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.     

Comparison of categories of resistance in wheat and barley genotypes against biotype 2 of the Russian wheat aphid, Diuraphis noxia (Kurdjumov)

The Russian wheat aphid Diuraphis noxia (Kurdjumov) (Homoptera: Aphididae) is a global pest of wheat and barley. This arthropod is difficult to manage with pesticides or biological control agents due to the aphid’s ability to seek shelter in rolled leaves and also to develop virulent biotypes. During the past 20 years, the use of aphid-resistant cereal cultivars has proven to be an economically and ecologically beneficial method of protecting crops from D. noxia damage. Our research reports the results of experiments to determine the categories of D. noxia biotype 2 resistance present in Cereal Introduction Triticeae (CItr) 2401, and a barley genotype (IBRWAGP4-7), compared to control resistant and susceptible wheat and barley genotypes. CItr2401 and IBRWAGP4-7 exhibit no antixenosis, but both genotypes demonstrated antibiosis to D. noxia in the form of reduced aphid populations. Reduced leaf dry weight change, a measure of plant tolerance of D. noxia feeding, was significantly less in CItr2401 and IBRWAGP4-7 plants than in plants of susceptible control varieties. However, tolerance was negated when a tolerance index was calculated to correct for differences in aphid populations. Barley IBRWAGP4-7 is a new source of D. noxia biotype 2 resistance. D. noxia foliar leaf damage and population growth were significantly less on IBRWAGP4-7 plants than on plants of the susceptible barley variety Morex. IBRWAGP4-7 plants were equal in resistance to plants of the resistant barley STARS 9301 and wheat genotype CItr2401. Handling editor: Heikki Hokkanen     

Resistance gene analogs in barley and their relationship to rust resistance genes.

Regions of amino acid conservation in the NBS domain of NBS-LRR resistance proteins facilitated the PCR isolation of eight resistance gene analog (RGA) sequences from genomic DNA of rice, barley, and Aegilops tauschii. These clones and other RGAs previously isolated from maize, rice, and wheat were assigned to 13 classes by DNA-sequence comparison and by their patterns of hybridisation to restricted barley DNA. Using a doubled-haploid mapping population, probes from 12 RGA classes were used to map 17 loci in the barley genome. Many of these probes have been used for mapping in wheat, and the collective data indicate that the positions of orthologous RGAs are conserved between barley and wheat. RGA loci were identified in the vicinity of barley leaf rust resistance loci Rph4, Rph7, and Rph10. Recombinants were identified between RGA loci and Rph7 and Rph10, while a cluster of RGA sequences detected by probe 5.2 cosegregated with Rph4 in 55 F2 lines.     

PCR markers for selection of adult plant leaf rust resistance in barley (Hordeum vulgare L.)

Adult plant resistance (APR) is considered potentially more durable for controlling barley leaf rust than seedling Rph (Resistance to Puccinia hordei) genes. A major gene for adult plant resistance to barley leaf rust has been mapped to the telomere region of chromosome 5HS. PCR-based molecular markers were developed for saturation of this region based on previously mapped simple sequence repeat, restriction fragment length polymorphism and Diversity Arrays Technology markers. In addition, defence gene homologue (DGH) and wheat expressed sequence tags mapped in specific bins were used to develop new PCR markers. Seventeen PCR-based markers were mapped to the short arm of chromosome 5H in 292 doubled haploid lines from a cross of Pompadour × Stirling, in which seven markers were mapped within 5 cM of the APR gene. The closest linked marker was about 0.7 cM from the APR gene. The wheat deletion bin map together with defence gene homologues was demonstrated to be an efficient tool for development of new molecular markers associated with the disease resistance gene. Four DGH markers were associated with the APR gene. The new molecular markers are a useful tool for marker-assisted selection of the APR gene and provided a better understanding of the molecular mechanism for leaf rust resistance.     

Cytological and molecular analysis of the Hordeum vulgare-Puccinia triticina nonhost interaction

Cultivated barley, Hordeum vulgare L., is considered to be a nonhost or intermediate host species for the wheat leaf rust fungus Puccinia triticina. Here, we have investigated, at the microscopic and molecular levels, the reaction of barley cultivars to wheat leaf rust infection. In the nonhost resistant cultivar Cebada Capa, abortion of fungal growth occurred at both pre- and posthaustorial stages, suggesting that defense genes are expressed throughout the development of the inappropriate fungus during the nonhost resistance reaction. In the two barley lines L94 and Bowman, a low level of prehaustorial resistance to P. triticina was observed and susceptibility was comparable to that of wheat control plants. Suppression subtractive hybridization was used to identify genes that are differentially expressed during the nonhost resistance reaction in Cebada Capa as well as during the successful establishment of the inappropriate wheat leaf rust fungus in L94. Northern analysis indicated that two candidate genes, including a barley ortholog of the rice resistance gene Xa21, are putatively involved in nonhost and non-race-specific resistance reactions. In addition, a new gene that is specifically induced during the successful development of the inappropriate fungus P. triticina in barley has been identified.     

