm in this region of the wheat genome. A similar pattern was observed with single nucleotide polymorphism analysis on a subset of the wEST derived clones and subclones from corresponding D genome BACs. One exception was a wEST derived clone that produced a consistent RFLP pattern that distinguished the Lr34/Yr18 genetic stocks and well-established cultivars known either to possess or lack Lr34/Yr18. Conversion of the RFLP to a codominant sequence tagged site (csLV34) revealed a bi-allelic locus, where a variant size of 79 bp insertion in an intron sequence was associated with lines or cultivars that lacked Lr34/Yr18. This association with Lr34/Yr18 was validated in wheat cultivars from diverse backgrounds. Genetic linkage between csLV34 and Lr34/Yr18 was estimated at 0.4 cM     

Effect of gene Lr34 in the enhancement of resistance to leaf rust of wheat

Summary Leaf rust resistance gene Lr34 is present in many wheat cultivars throughout the world that have shown durable resistance to leaf rust. Fourteen pair-wise combinations of Lr34 and seedling leaf rust resistance genes were developed by intercrossing near isogenic Thatcher lines. In both seedling and adult plant tests homozygous paired combinations of specific resistance genes with Lr34 had enhanced resistance relative to either parent to different numbers of isolates that were avirulent to the additional resistance genes. The TcLr34, 18 line also expressed enhanced resistance to specific isolates virulent to Lr18 in seedling and adult plant stages. In rust nursery tests, homozygous lines were more resistant than either parent, if the additional leaf rust gene conditioned an effective of resistance when present singly. The ability of Lr34 to interact with other genes conditioning effective resistance may contribute to the durability of leaf rust resistance in cultivars with Lr34. Contribution 1453 Agriculture Canada     

Development of PCR markers for the wheat leaf rust resistance gene Lr47

The leaf rust resistance gene Lr47 confers resistance to a wide spectrum of leaf rust strains. This gene was recently transferred from chromosome 7 S of Triticum speltoides to chromosome 7 A of hexaploid wheat Triticum aestivum. To facilitate the transfer of Lr47 to commercial varieties, the completely linked restriction fragment length polymorphism (RFLP) locus Xabc465 was converted into a PCR-based marker. Barley clone ABC465 is orthologous to the type-I wheat sucrose synthase gene and primers were designed for the conserved regions between the two sequences. These conserved primers were used to amplify, clone and sequence different alleles from T. speltoides and T. aestivum. This sequence information was then used to identify the T. speltoides sequence, detect allele-specific mutations, and design specific primers. Cosegregation of the PCR product of these primers and the T. speltoides chromosome segment was confirmed in four backcross-populations. To complement this dominant marker, a cleavage amplified polymorphic sequence (CAPS) was developed for the 7 A allele of Xabc465. This CAPS marker is useful to select homozygous Lr47 plants from F₂or backcross-F₂ segregating populations, and in combination with the T. speltoides-specific primers is expected to facilitate the deployment of Lr47 in new bread wheat varieties. Received: 7 June 1999 / Accepted: 30 September 1999     

Cytogenetic and molecular mapping of the leaf rust resistance gene Lr39 in wheat

Leaf rust, caused by the fungus Puccinia triticina Eriks,is one of the most serious diseases of wheat (Triticum aestivum AABBDD, 2n=6x=42) worldwide. Growing resistant cultivars is an efficient and economical method of reducing losses to leaf rust. Here we report a new leaf rust resistance gene, Lr39, transferred from Aegilops tauschii into common wheat. Lr39 conditions both seedling and adult plant resistance to the leaf rust pathogen. The inter- and intra-chromosomal mapping of the Lr39 gene showed that it is different from all previously described Lr genes. We used monosomic analysis for the inter-chromosomal mapping and wheat microsatellite markers for the intra-chromosomal mapping. The monosomic and ditelosomic analysis indicated that Lr39 is independent of the centromere on the short arm of chromosome 2D. Eight microsatellite markers for 2DS were used for linkage analysis on a population of 57 F₂ plants derived from a cross of an Ae. tauschii-derived wheat, cv. Wichita line TA4186 (possessing Lr39), with Wichita monosomics for the D-genome chromosomes. The microsatellite marker analysis confirmed the location of the gene on 2DS. Three markers were polymorphic and linked to the gene. The closest marker Xgwm210 mapped 10.7 cM from Lr39. The location of Lr39 near the telomere of 2DS distinguishes it from the Lr2 and Lr22 loci, which are located on 2DS proximal to Xgwm210. Received: 19 April 2000 / Accepted: 15 May 2000     

An interchromosomal reciprocal translocation in wheat involving leaf rust resistance gene Lr34.

