The maize rp1 rust resistance gene identifies homologues in barley that have been subjected to diversifying selection

A number of agronomically important grasses (sorghum, wheat, panicum, sugar cane, oats, rice and barley) are shown to contain sequences homologous to rp1, a maize gene that confers race-specific resistance to the rust fungus Puccinia sorghi. Mapping of rp1-related sequences in barley identified three unlinked loci on chromosomes 1HL, 3HL and 7HS. The locus located on chromosome 7HS comprises a small gene family of at least four members, two of which were isolated and are predicted to encode nucleotide binding site-leucine-rich repeat (NBS-LRR) proteins that are respectively 58% and 60% identical to the maize rp1 protein. Evidence of positive selection for sequence diversification acting upon these two barley genes was observed; however, diversifying selection was restricted to the carboxy terminal half of the LRR domain. One of these rp1 homologous genes cosegregated with the barley Rpg1 stem rust resistance gene amongst 148 members of the Steptoe × Morex double haploid mapping family. Three other unrelated resistance gene-like sequences, potentially encoding NBS-LRR proteins, are also shown to be linked to the Rpg1 locus but not cosegregating with the gene. Received: 2 August 1999 / Accepted: 28 September 1999     

Parallel expression profiling of barley–stem rust interactions

The dominant barley stem rust resistance gene Rpg1 confers resistance to many but not all pathotypes of the stem rust fungus Puccinia graminis f. sp. tritici (Pgt). Transformation of Rpg1 into susceptible cultivar Golden Promise rendered the transgenic plants resistant to Pgt pathotype MCC but not to Pgt pathotype QCC. Our objective was to identify genes that are induced/repressed during the early stages of pathogen infection to elucidate the molecular mechanisms and role of Rpg1 in defense. A messenger ribonucleic acid expression analysis using the 22K Barley1 GeneChip was conducted in all pair-wise combinations of two isolines (cv. Golden Promise and Rpg1 transgenic line G02-448F-3R) and two Pgt pathotypes (MCC and QCC) across six time points. Analysis showed that a total of 34 probe sets exhibited expression pattern differences between Golden Promise (susceptible) and G02-448F-3R (resistant) infected with Pgt-MCC. A total of 14 probe sets exhibited expression pattern differences between Pgt-MCC (avirulent) and Pgt-QCC (virulent) inoculated onto G02-448F-3R. These differentially expressed genes were activated during the early infection process, before the hypersensitive response or fungal growth inhibition occurred. Our analysis provides a list of candidate signaling components, which can be analyzed for function in Rpg1-mediated disease resistance.     

Rpr1, a gene required for Rpg1-dependent resistance to stem rust in barley

Rpg1 is a stem rust resistance gene that has protected barley from severe losses for over 60 years in the US and Canada. It confers resistance to many, but not all, pathotypes of the stem rust fungus Puccinia graminis f. sp. tritici. A fast neutron induced deletion mutant, showing susceptibility to stem rust pathotype Pgt-MCC, was identified in barley cv. Morex, which carries Rpg1. Genetic and Rpg1 mRNA and protein expression level analyses showed that the mutation was a suppressor of Rpg1 and was designated Rpr1 (Required for P. graminis resistance). Genome-wide expression profiling, using the Affymetrix Barley1 GeneChip containing ∼22,840 probe sets, was conducted with Morex and the rpr1 mutant. Of the genes represented on the Barley1 microarray, 20 were up-regulated and 33 were down-regulated by greater than twofold in the mutant, while the Rpg1 mRNA level remained constant. Among the highly down-regulated genes (greater than fourfold), genomic PCR, RT-PCR and Southern analyses identified that three genes (Contig4901_s_at, HU03D17U_s_at, and Contig7061_s_at), were deleted in the rpr1 mutant. These three genes mapped to chromosome 4(4H) bin 5 and co-segregated with the rpr1-mediated susceptible phenotype. The loss of resistance was presumed to be due to a mutation in one or more of these genes. However, the possibility exists that there are other genes within the deletions, which are not represented on the Barley1 GeneChip. The Rpr1 gene was not required for Rpg5- and rpg4-mediated stem rust resistance, indicating that it shows specificity to the Rpg1-mediated resistance pathway.     

