NBS-LRR sequence family is associated with leaf and stripe rust resistance on the end of homoeologous chromosome group 1S of wheat

A detailed RFLP map was constructed of the distal end of the short arm of chromosome 1D of Aegilops tauschii and wheat. At least two unrelated resistance-gene analogs (RGAs) mapped close to known leaf rust resistance genes (Lr21 and Lr40) located distal to seed storage protein genes on chromosome 1DS. One of the two RGA clones, which was previously shown to be part of a candidate gene for stripe rust resistance (Yr10) located within the homoeologous region on 1BS, identified at least three gene family members on chromosome 1DS of Ae. tauschii. One of the gene members co-segregated with the leaf rust resistance genes, Lr21 and Lr40, in Ae. tauschii and wheat segregating families. Hence, a RGA clone derived from a candidate gene for stripe rust resistance located on chromosome 1BS detected candidate genes for leaf rust resistance located in the corresponding region on 1DS of wheat. Received: 10 January 2000 / Accepted: 25 March 2000     

Identification and genetic characterization of an <Emphasis Type="Italic">Aegilops tauschii</Emphasis> ortholog of the wheat leaf rust disease resistance gene <Emphasis Type="Italic">Lr1</Emphasis>

Aegilops tauschii (goat grass) is the progenitor of the D genome in hexaploid bread wheat. We have screened more than 200 Ae. tauschii accessions for resistance against leaf rust (Puccinia triticina) isolates, which are avirulent on the leaf rust resistance gene Lr1. Approximately 3.5% of the Ae. tauschii accessions displayed the same low infection type as the tester line Thatcher Lr1. The accession Tr.t. 213, which showed resistance after artificial infection with Lr1 isolates both in Mexico and in Switzerland, was chosen for further analysis. Genetic analysis showed that the resistance in this accession is controlled by a single dominant gene, which mapped at the same chromosomal position as Lr1 in wheat. It was delimited in a 1.3-cM region between the restriction fragment length polymorphism (RFLP) markers ABC718 and PSR567 on chromosome 5DL of Ae. tauschii. The gene was more tightly linked to PSR567 (0.47 cM) than to ABC718 (0.79 cM). These results indicate that the resistance gene in Ae. tauschii accession Tr.t. 213 is an ortholog of the leaf rust resistance gene Lr1 of bread wheat, suggesting that Lr1 originally evolved in diploid goat grass and was introgressed into the wheat D genome during or after domestication of hexaploid wheat. Compared to hexaploid wheat, higher marker polymorphism and recombination frequencies were observed in the region of the Lr1 ortholog in Ae. tauschii. The identification of Lr1^Ae, the orthologous gene of wheat Lr1, in Ae. tauschii will allow map-based cloning of Lr1 from this genetically simpler, diploid genome.Hong-Qing Ling and Jiwen Qiu have contributed equally to this work     

Two different CC‐NBS‐LRR genes are required for Lr10‐mediated leaf rust resistance in tetraploid and hexaploid wheat

Comparative study of disease resistance genes in crop plants and their relatives provides insight on resistance gene function, evolution and diversity. Here, we studied the allelic diversity of the Lr10 leaf rust resistance gene, a CC‐NBS‐LRR coding gene originally isolated from hexaploid wheat, in 20 diploid and tetraploid wheat lines. Besides a gene in the tetraploid wheat variety ‘Altar’ that is identical to the hexaploid wheat Lr10, two additional, functional resistance alleles showing sequence diversity were identified by virus‐induced gene silencing in tetraploid wheat lines. In contrast to most described NBS‐LRR proteins, the N‐terminal CC domain of LR10 was found to be under strong diversifying selection. A second NBS‐LRR gene at the Lr10 locus, RGA2, was shown through silencing to be essential for Lr10 function. Interestingly, RGA2 showed much less sequence diversity than Lr10. These data demonstrate allelic diversity of functional genes at the Lr10 locus in tetraploid wheat, and these new genes can now be analyzed for agronomic relevance. Lr10‐based resistance is highly unusual both in its dependence on two, only distantly, related CC‐NBS‐LRR proteins, as well as in the pattern of diversifying selection in the N‐terminal domain. This indicates a new and complex molecular mechanism of pathogen detection and signal transduction.     