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.     

In vitro selection of barley and wheat for resistance against Helminthosporium sativum

Summary Calli derived from immature embryos of barley and wheat genotypes were screened for their resistance to purified culture filtrate produced by the fungus Helminthosporium sativum P.K. and B. Two selection methods were used: a continuous method in which four cycles of selection were performed one after another on toxic medium and a discontinuous method in which a pause on non-toxic medium was given after the second or third cycle of selection. The latter was superior as it allowed the calli to regain their regeneration ability. About 3,000 calli from two barley genotypes and 2,000 from two wheat genotypes were used for selection. The selection with the pathotoxins resulted in 6% to 17% surviving calli. Toxin tolerant callus lines of barley were characterised by protein isozymes. Zymograms showed one more isozyme than with the unselected sensitive callus. Barley and wheat plants have been regenerated from callus lines surviving the toxin treatment and in vivo testing against pathogen revealed that the majority of these plants were less sensitive.     

Cloning and characterisation of a family of disease resistance gene analogs from wheat and barley

 The most common class of plant disease resistance (R) genes cloned so far belong to the NBS-LRR group which contain nucleotide-binding sites (NBS) and a leucine-rich repeat (LRR). Specific primer sequences derived from a previously isolated NBS-LRR sequence at the Cre3 locus, which confers resistance to cereal cyst nematode (CCN) in wheat (Triticum aestivum L.) were used in isolating a family of resistance gene analogs (RGA) through a polymerase chain reaction (PCR) cloning approach. The cloning, analysis and genetic mapping of a family of RGAs from wheat (cv ‘Chinese Spring’) and barley (Hordeum vulgare L. cvs ‘Chebec’ and ‘Harrington’) are presented. The wheat and barley RGAs contain other conserved motifs present in known R genes from other plants and share between 55–99% amino acid sequence identity to the NBS-LRR sequence at the Cre3 locus. Phylogenetic analysis of the RGAs with other cloned R genes and RGAs from various plant species indicate that they belong to a superfamily of NBS-containing genes. Two of the barley derived RGAs were mapped onto loci on chromosomes 2H (2), 5H (7) and 7H (1) using barley doubled haploid (DH) mapping populations. Some of these loci identified are associated with regions carrying resistance to CCN and corn leaf aphid. Received: 6 January 1998 / Accepted: 1 April 1998     

Haustorium Formation and Cell Wall Appositions in Susceptible and Partially Resistant Wheat and Barley Seedlings Infected with Wheat Leaf Rust

Phase contrast light microscopy observations of wheat and barley seedlings infected with wheat leaf rust spores suggested that cell wall appositions are structural barriers against haustorium formation leading to abortion of infection structures. Nearly equal numbers of cell wall appositions per infection structure were detected in seedlings of susceptible and partially resistant wheat genotypes. Differences between susceptible and partially resistant genotypes became evident after the first haustorium had been formed. This again indicates the presence of a post-haustorial effect of partial resistance. Some factors influencing nutrient uptake are discussed. Wheat leaf rust colonies hardly formed haustoria in barley seedlings, the few not aborted infection structures were accompanied by cell collapse. The mechanisms of partial resistance in wheat and barley to their respective leaf rust fungi seem different, but their non-host reactions appear similar.     

Abundance, variability and chromosomal location of microsatellites in wheat

The potential of microsatellite sequences as genetic markers in hexaploid wheat (Triticum aestivum) was investigated with respect to their abundance, variability, chromosomal location and usefulness in related species. By screening a lambda phage library, the total number of (GA)_n blocks was estimated to be 3.6 x 10⁴ and the number of (GT)_n blocks to be 2.3 x 10⁴ per haploid wheat genome. This results in an average distance of approximately 270 kb between these two microsatellite types combined. Based on sequence analysis data from 70 isolated microsatellites, it was found that wheat microsatellites are relatively long containing up to 40 dinucleotide repeats. Of the tested primer pairs, 36% resulted in fragments with a size corresponding to the expected length of the sequenced microsatellite clone. The variability of 15 microsatellite markers was investigated on 18 wheat accessions. Significantly, more variation was detected with the microsatellite markers than with RFLP markers with, on average, 4.6 different alleles per microsatellite. The 15 PCR-amplified microsatellites were further localized on chromosome arms using cytogenetic stocks of Chinese Spring. Finally, the primers for the 15 wheat microsatellites were used for PCR amplification with rye (Secale cereale) and barley accessions (Hordeum vulgare, H. spontaneum). Amplified fragments were observed for ten primer pairs with barley DNA and for nine primer pairs with rye DNA as template. A microsatellite was found by dot blot analysis in the PCR products of barley and rye DNA for only one primer pair.     