The barley serine/threonine kinase gene Rpg1 providing resistance to stem rust belongs to a gene family with five other members encoding kinase domains

The barley (Hordeum vulgare L.) stem rust (Puccinia graminis f. sp. tritici) resistance gene Rpg1 encodes a serine/threonine protein kinase with two tandem kinase domains. The Rpg1 gene family was identified from the cv. Morex and consists of five additional members with divergent homology to Rpg1. All family members encode serine/threonine kinase-like proteins with at least one predicted catalytically active kinase domain. The five family members were sequenced from cDNA and genomic DNA and genetically mapped. The family member most closely related to Rpg1, ABC1037, is located on chromosome 1(7H) bin 01, very near (∼50 kb) but not co-segregating with Rpg1. Two others, ABC1036 and ABC1040, are closely related to each other and tightly linked on chromosome 7(5H) bin 07. ABC1041 mapped to chromosome 7(5H) bin 13, tightly linked to the rust resistance genes rpg4 and Rpg5 providing resistance to barley stem rust pathotype QCC and rye stem rust pathotype 92-MN-90, respectively, but segregated away in a high-resolution population. ABC1063 was localized to chromosome 4(4H) bin 6. An interesting Rpg1 allele that appears to be the result of unequal recombination between Rpg1 and ABC1037 was characterized. No known resistance loci cosegregated with any family members, however characterization of the Rpg1 family has provided insight into the evolution of this novel gene family and may present tools for understanding the functional domains of Rpg1. The genetic mapping, gene structures, and analysis of amino-acid sequences of the Rpg1 gene family members are presented.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Allele-specific amplification of polymorphic sites for the detection of powdery mildew resistance loci in cereals

Primers for the polymerase chain reaction (PCR) were tailored to selectively amplify RFLP marker alleles associated with resistance and susceptibility for powdery mildew in cereals. The differentiation between marker alleles for susceptible and resistant genotypes is based on the discrimination of a single nucleotide by using allele-specific oligonucleotides as PCR primers. The PCR assays developed are diagnostic for RFLP alleles at the loci MWG097 in the barley genome and Whs350 in the wheat genome. The first marker locus is closely linked to MlLa resistance in barley, while the latter is linked to Pm2 resistance locus in wheat. PCR analysis of 31 barley and 30 wheat cultivars, with some exceptions, verified the presence or absence of the resistance loci investigated. These rapid PCR-based approaches are proposed as an efficient alternative to conventional procedures for selecting powdery mildew-resistant genotypes in breeding programs.     

Divergence and allelomorphic relationship of a soybean virus resistance gene based on tightly linked DNA microsatellite and RFLP markers

The use of genetically diverse resistance sources is important in breeding for durable disease resistance. Detection and evaluation of resistance genes by conventional inheritance experiments, however, often require laborious screening and genetic testing. In the present study, a marker-assisted screening for resistance sources was initiated in soybean [Glycine max (L.) Merr] using one DNA microsatellite and two RFLP markers tightly linked to a soybean mosaic virus (SMV) resistance gene (Rsv1). The three marker loci were used to screen 67 diverse soybean cultivars, breeding lines, and plant introductions. Five variants were found at the microsatellite locus (HSP176L), and the two RFLP loci (pA186 and pK644a) near Rsv1 show a remarkably higher level of restriction polymorphism than Rsv1-independent RFLP loci. Several specific variants at the three marker loci were found to be correlated with virus resistance, among which HSP176L-2 can be detected by PCR, thus may be useful for germplasm screening. The grouping of the 67 accessions according to their multilocus marker variants agrees with the available pedigree information. When all, or most, of the cultivars within a given group with the same Rsv1-linked marker variant are resistant, their SMV resistance is most likely conferred by Rsv1. These putatively Rsv1-carrying groups contain a total of 38 SMV-resistant lines including six differential cultivars that are known to carry Rsv1. The remaining seven resistant accessions (Columbia, Holladay, Peking, Virginia, FFR-471, PI 507403, and PI 556949) do not carry resistance marker variants, and at least some of them could be sources of resistance genes independent of Rsv1.     