An RGA – like marker detects all known Lr21 leaf rust resistance gene family members in Aegilops tauschii and wheat

Leaf rust is one of the most important diseases of wheat worldwide, particularly in the Great Plains region of the USA. One long-term strategy for the control of this disease may be through durable genetic resistance by gene pyramiding. An important step in this strategy is identifying molecular markers linked to different leaf rust-resistance genes. Here we report the molecular tagging of a leaf rust-resistance gene that may have the potential for durable resistance through further genetic manipulation and gene pyramiding. Lr39 was previously designated for a leaf rust-resistance gene introgressed from Aegilops tauschii accession TA1675 into the common wheat germplasm WGRC2. Lr40 was designated for a gene derived from Ae. tauschii accession TA1649 and is present in germplasm WGRC7. These genes are now believed to be allelic to Lr21, which was transferred to wheat from a different accession of Ae. tauschii. Molecular mapping of Lr39 and Lr40 indicates that both genes come from TA1649. WGRC2 and WRGC7 also have a similar infection type against rust culture PRTUS6. We suggest the designation of the gene in WGRC2 should be changed to Lr40. RFLP marker KSUD14 (locus Xksud14) was found 0.2-cM proximal to Lr40 in a WGRC2/Wichita F₂ population (218 individuals), and co-segregated with the gene in a WGRC7/ Wichita F₂ population (165 individuals). A PCR-based molecular marker developed from the sequence-tagged-site (STS) of Xksud14 was mapped to the same locus as the RFLP marker KSUD14 in both populations. KSUD14 has the structure of a resistance gene analog (RGA) including kinase2a and kinase3 domains similar to the Cre3 gene of wheat and the rust resistance gene Rp1-D of maize. When the PCR products amplified from KSU14 STS were cleaved with restriction enzyme MspI, an 885-bp fragment was found in WGRC2, WGRC7, the Lr21 near-isogenic line, and eight accessions of Ae. tauschii shown to have resistance gene alleles at the Lr21 locus. The KSUD14 PCR-based assay provides an excellent marker for Lr40 and Lr21 in diverse wheat breeding and wild Ae. tauschii populations. Received: 22 December 2000 / Accepted: 12 February 2001     

Microsatellite mapping of adult-plant leaf rust resistance gene <Emphasis Type="Italic">Lr22a</Emphasis> in wheat

This study was conducted to identify microsatellite markers (SSR) linked to the adult-plant leaf rust resistance gene Lr22a and examine their cross-applicability for marker-assisted selection in different genetic backgrounds. Lr22a was previously introgressed from Aegilops tauschii Coss. to wheat (Triticum aestivum L.) and located to chromosome 2DS. Comparing SSR alleles from the donor of Lr22a to two backcross lines and their recurrent parents showed that between two and five SSR markers were co-introgressed with Lr22a and the size range of the Ae. tauschii introgression was 9–20 cM. An F₂ population from the cross of 98B34-T4B × 98B26-N1C01 confirmed linkage between the introgressed markers and Lr22a on chromosome 2DS. The closest marker, GWM296, was 2.9 cM from Lr22a. One hundred and eighteen cultivars and breeding lines of different geographical origins were tested with GWM296. In total 14 alleles were amplified, however, only those lines predicted or known to carry Lr22a had the unique Ae. tauschii allele at GWM296 with fragments of 121 and 131 bp. Thus, GWM296 is useful for selecting Lr22a in diverse genetic backgrounds. Genotypes carrying Lr22a showed strong resistance to leaf rust in the field from 2002 to 2006. Lr22a is an ideal candidate to be included in a stack of leaf rust resistance genes because of its strong adult-plant resistance, low frequency of commercial deployment, and the availability of a unique marker. An erratum to this article can be found at     

Leaf rust resistance gene Lr1, isolated from bread wheat (Triticum aestivum L.) is a member of the large psr567 gene family