Prospects for exploitation of disease resistance from Hordeum chilense in cultivated cereals

Hordeum chilense is a South American wild barley with high potential for cereal breeding given its high crossability with other members of the Triticeae. In the present paper we consider the resistance of H. chilense to several fungal diseases and the prospects for its transference to cultivated cereals. All H. chilense accessions studied are resistant to the barley, wheat and rye brown rusts, the powdery mildews of wheat, barley, rye and oat, to Septoria leaf blotch, common bunt and to loose smuts, which suggests that H. chilense is a non-host of these diseases. There are also lines resistant to wheat and barley yellow rust, stem rust and to Agropyron leaf rust, as well as lines giving moderate levels of resistance to Septoria glume blotch, tan spot and Fusarium head blight. Some H. chilense lines display pre-appressorial avoidance to brown rust. Lines differ in the degree of haustorium formation by rust and mildew fungi they permit, and in the degree to which a hypersensitive response occurs after haustoria are formed. Unfortunately, resistance of H. chilense to rust fungi is not expressed in tritordeum hybrids, nor in chromosome addition lines in wheat. In tritordeum, H. chilense contributes quantitative resistance to wheat powdery mildew, tan spot and loose smut. The resistance to mildew, expressed as a reduced disease severity, is not associated with macroscopically visible necrosis. Hexaploid tritordeums are immune to Septoria leaf blotch and to common bunt although resistance to both is slightly diluted in octoploid tritordeums. Studies with addition lines in wheat indicate that the resistance of H. chilense to powdery mildew, Septoria leaf blotch and common bunt is of broad genetic basis, conferred by genes present on various chromosomes.     

Using real-time PCR to determine transgene copy number in wheat

Transgene copy number is usually determined by means of Southern blot analysis which can be time consuming and laborious. In this study, quantitative real-time PCR was developed to determine transgene copy number in transgenic wheat. A conserved wheat housekeeping gene,puroindoline-b, was used as an internal control to calculate transgene copy number. Estimated copy number in transgenic lines using real-time quantitative PCR was correlated with actual copy number based on Southern blot analysis. Real-time PCR can analyze hundreds of samples in a day, making it an efficient method for estimating copy number in transgenic wheat.     

Mapping multiple disease resistance genes using a barley mapping population evaluated in Peru, Mexico, and the USA

We used a well-characterized barley mapping population (BCD 47 × Baronesse) to determine if barley stripe rust (BSR) resistance quantitative trait loci (QTL) mapped in Mexico and the USA were effective against a reported new race in Peru. Essentially the same resistance QTL were detected using data from each of the three environments, indicating that these resistance alleles are effective against the spectrum of naturally occurring races at these sites. In addition to the mapping population, we evaluated a germplasm array consisting of lines with different numbers of mapped BSR resistance alleles. A higher BSR disease severity on CI10587, which has a single qualitative resistance gene, in Peru versus Mexico suggests there are differences in pathogen virulence between the two locations. Confirmation of a new race in Peru will require characterization using a standard set of differentials, an experiment that is underway. The highest levels of resistance in Peru were observed when the qualitative resistance gene was pyramided with quantitative resistance alleles. We also used the mapping population to locate QTL conferring resistance to barley leaf rust and barley powdery mildew. For mildew, we identified resistance QTL under field conditions in Peru that are distinct from the Mla resistance that we mapped using specific isolates under controlled conditions. These results demonstrate the long-term utility of a reference mapping population and a well-characterized germplasm array for locating and validating genes conferring quantitative and qualitative resistance to multiple pathogens.     