Molecular marker analyses of powdery mildew resistance in barley

Powdery mildew, caused byEryisphe graminis f. sp.hordei, is one of the most important diseases of barley (Hordeum vulgare). A number of loci conditioning resistance to this disease have been reported previously. The objective of this study was to use molecular markers to identify chromosomal regions containing genes for powdery mildew resistance and to estimate the resistance effect of each locus. A set of 28 F₁ hybrids and eight parental lines from a barley diallel study was inoculated with each of five isolates ofE. graminis. The parents were surveyed for restriction fragment length polymorphisms (RFLPs) at 84 marker loci that cover about 1100 cM of the barley genome. The RFLP genotypes of the F₁s were deduced from those of the parents. A total of 27 loci, distributed on six of the seven barley chromosomes, detected significant resistance effects to at least one of the five isolates. Almost all the chromosomal regions previously reported to carry genes for powdery mildew resistance were detected, plus the possible existence of 1 additional locus on chromosome 7. The analysis indicated that additive genetic effects are the most important component in conditioning powdery mildew resistance. However, there is also a considerable amount of dominance effects at most loci, and even overdominance is likely to be present at a number of loci. These results suggest that quantitative differences are likely to exist among alleles even at loci which are considered to carry major genes for resistance, and minor effects may be prevalent in cultivars that are not known to carry major genes for resistance.     

A novel gene for rust resistance

The Rpg1 gene, which has provided North American cultivars of barley with resistance to the stem rust fungus Puccinia graminis f.sp. tritici for more than 60 years, has been cloned. A single copy of the gene can confer resistance to a susceptible barley variety. Although unexplained, the progeny are consistently more resistant than the variety from which the gene was obtained. The gene might represent a new class of plant resistance genes.     

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     

Molecular mapping of the Rph7.g leaf rust resistance gene in barley (Hordeum vulgare L.)

In many temperate areas of the world, leaf rust is becoming an important disease of barley. In the last decade, new races of Puccinia hordei G. Otth have emerged which are virulent against the so-far most-effective race-specific resistance genes, such as Rph7. Marker-assisted selection greatly facilitates the pyramidization of two or more resistance genes in a single variety in order to achieve a more comprehensive resistance. Such a strategy requires the development of efficient and reliable markers. Here, we have developed a linkage map and found RFLP markers closely linked to the Rph7.g resistance gene on chromosome 3HS of barley. The receptor-like kinase gene Hv3Lrk that maps at 3.2 cM from Rph7.g was used to develop a PCR-based marker by exploiting a single nucleotide polymorphism. This marker was detected in 11 out of 12 (92%) barley lines having Rph7 and represents a valuable tool for marker-assisted selection. In addition, the identification of markers flanking Rph7.g provides the basis for positional cloning of this gene. Received: 1 December 1999 / Accepted: 28 February 2000     

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.     

The use of bulk segregant analysis to identify a RAPD marker linked to leaf rust resistance in barley

An F₂ population from a cross between barley accession Q21861 and the Australian barley variety Galleon was used to develop RAPD markers for resistance to barley leaf rust (Puccinia hordei). Resistant and susceptible DNA bulks were constructed following the classification of F₂ plants by leaf rust infection type. Bulked segregant analysis was then used to identify a 2.7-kb marker, designated OU02₂₇₀₀ and located approximately 12cM from RphQ, a leaf rust resistance gene in Q21861. The marker was generated by PCR with the oligonucleotide primer OPU-02 (Operon). Infection types of F₃ progeny were used to confirm assignment of F₂ genotypes. OU02₂₇₀₀ was shown, retrospectively, to be useful in the identification of individual F₂ plants that had been originally misclassified as having susceptible infection types. Both the RAPD marker and RphQ will be potentially useful in the development of new barley cultivars.     

Genetic mapping of a leaf rust resistance gene in the former Yugoslavian barley landrace MBR1012

Leaf rust, caused by Puccinia hordei Otth, is an important disease of barley (Hordeum vulgare L.) in many areas of the world. The appearance of new virulent races necessitates the identification of new resistance genes in barley. Screening of spring barley landraces from former Yugoslavia led to the identification of an accession (MBR1012) carrying resistance to the most widespread virulent leaf rust pathotypes in Europe. Ninety-one doubled haploid lines derived from a cross between landrace MBR1012 and the susceptible German cultivar Scarlett were evaluated for resistance to P. hordei isolate I-80 and segregated 48 resistant : 43 susceptible ( $ \chi_{1:1}^{2} = \, 0.29, $ p?=?0.6), indicating a monogenic inheritance of resistance. Using simple sequence repeats (SSR) and single nucleotide polymorphism (SNP) markers, the resistance gene in MBR1012 was mapped to the telomeric region of chromosome 1HS. This gene is assigned the temporary locus designation of Rph _MBR1012 until it can be unequivocally determined to be different from all previously reported resistance genes. The closest flanking markers for Rph _MBR1012 are located 0.8?cM distal (SNP marker GBS546 and SSR marker GBMS187) and 6.0?cM proximal (SSR marker GMS21). The diagnostic value of the closest linked markers was assessed in a genetically diverse collection of 51 susceptible and resistant barley lines and cultivars. The SSR GBMS187 predicted the presence of Rph _MBR1012 with 100% accuracy. However, this marker could not be used singly for the rapid incorporation of resistance into high yielding barley cultivars, since it detects a null allele in MBR1012. Therefore, simultaneous use of the markers closely linked to Rph _MBR1012 is needed for transferring Rph _MBR1012 into adapted cultivars.     