In hexaploid wheat, leaf rust resistance gene Lr1 is located at the distal end of the long arm of chromosome 5D. To clone this gene, an F₁-derived doubled haploid population and a recombinant inbred line population from a cross between the susceptible cultivar AC Karma and the resistant line 87E03-S2B1 were phenotyped for resistance to Puccinia triticina race 1-1 BBB that carries the avirulence gene Avr1. A high-resolution genetic map of the Lr1 locus was constructed using microsatellite, resistance gene analog (RGA), BAC end (BE), and low pass (LP) markers. A physical map of the locus was constructed by screening a hexaploid wheat BAC library from cultivar Glenlea that is known to have Lr1. The locus comprised three RGAs from a gene family related to RFLP marker Xpsr567. Markers specific to each paralog were developed. Lr1 segregated with RGA567-5 while recombinants were observed for the other two RGAs. Transformation of the susceptible cultivar Fielder with RGA567-5 demonstrated that it corresponds to the Lr1 resistance gene. In addition, the candidate gene was also confirmed by virus-induced gene silencing. Twenty T ₁ lines from resistant transgenic line T ₀-938 segregated for resistance, partial resistance and susceptibility to Avr1 corresponding to a 1:2:1 ratio for a single hemizygous insertion. Transgene presence and expression correlated with the phenotype. The resistance phenotype expressed by Lr1 seemed therefore to be dependant on the zygosity status. T ₃-938 sister lines with and without the transgene were further tested with 16 virulent and avirulent rust isolates. Rust reactions were all as expected for Lr1 thereby providing additional evidence toward the Lr1 identity of RGA567-5. Sequence analysis of Lr1 indicated that it is not related to the previously isolated Lr10 and Lr21 genes and unlike these genes, it is part of a large gene family. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. The Canadian Crown's right to retain a non-exclusive, royalty-free licence in and to any copyright is acknowledged.     

High-resolution mapping of the leaf rust disease resistance gene <Emphasis Type="Italic">Lr1</Emphasis> in wheat and characterization of BAC clones from the <Emphasis Type="Italic">Lr1</Emphasis> locus

Leaf rust is the most common disease in wheat production. There are more than 45 specific resistance genes described and used in wheat breeding to control epidemics of leaf rust, but none of them has been cloned. The leaf rust disease resistance gene 1 ( Lr1) is a good model gene for isolation by map-based cloning because it is a single, dominant gene which is located in the distal region of chromosome 5DL of wheat. As the first step towards the isolation of this gene we constructed a high-resolution genetic map in the region of the Lr1 locus by saturation mapping of two large segregating F(2) populations (Thatcher Lr1 x Thatcher, Thatcher Lr1 x Frisal). The resistance gene Lr1 was delimited in a 0.16-cM region between the RFLP markers ABC718 and PSR567 (0.12 cM from ABC718 and 0.04 cM from PSR567). A genomic BAC library of Aegilops tauschii (D genome) was screened using the RFLP markers ABC718 and PSR567. Five positive BAC clones were identified by ABC718 and four clones by PSR567. Two NBS-LRR type of resistance gene analogs, which encode proteins highly homologous to the bacterial blight disease resistance protein Xa1 of rice, were identified on BAC clones isolated with PSR567. Polymorphic BAC end probes were isolated from both ends of a 105-kb large BAC clone identified by ABC718. The end probes were mapped at the same locus as ABC718, and no recombination event was found within 105 kb around ABC718 in our analysis of more than 4,000 gametes.     

<Emphasis Type="Italic">Lr41, Lr39,</Emphasis> and a leaf rust resistance gene from<Emphasis Type="Italic"> Aegilops cylindrica</Emphasis> may be allelic and are located on wheat chromosome 2DS

The leaf rust resistance gene Lr41 in wheat germplasm KS90WGRC10 and a resistance gene in wheat breeding line WX93D246-R-1 were transferred to Triticum aestivum from Aegilops tauschii and Ae. cylindrica, respectively. The leaf rust resistance gene in WX93D246-R-1 was located on wheat chromosome 2D by monosomic analysis. Molecular marker analysis of F₂ plants from non-critical crosses determined that this gene is 11.2 cM distal to marker Xgwm210 on the short arm of 2D. No susceptible plants were detected in a population of 300 F₂ plants from a cross between WX93D246-R-1 and TA 4186 (Lr39), suggesting that the gene in WX93D246-R-1 is the same as, or closely linked to, Lr39. In addition, no susceptible plants were detected in a population of 180 F₂ plants from the cross between KS90WGRC10 and WX93D246-R-1. The resistance gene in KS90WGRC10, Lr41, was previously reported to be located on wheat chromosome 1D. In this study, no genetic association was found between Lr41 and 51 markers located on chromosome 1D. A population of 110 F₃ lines from a cross between KS90WGRC10 and TAM 107 was evaluated with polymorphic SSR markers from chromosome 2D and marker Xgdm35 was found to be 1.9 cM proximal to Lr41. When evaluated with diverse isolates of Puccinia triticina, similar reactions were observed on WX93D246-R-1, KS90WGRC10, and TA 4186. The results of mapping, allelism, and race specificity test indicate that these germplasms likely have the same gene for resistance to leaf rust.Contribution number 03-348-J from the Kansas Agricultural Experimental Station, Manhattan, KansasCommunicated by J. Dvorak     