Effect of Host Plant Resistance on Haustorium Formation in Cereal Rust Fungi

Haustoria of Puccinia triticina (wheat leaf rust fungus) and P. hordei (barley leaf rust fungus) were isolated from susceptible and partially resistant wheat lines, and susceptible, hypersensitive and partially resistant barley lines. Haustoria were counted and measured. The size of haustoria was similar in the partially resistant and susceptible genotypes but haustoria were smaller in the hypersensitive barley line L94+Pa7. The number of haustoria was reduced in both partially and hypersensitive lines when compared with susceptible ones. Therefore it seems that the reduction in the number of haustoria is a consequence of the resistance that can be attributable either to early abortion of infection units or reduced colony growth. The reduction of the number of haustoria was more pronounced in the adult plant stage.     

Concentration of ergosterol in small-grained naturally contaminated and inoculated cereals

Ergosterol (ERG) is a major sterol constituent of most fungi. Its concentration is negligible in higher plants, but can be used as a chemical marker of the presence of fungal contaminations. In this study, ERG concentration was assessed in randomly collected samples of naturally contaminated grain (wheat, barley and oat) and in samples of grain (wheat, barley, triticale and oat) harvested after inoculation of heads with conidia of different Fusarium species. Wheat samples were analysed at three stages of grain development. The lowest ERG concentration was found in non-inoculated samples at the first stage of grain development. This concentration was increasing with grain ripening. In naturally contaminated samples collected after harvest, ERG concentration was lower in wheat than in barley and oat. ERG concentrations in inoculated samples varied significantly, but were always significantly higher than in naturally contaminated samples. In the above cereal samples it was much lower than the levels assayed in laboratory cultures inoculated with fungi from genus Fusarium. The content of ERG was also analyzed in milling products of small-grained cereals and other foodstuffs, where a considerable variation was observed. The lowest ERG amounts were assayed in flours with a high degree of purification, while the highest ones in case of flours and products with a low purification rate. The results indicate the potential application of HPLC combined with microwave-assisted extraction both when assaying samples with low ERG concentrations (naturally contaminated) and those characterized with high contents of fungal biomass (strongly infected, artificially inoculated). It also facilitates analyses of fungal biomass in technological processes, where results may be expected to vary considerably.     

A new technique for monoconidial culture of the most aggressive isolate in a given population of Bipolaris sorokiniana, cause of foliar spot blotch in wheat and barley

We developed a new technique for monoconidial culture of the most aggressive isolate in a given population of Bipolaris sorokiniana, to facilitate the evaluation of spot blotch resistance in wheat and barley. Blotched portions of infected barley leaves were placed on a glass slide in a moist chamber for production of conidia by associated fungal hyphae. Conidia were collected separately and grown on water agar discs. Individual water agar discs having conidium growth were inoculated on barley leaves. The conidium producing the earliest symptom with the largest lesion was considered most aggressive. This lesion was incubated in a moist chamber and the conidial offspring were tested for pathogenicity. When a uniform infection was observed, a small piece of the lesion was cut using a sterilized scalpel, surface sterilized with NaOCl, and inoculated in the centre of Petri dishes containing potato dextrose agar medium. The inoculated Petri dishes were incubated at 25 ± 1 °C to yield monoconidial cultures of the most aggressive isolate. Variability in symptom expression caused by the most aggressive isolate of a given population was much less than variability in symptom expression caused by all isolates collectively. The techniques will be useful for plant pathologists and breeders in screening for spot blotch resistance in wheat and barley.     

Quantification of Fusarium culmorum in Wheat and Barley Tissues Using Real-Time PCR in Comparison with DON Content

A real‐time polymerase chain reaction (PCR) assay was developed for the specific detection of Fusarium culmorum in infected seeds. Primers and TaqMan minor groove binder probe were derived from the sequences of a F. culmorum specific PCR product. The specificity of the assay was confirmed by test in seven Fusarium species and 21 non‐Fusarium fungal species. With serial dilutions of purified genomic DNA from F. culmorum isolate B as the template, the detection limit of the assay was found to be 0.9 pg of fungal genomic DNA per reaction. A significant correlation ( = 0.982) and collinearity was found between DNA concentration and Ct (cycle threshold) values of real‐time PCR assay with serial diluted DNAs extracted from three seed samples with different deoxynivalenol (DON) content. Eight barley and nine wheat varieties infected by F. culmorum isolate B were evaluated in 1 (barley samples) and in 4 years (wheat samples). The results of real‐time PCR analysis and enzyme‐linked immunosorbent assay testing for DON content were compared and a significant correlation was found for barley samples (r² = 0.935). Concerning wheat we found rather complicated relationship between Ct values and DON contents influenced by environmental conditions of field trials. The real‐time PCR assay was found to be highly specific and sensitive. It could be used in phytopathological studies and praxis.