'Thatcher' backcross lines RL6058 and RL6077 have adult-plant leaf rust resistance and were believed to have Lr34. However, genetic analysis revealed that the genes in the two lines were independent of each other. Previous work demonstrated that Lr34 is located on chromosome 7D. The leaf rust resistance gene in RL6058 must be on chromosome 7DS because no recombinants were observed between it and gene Lr29, known to be on chromosome 7DS. It was also linked with Rc3 (30.25 +/- 2.88%), a gene for purple coleoptile on chromosome 7DS. It was independent of Lr19 and NS1 (nonsuppressor mutant), which are located on 7DL. The leaf rust resistance gene in RL6077 was independent of genes Lr19 and Lr29. The presence of quadrivalents in pollen mother cells of the RL6058/RL6077 hybrid indicates that the Lr34 gene in RL6077 may have been translocated onto another chromosome. Lr34 from RL6058 and RL6077 may have been combined in four F3 lines derived from their intercross.     

Identification of a STS marker linked to the Aegilops speltoides-derived leaf rust resistance gene Lr28 in wheat

 A sequence-tagged-site (STS) marker is reported linked to Lr28, a leaf rust resistance gene in wheat. RAPD (random amplified polymorphic DNA) analysis of near-isogenic lines (NILs) of Lr28 in eight varietal backgrounds was carried out using random primers. Genomic DNA enriched for low-copy sequences was used for RAPD analysis to overcome the lack of reproducibility due to the highly repetitive DNA sequences present in wheat. Of 80 random primers tested on the enriched DNA, one RAPD marker distinguished the NILs and the donor parent from the susceptible recurrent parents. The additional band present in resistant lines was cloned, sequenced, and STS primers specific for Lr28 were designed. The STS marker (Indian patent pending: 380 Del98) was further confirmed by bulk segregation analysis of F₃ families. It was consistently present in the NILs, the resistant F₃ bulk and the resistant F₃ lines, but was absent in recurrent parents, the susceptible F₃ bulk and the susceptible F₃ lines. Received: 20 February 1998 / Accepted: 4 March 1998     

A candidate for Lr19, an exotic gene conditioning leaf rust resistance in wheat

Lr19, one of the few widely effective genes conferring resistance to leaf rust in wheat, was transferred from the wild relative Thinopyrum ponticum to durum wheat. Since Lr19 confers a hypersensitive response to the pathogen, it was considered likely that the gene would be a member of the major nucleotide-binding site (NBS)-leucine-rich repeat (LRR) plant R gene family. NBS profiling, based on PCR amplification of conserved NBS motifs, was applied to durum wheat–Th. ponticum recombinant lines involving different segments of the alien 7AgL chromosome arm, carrying or lacking Lr19. Differential PCR products were isolated and sequenced. From one such sequence (AG15), tightly linked to Lr19, a 4,121-bp full-length cDNA was obtained. Its deduced 1,258 amino acid sequence has the characteristic NBS-LRR domains of plant R gene products and includes a coiled-coil (CC) region typical of monocots. The genomic DNA sequence showed the presence of two exons and a short intron upstream of the predicted stop codon. Homology searches revealed considerable identity of AG15 with the cloned wheat resistance gene Pm3a and a lower similarity with wheat Lr1, Lr21, and Lr10. Quantitative PCR on leaf-rust-infected and non-infected Lr19 carriers proved AG15 to be constitutively expressed, as is common for R genes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Identification and validation of molecular markers linked to the leaf rust resistance gene Lr19 in wheat

A leaf rust resistance gene Lr19 on the chromosome 7DL of wheat derived from Agropyron elongatum was tagged with random amplified polymorphic DNA (RAPD) and microsatellite markers. The F₂ population of 340 plants derived from a cross between the leaf rust resistant near-isogenic line (NIL) of Thatcher (Tc + Lr19) and leaf rust susceptible line Agra Local that segregated for dominant monogenic leaf rust resistance was utilized for generating the mapping population. The molecular markers were mapped in the F₂ derived F₃ homozygous population of 140 seedlings. Sixteen RAPD markers were identified as linked to the alien gene Lr19 among which eight were in a coupling phase linkage. Twelve RAPD markers co-segregated with Lr19 locus. Nine microsatellite markers located on the long arm of chromosome 7D were also mapped as linked to the gene Lr19, including 7 markers which co-segregated with Lr19 locus, thus generating a saturated region carrying 25 molecular markers linked to the gene Lr19 within 10.2 ± 0.062 cM on either side of the locus. Two RAPD markers S265₅₁₂ and S253₇₃₇ which flanked the locus Lr19 were converted to sequence characterized amplified region markers SCS265₅₁₂ and SCS253₇₃₆, respectively. The marker SCS265₅₁₂ was linked with Lr19 in a coupling phase and the marker SCS253₇₃₆ was linked in a repulsion phase, which when used together mimicked one co-dominant marker capable of distinguishing the heterozygous resistant seedlings from the homozygous resistant. The molecular markers were validated on NILs mostly in Thatcher background isogenic for 44 different Lr genes belonging to both native and alien origin. The validation for polymorphism in common leaf rust susceptible cultivars also confirmed the utility of these tightly linked markers to the gene Lr19 in marker-assisted selection.     