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     

Microsatellite tagging of the leaf rust resistance gene <Emphasis Type="Italic">Lr16</Emphasis> on wheat chromosome 2BSc

Leaf rust, caused by Puccinia triticina, is one of the most damaging diseases of wheat worldwide. Lr16 is a widely deployed leaf rust resistance gene effective at the seedling stage. Although virulence to Lr16 exists in the Canadian P. triticina population, Lr16 provides a level of partial resistance in the field. The primary objective of this study was to identify markers linked to Lr16 that are suitable for marker-assisted selection (MAS). Lr16 was tagged with microsatellite markers on the distal end of chromosome 2BS in three mapping populations. Seven microsatellite loci mapped within 10 cM of Lr16, with the map distances varying among populations. Xwmc764 was the closest microsatellite locus to Lr16, and mapped 1, 9, and 3 cM away in the RL4452/AC Domain, BW278/AC Foremost, and HY644/McKenzie mapping populations, respectively. Lr16 was the terminal locus mapped in all three populations. Xwmc764, Xgwm210, and Xwmc661 were the most suitable markers for selection of Lr16 because they had simple PCR profiles, numerous alleles, high polymorphism information content (PIC), and were tightly linked to Lr16. Twenty-eight spring wheat lines were evaluated for leaf rust reaction with the P. triticina virulence phenotypes MBDS, MBRJ, and MGBJ, and analyzed with five microsatellite markers tightly linked to Lr16. There was good agreement between leaf rust infection type (IT) data and the microsatellite allele data. Microsatellite markers were useful for postulating Lr16 in wheat lines with multiple leaf rust resistance genes.     

Identification of microsatellite markers linked to leaf rust resistance gene <Emphasis Type="Italic">Lr25</Emphasis> in wheat

The leaf rust resistance gene Lr25, transferred from Secale cereale L. into wheat and located on chromosome 4B, imparts resistance to all pathotypes of leaf rust in South-East Asia. In an F₂-derived F₃ population, created by crossing TcLr25 that carries the gene Lr25 for leaf rust resistance with leaf rust-susceptible parent Agra Local, three microsatellite markers located on the long arm of chromosome 4B were found to be linked to the Lr25 locus. The donor parent TcLr25 is a near-isogenic line derived from the variety Thatcher. The most virulent pathotype of leaf rust in the South-East Asian region, designated 77–5 (121R63-1), was used for challenging the population under artificially controlled conditions. The marker Xgwm251 behaved as a co-dominant marker placed 3.8 cM away from the Lr25 locus on 4BL. Two null allele markers, Xgwm538 and Xgwm6, in the same linkage group were located at a distance of 3.8 cM and 16.2 cM from the Lr25 locus, respectively. The genetic sequence of Xgwm251, Lr25, Xgwm538, and Xgwm6 covered a total length of 20 cM on 4BL. The markers were validated for their specificity to Lr25 resistance in a set of 43 wheat genetic stocks representing 43 other Lr genes.     

Development of simple sequence repeat markers specific for the Lr34 resistance region of wheat using sequence information from rice and Aegilops tauschii

Hexaploid wheat (Triticum aestivum L.) originated about 8,000 years ago from the hybridization of tetraploid wheat with diploid Aegilops tauschii Coss. containing the D-genome. Thus, the bread wheat D-genome is evolutionary young and shows a low degree of polymorphism in the bread wheat gene pool. To increase marker density around the durable leaf rust resistance gene Lr34 located on chromosome 7DS, we used molecular information from the orthologous region in rice. Wheat expressed sequence tags (wESTs) were identified by homology with the rice genes in the interval of interest, but were monomorphic in the ‘Arina’ × ‘Forno’ mapping population. To derive new polymorphic markers, bacterial artificial chromosome (BAC) clones representing a total physical size of ∼1 Mb and belonging to four contigs were isolated from Ae. tauschii by hybridization screening with wheat ESTs. Several BAC clones were low-pass sequenced, resulting in a total of ∼560 kb of sequence. Ten microsatellite sequences were found, and three of them were polymorphic in our population and were genetically mapped close to Lr34. Comparative analysis of marker order revealed a large inversion between the rice genome and the wheat D-genome. The SWM10 microsatellite is closely linked to Lr34 and has the same allele in the three independent sources of Lr34: ‘Frontana’, ‘Chinese Spring’, and ‘Forno’, as well in most of the genotypes containing Lr34. Therefore, SWM10 is a highly useful marker to assist selection for Lr34 in breeding programs worldwide.     