New disease resistance genes in soybean against Pseudomonas syringae pv glycinea: evidence that one of them interacts with a bacterial elicitor

Summary Soybean [Glycine max (L.) Merr.] cultivars Flambeau and Merit differed in their resistance to Pseudomonas syringae pv glycinea (Psg) race 4, carrying each of four different avirulence (avr) genes cloned from Psg or the related bacterium, Pseudomonas syringae pv tomato. Segregation data for F₂ and F₃ progeny of Flambeau x Merit crosses indicated that single dominant and nonallelic genes account for resistance to Psg race 4, carrying avirulence genes avrA, avrB, avrC, or avrD. Segregants were also recovered that carried all four or none of the disease resistance genes. One of the disease resistance genes (Rpg1, complementing bacterial avirulence gene B) had been described previously, but the other three genes — designated Rpg2, Rpg3, and Rpg4 — had not here to fore been defined. Rpg3 and Rpg4 are linked (40.5 ± 3.2 recombination units). Rpg4 complements avrD, cloned from Pseudomonas syringae pv tomato, but a functional copy of this avirulence gene has not thus far been observed in Pseudomonas syringae pv glycinea. Resistance gene Rpg4 therefore may account in part for the resistance of soybean to Pseudomonas syringae pv tomato and other pathogens harboring avrD.     

Evaluation of Pakistan wheat germplasms for stripe rust resistance using molecular markers

Wheat production in Pakistan is seriously constrained due to rust diseases and stripe rust (yellow) caused by Puccinia striiformis f. sp. tritici, which could limit yields. Thus development and cultivation of genetically diverse and resistant varieties is the most sustainable solution to overcome these diseases. The first objective of the present study was to evaluate 100 Pakistan wheat cultivars that have been grown over the past 60 years. These cultivars were inoculated at the seedling stage with two virulent stripe rust isolates from the United States and two from Pakistan. None of the wheat cultivars were resistant to all tested stripe rust isolates, and 16% of cultivars were susceptible to the four isolates at the seedling stage. The data indicated that none of the Pakistan wheat cultivars contained either Yr5 or Yr15 genes that were considered to be effective against most P. striiformis f. sp. tritici isolates from around the world. Several Pakistan wheat cultivars may have gene Yr10, which is effective against isolate PST-127 but ineffective against PST-116. It is also possible that these cultivars may have other previously unidentified genes or gene combinations. The second objective was to evaluate the 100 Pakistan wheat cultivars for stripe rust resistance during natural epidemics in Pakistan and Washington State, USA. It was found that a higher frequency of resistance was present under field conditions compared with greenhouse conditions. Thirty genotypes (30% of germplasms) were found to have a potentially high temperature adult plant (HTAP) resistance. The third objective was to determine the genetic diversity in Pakistan wheat germplasms using molecular markers. This study was based on DNA fingerprinting using resistance gene analog polymorphism (RGAP) marker analysis. The highest polymorphism detected with RGAP primer pairs was 40%, 50% and 57% with a mean polymorphism of 36%. A total of 22 RGAP markers were obtained in this study. RGAP, simple sequence repeat (SSR) and sequence tagged site (STS) markers were used to determine the presence and absence of some important stripe rust resistance genes, such as Yr5, Yr8, Yr9, Yr15 and Yr18. Of the 60 cultivars analyzed, 17% of cultivars showed a RGAP marker band for Yr9 and 12% of cultivars exhibited the Yr18 marker band. No marker band was detected for Yr5, Yr8 and Yr15, indicating a likely absence of these genes in the tested Pakistan wheat cultivars. Cluster analysis based on molecular and stripe rust reaction data is useful in identifying considerable genetic diversity among Pakistan wheat cultivars. The resistant germplasms identified with 22 RGAP markers and from the resistance evaluations should be useful in developing new wheat cultivars with stripe rust resistance.     