Identification of molecular markers linked to the Agropyron elongatum-derived leaf rust resistance gene Lr24 in wheat

The objective of this study was to identify molecular markers linked to the wheat leaf rust resistance gene Lr24 derived from Agropyron elongatum (3DL/3Ag translocation). Two near isogenic lines (NILs), ‘Arina’ and Lr24/7 ^* “Arina”, were screened for polymorphism at the DNA level with 115 RFLP probes. Twenty-one of these probes map to the homoeologous group 3. In addition, 360 RAPD primers were tested on the NILs. Six RFLP probes showed polymorphism between the NILs, and 11 RAPD primers detected one additional band in the resistant NIL. The genetic linkage of the polymorphic markers with Lr24 was tested on a segregating F₂ population (150 plants) derived from a cross between the leaf rust resistant Lr24/7 ^* “Arina” and the susceptible spelt (Triticum spelta) variety ‘Oberkulmer’. All 6 RFLP markers were completely linked to Lr24: one was inherited as a codominant marker (PSR1205), one was in coupling phase (PSR1203) and 4 were in repulsion phase (PSR388, PSR904, PSR931, PSR1067) with Lr24. The localization of these probes on chromosome 3D was confirmed by nulli-tetrasomic analysis. Distorted genotypic segregation was found for the Codominant RFLP marker PSR1205. This distortion can be explained by the occurrence of hemizygous plants. One of the 11 RAPD markers (OPJ-09) also showed complete linkage to theLr24 resistance gene. The polymorphic RAPD fragment was cloned and sequenced. Specific primers were synthesized, and they produced an amplification product only in the resistant plants. This specific marker allows a reliable and rapid screening of a large number of genotypes in practical breeding. Analysis of 6 additional lines containing Lr24 revealed that 3 lines have a smaller chromosomal segment of A. elongatum than lines derived from ‘Agent’, a commonly used gene donor for the Lr24 resistance gene.     

Development of a molecular marker for the adult plant leaf rust resistance gene Lr35 in wheat

The objective of this work was to develop a marker for the adult plant leaf rust resistance gene Lr35. The Lr35 gene was originally introgressed into chromosome 2B from Triticum speltoides, a diploid relative of wheat. A segregating population of 96 F ₂ plants derived from a cross between the resistant line ThatcherLr35 and the susceptible variety Frisal was analysed. Out of 80 RFLP probes previously mapped on wheat chromosome 2B, 51 detected a polymorphism between the parents of the cross. Three of them were completely linked with the resistance gene Lr35. The co-segregating probe BCD260 was converted into a PCR-based sequence-tagged-site (STS) marker. A set of 48 different breeding lines derived from several European breeding programs was tested with the STS marker. None of these lines has a donor for Lr35 in its pedigree and all of them reacted negatively with the STS marker. As no leaf rust races virulent on Lr35 have been found in different areas of the world, the STS marker for the Lr35 resistance gene is of great value to support the introgression of this gene in combination with other leaf rust (Lr) genes into breeding material by marker-assisted selection. Received: 14 December 1998 / Accepted: 30 January 1999     

Identification and localization of molecular markers linked to the Lr9 leaf rust resistance gene of wheat

Near-isogenic lines (NILs) for the leaf rust resistance gene Lr9 were screened for polymorphisms at the molecular level. RAPD (random amplified polymorphic DNA) primers as well as RFLP (restriction fragment length polymorphism) markers were used. Out of 395 RAPD primers tested, three showed polymorphisms between NILs, i.e., an additional band was found in resistant lines. One of these polymorphic bands was cloned and sequenced. Specific primers were synthesized, and after amplification only resistant lines showed an amplified product. Thus, these primers define a sequence-tagged site that is specific for the translocated fragment carrying the Lr9 gene. A cross between a resistant NIL and the spelt (Triticum spelta) variety Oberkulmer was made, and F₂ plants were analyzed for genetic linkage. All three polymorphisms detected by the PCR (polymerase chain reaction) and one RFLP marker (cMWG684) showed complete linkage to the Lr9 gene in 156 and 133 plants analyzed, respectively. A second RFLP marker (PSR546) was closely linked (8±2.4 cM) to the Lr9 gene and the other four DNA markers. As this marker maps to the distal part of the long arm of chromosome 6B of wheat, Lr9 and the other DNA markers also map to the distal region of 6BL. All three PCR markers detected the Lr9 gene in independently derived breeding lines and varieties, thus proving their general applicability in wheat breeding programs.