<Emphasis Type="Italic">Aegilops-Secale</Emphasis> amphiploids: chromosome categorisation,pollen viability and identification of fungal disease resistance genes

The aim of this study was to assess the potential breeding value of goatgrass-rye amphiploids, which we are using as a “bridge” in a transfer of Aegilops chromatin (containing, e.g. leaf rust resistance genes) into triticale. We analysed the chromosomal constitution (by genomic in situ hybridisation, GISH), fertility (by pollen viability tests) and the presence of leaf rust and eyespot resistance genes (by molecular and endopeptidase assays) in a collection of 6× and 4× amphiploids originating from crosses between five Aegilops species and Secale cereale. In the five hexaploid amphiploids Aegilops kotschyi × Secale cereale (genome UUSSRR), Ae. variabilis × S. cereale (UUSSRR), Ae. biuncialis × S. cereale (UUMMRR; two lines) and Ae. ovata × S. cereale (UUMMRR), 28 Aegilops chromosomes were recognised, while in the Ae. tauschii × S. cereale amphiploid (4×; DDRR), only 14 such chromosomes were identified. In the materials, the number of rye chromosomes varied from 14 to 16. In one line of Ae. ovata × S. cereale, the U-R translocation was found. Pollen viability varied from 24.4 to 75.4%. The leaf rust resistance genes Lr22, Lr39 and Lr41 were identified in Ae. tauschii and the 4× amphiploid Ae. tauschii × S. cereale. For the first time, the leaf rust resistance gene Lr37 was found in Ae. kotschyi, Ae. ovata, Ae. biuncialis and amphiploids derived from those parental species. No eyespot resistance gene Pch1 was found in the amphiploids.     

Fine genetic mapping of greenbug aphid-resistance gene <Emphasis Type="Italic">Gb3</Emphasis> in <Emphasis Type="Italic">Aegilops tauschii</Emphasis>

The greenbug, Schizaphis graminum (Rondani), is an important aphid pest of small grain crops especially wheat (Triticum aestivum L., 2n = 6x = 42, genomes AABBDD) in many parts of the world. The greenbug-resistance gene Gb3 originated from Aegilops tauschii Coss. (2n = 2x = 14, genome D^tD^t) has shown consistent and durable resistance against prevailing greenbug biotypes in wheat fields. We previously mapped Gb3 in a recombination-rich, telomeric bin of wheat chromosome arm 7DL. In this study, high-resolution genetic mapping was carried out using an F_2:3 segregating population derived from two Ae. tauschii accessions, the resistant PI 268210 (original donor of Gb3 in the hexaploid wheat germplasm line ‘Largo’) and susceptible AL8/78. Molecular markers were developed by exploring bin-mapped wheat RFLPs, SSRs, ESTs and the Ae. tauschii physical map (BAC contigs). Wheat EST and Ae. tauschii BAC end sequences located in the deletion bin 7DL3-0.82–1.00 were used to design STS (sequence tagged site) or CAPS (Cleaved Amplified Polymorphic Sequence) markers. Forty-five PCR-based markers were developed and mapped to the chromosomal region spanning the Gb3 locus. The greenbug-resistance gene Gb3 now was delimited in an interval of 1.1 cM by two molecular markers (HI067J6-R and HI009B3-R). This localized high-resolution genetic map with markers closely linked to Gb3 lays a solid foundation for map based cloning of Gb3 and marker-assisted selection of this gene in wheat breeding.     