GA-20 oxidase as a candidate for the semidwarf gene sdw1/denso in barley

The barley sdw1/denso gene not only controls plant height but also yield and quality. The sdw1/denso gene was mapped to the long arm of chromosome 3H. Comparative genomic analysis revealed that the sdw1/denso gene was located in the syntenic region of the rice semidwarf gene sd1 on chromosome 1. The sd1 gene encodes a gibberellic acid (GA)-20 oxidase enzyme. The gene ortholog of rice sd1 was isolated from barley using polymerase chain reaction. The barley and rice genes showed a similar gene structure consisting of three exons and two introns. Both genes share 88.3% genomic sequence similarity and 89% amino acid sequence identity. A single nucleotide polymorphism was identified in intron 2 between barley varieties Baudin and AC Metcalfe with Baudin known to contain the denso semidwarf gene. The single nucleotide polymorphism (SNP) marker was mapped to chromosome 3H in a doubled haploid population of Baudin × AC Metcalfe with 178 DH lines. Quantitative trait locus analysis revealed that plant height cosegregated with the SNP. The sdw1/denso gene in barley is the most likely ortholog of the sd1 in rice. The result will facilitate understanding of the molecular mechanism controlling semidwarf phenotype and provide a diagnostic marker for selection of semidwarf gene in barley. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Rpg1, a soybean gene effective against races of bacterial blight, maps to a cluster of previously identified disease resistance genes

 Alleles, or tightly linked genes, at the soybean (Glycine max L. Merr.) Rpg1 locus confer resistance to races of Pseudomonas syringae pv. glycinea that express the avirulence genes avrB or avrRpm1. In this study we demonstrate that Rpg1 maps to a cluster of previously identified resistance genes, including those effective against fungal, viral and nematode pathogens. Rpg1 is in molecular linkage group (MLG) F, flanked by the markers K644 and B212. The RFLP markers R45, php2265 and php2385 cosegregated with Rpg1, as did the marker nbs61, which encodes a protein related to previously isolated resistance genes. Received: 7 July 1997 / Accepted: 6 October 1997     

A barley gene family homologous to the maize rust resistance gene <Emphasis Type="Italic">Rp1-D</Emphasis>

Many characterized plant disease resistance genes encode proteins which have conserved motifs such as the nucleotide binding site. Conservation extends across different species, therefore resistance genes from one species can be used to isolate homologous regions from another by employing DNA sequences encoding conserved protein motifs as probes. Here we report the isolation and characterization of a barley (Hordeum vulgare L.) resistance gene analog family consisting of nine members homologous to the maize rust resistance gene Rp1-D. Five barley Rp1-D homologues are clustered within approximately 400 kb on chromosome 1(7H), near, but not co-segregating with, the barley stem rust resistance gene Rpg1; while others are localized on chromosomes 3(3H), 5(1H), 6(6H) and 7(5H). Analyses of predicted amino-acid sequences of the barley Rp1-D homologues and comparison with known plant disease resistance genes are presented.     

SSR Markers Closely Linked to the Pi-z Locus are Useful for Selection of Blast Resistance in a Broad Array of Rice Germplasm

Pi-z is a disease resistance gene that has been effectively used to combat a broad-spectrum of races of the rice blast fungus Magnaporthe grisea. Although DNA markers have been reported for selection of the Pi2(t) and Pi-z resistance genes at the Pi-z locus, markers that are more tightly linked to the Pi-z locus would benefit rapid and effective cultivar development. Analysis of the publicly available genome sequence of Nipponbare near the Pi-z locus revealed numerous SSRs that could be converted into markers. Three SSRs on rice PAC AP005659 were found to be very tightly linked to the Pi-z locus, with one marker, AP5659-3, co-segregating with the Pi-z resistance reaction. The Pi-z factor conferring resistance to two races of blast was mapped to a 57 kb region on the physical map of Nipponbare in a location where the Pi2(t) gene was physically mapped. Two SSR marker haplotypes were unique for cultivars carrying the Pi-z gene, which indicates these markers are useful for selection of resistance genes at the Pi-z locus in rice germplasm.