Phenotypic and genetic diversity of leaf rust resistance in wheat wild relatives

Puccinia triticina (Pt), the causal agent of leaf rust evolves through forming new pathotypes that adversely affect the growth and yield of wheat cultivars. Therefore, continued production of resistant varieties through exploring novel sources of resistance in wild relatives which are abundantly found in Iran and the neighbouring regions is a major task in wheat breeding programs. The aim of the present study was to explore 60 wild wheat genotypes selected from the species Triticum monococcum, Aegilops tauschii, Ae. neglecta, Ae. cylindrica, Ae. triuncialis, Ae. umbellulata, Ae. speltoides, Ae. columnaris, Ae. crassa and Ae. ventricosa for resistance to leaf rust. The cultivar ‘Boolani’ and Thatcher near-isogenic lines were used as controls. Two-week-old seedlings were inoculated using 10 Pt pathotypes, and the infection types were recorded. The genotypes were also analysed for polymorphism using six sequence-tagged sites (STS) and sequence characterized amplified region (SCAR) markers. Forty-eight genotypes produced high infection types (3⁺) for two pathotypes, but the remaining genotypes produced low infection types of ‘0; ^=’ to ‘1⁺CN’ to all pathotypes. The latter included three accessions of Ae. tauschii, two accessions of each Ae. umbellulata, Ae. columnaris and Triticum monococcum, and one accession from each Ae. triuncialis, Ae. ventricosa and Ae. neglecta. Analysis for STS and SCAR markers suggested several genotypes could carry the genes Lr9, Lr10, Lr19, Lr24, Lr26 and Lr37 or their potential orthologs in addition to unknown resistance genes. In conclusion, the identified resistant genotypes could be further characterized and used in wheat breeding programs for leaf rust resistance.     

Isolation of TIR and nonTIR NBS–LRR resistance gene analogues and identification of molecular markers linked to a powdery mildew resistance locus in chestnut rose (Rosa roxburghii Tratt)

Toll and interleukin-1 receptor (TIR) and nonTIR nucleotide binding site–leucine rich repeat (NBS–LRR) resistance gene analogues (RGAs) were obtained from chestnut rose (Rosa roxburghii Tratt) by two PCR-based amplification strategies (direct amplification and overlap extension amplification) with degenerate primers designed to the conserved P-loop, kinase-2, and Gly-Leu-Pro-Leu (GLPL) motifs within the NBS domain of plant resistance gene (R gene) products. Thirty-four of 65 cloned PCR fragments contained a continuous open reading frame (ORF) and their predicted protein products showed homology to the NBS–LRR class R proteins in the GenBank database. These 34 predicted protein sequences exhibited a wide range (19.5–99.4%) of sequence identity among them and were classified into two distinct groups by phylogenetic analysis. The first group consisted of 23 sequences and seemed to belong to the nonTIR NBS–LRR RGAs, since they contained group specific motifs (RNBS-A-nonTIR motif) that are often present in the coiled-coil domain of the nonTIR NBS–LRR class R genes. The second group comprised 11 sequences that contained motifs found in the TIR domain of TIR NBS–LRR class R genes. Restriction fragment length polymorphic (RFLP) markers were developed from some of the RGAs and used for mapping powdery mildew resistance genes in chestnut rose. Three markers, RGA22C, RGA4A, and RGA7B, were identified to be linked to a resistance gene locus, designated CRPM1 for chestnut rose powdery mildew resistance 1, which accounted for 72% of the variation in powdery mildew resistance phenotype in an F1 segregating population. To our knowledge, this is the first report on isolation, phylogenetic analysis and potential utilization as genetic markers of RGAs in chestnut rose.     

Characterisation of a new stripe rust resistance gene <Emphasis Type="Italic">Yr47</Emphasis> and its genetic association with the leaf rust resistance gene <Emphasis Type="Italic">Lr52</Emphasis>

Two Iranian common wheat landraces AUS28183 and AUS28187 from the Watkins collection showed high levels of seedling resistance against Australian pathotypes of leaf rust and stripe rust pathogens. Chi-squared analyses of rust response segregation among F₃ populations derived from crosses of AUS28183 and AUS28187 with a susceptible genotype AUS27229 revealed monogenic inheritance of leaf rust and stripe rust resistance. As both genotypes produced similar leaf rust and stripe rust infection types, they were assumed to carry the same genes. The genes were temporarily named as LrW1 and YrW1. Molecular mapping placed LrW1 and YrW1 in the short arm of chromosome 5B, about 10 and 15 cM proximal to the SSR marker gwm234, respectively, and the marker cfb309 mapped 8–12 cM proximal to YrW1. LrW1 mapped 3–6 cM distal to YrW1 in two F₃ populations. AUS28183 corresponded to the accession V336 of the Watkins collection which was the original source of Lr52. Based on the genomic location and accession records, LrW1 was concluded to be Lr52. Because no other seedling stripe rust resistance gene has previously been mapped in chromosome 5BS, YrW1 was permanently named as Yr47. A combination of flanking markers gwm234 and cfb309 with phenotypic assays could be used to ascertain the presence of Lr52 and Yr47 in segregating populations. This investigation characterised a valuable source of dual leaf rust and stripe rust resistance for deployment in new wheat cultivars. Transfer of Lr52 and Yr47 into current Australian wheat backgrounds is in progress.     

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.     

Fine scale genetic and physical mapping using interstitial deletion mutants of <Emphasis Type="Italic">Lr34</Emphasis><Emphasis Type="Italic">/Yr18</Emphasis>: a disease resistance locus effective against multiple pathogens in wheat

The Lr34/Yr18 locus has contributed to durable, non-race specific resistance against leaf rust (Puccinia triticina) and stripe rust (P. striiformis f. sp. tritici) in wheat (Triticum aestivum). Lr34/Yr18 also cosegregates with resistance to powdery mildew (Pm38) and a leaf tip necrosis phenotype (Ltn1). Using a high resolution mapping family from a cross between near-isogenic lines in the “Thatcher” background we demonstrated that Lr34/Yr18 also cosegregated with stem rust resistance in the field. Lr34/Yr18 probably interacts with unlinked genes to provide enhanced stem rust resistance in “Thatcher”. In view of the relatively low levels of DNA polymorphism reported in the Lr34/Yr18 region, gamma irradiation of the single chromosome substitution line, Lalbahadur(Parula7D) that carries Lr34/Yr18 was used to generate several mutant lines. Characterisation of the mutants revealed a range of highly informative genotypes, which included variable size deletions and an overlapping set of interstitial deletions. The mutants enabled a large number of wheat EST derived markers to be mapped and define a relatively small physical region on chromosome 7DS that carried Lr34/Yr18. Fine scale genetic mapping confirmed the physical mapping and identified a genetic interval of less than 0.5 cM, which contained Lr34/Yr18. Both rice and Brachypodium genome sequences provided useful information for fine mapping of ESTs in wheat. Gene order was more conserved between wheat and Brachypodium than with rice but these smaller grass genomes did not reveal sequence information that could be used to identify a candidate gene for rust resistance in wheat. We predict that Lr34/Yr18 is located within a large insertion in wheat not found at syntenic positions in Brachypodium and rice. W. Spielmeyer and R. P. Singh contributed equally to the study through the “Thatcher” and “Lalbahadur” genetic stocks, respectively.     

Microsatellite mapping of adult-plant leaf rust resistance gene <Emphasis Type="Italic">Lr22a</Emphasis> in wheat

This study was conducted to identify microsatellite markers (SSR) linked to the adult-plant leaf rust resistance gene Lr22a and examine their cross-applicability for marker-assisted selection in different genetic backgrounds. Lr22a was previously introgressed from Aegilops tauschii Coss. to wheat (Triticum aestivum L.) and located to chromosome 2DS. Comparing SSR alleles from the donor of Lr22a to two backcross lines and their recurrent parents showed that between two and five SSR markers were co-introgressed with Lr22a and the size range of the Ae. tauschii introgression was 9-20 cM. An F(2) population from the cross of 98B34-T4B x 98B26-N1C01 confirmed linkage between the introgressed markers and Lr22a on chromosome 2DS. The closest marker, GWM296, was 2.9 cM from Lr22a. One hundred and eighteen cultivars and breeding lines of different geographical origins were tested with GWM296. In total 14 alleles were amplified, however, only those lines predicted or known to carry Lr22a had the unique Ae. tauschii allele at GWM296 with fragments of 121 and 131 bp. Thus, GWM296 is useful for selecting Lr22a in diverse genetic backgrounds. Genotypes carrying Lr22a showed strong resistance to leaf rust in the field from 2002 to 2006. Lr22a is an ideal candidate to be included in a stack of leaf rust resistance genes because of its strong adult-plant resistance, low frequency of commercial deployment, and the availability of a unique marker.