Hugh Wallwork

Sodium exclusion QTL associated with improved seedling growth in bread wheat under salinity stress

Worldwide, dryland salinity is a major limitation to crop production. Breeding for salinity tolerance could be an effective way of improving yield and yield stability on saline-sodic soils of dryland agriculture. However, this requires a good understanding of inheritance of this quantitative trait. In the present study, a doubled-haploid bread wheat population (Berkut/Krichauff) was grown in supported hydroponics to identify quantitative trait loci (QTL) associated with salinity tolerance traits commonly reported in the literature (leaf symptoms, tiller number, seedling biomass, chlorophyll content, and shoot Na+ and K+ concentrations), understand the relationships amongst these traits, and determine their genetic value for marker-assisted selection. There was considerable segregation within the population for all traits measured. With a genetic map of 527 SSR-, DArT- and gene-based markers, a total of 40 QTL were detected for all seven traits. For the first time in a cereal species, a QTL interval for Na+ exclusion (wPt-3114-wmc170) was associated with an increase (10%) in seedling biomass. Of the five QTL identified for Na+ exclusion, two were co-located with seedling biomass (2A and 6A). The 2A QTL appears to coincide with the previously reported Na+ exclusion locus in durum wheat that hosts one active HKT1;4 (Nax1) and one inactive HKT1;4 gene. Using these sequences as template for primer design enabled mapping of at least three HKT1;4 genes onto chromosome 2AL in bread wheat, suggesting that bread wheat carries more HKT1;4 gene family members than durum wheat. However, the combined effects of all Na+ exclusion loci only accounted for 18% of the variation in seedling biomass under salinity stress indicating that there were other mechanisms of salinity tolerance operative at the seedling stage in this population. Na+ and K+ accumulation appear under separate genetic control. The molecular markers wmc170 (2A) and cfd080 (6A) are expected to facilitate breeding for salinity tolerance in bread wheat, the latter being associated with seedling vigour.

Survey of Fusarium species associated with crown rot of wheat and barley in eastern Australia

Fusarium species associated with crown rot were isolated and identified from 409 wheat, barley or durum wheat crops from the eastern Australian grain belt between 1996 and 1999. Fusarium pseudograminearum was almost the only species isolated from crops in Queensland and New South Wales. F pseudograminearum was also the most common species in Victoria and South Australia, but F. culmorum was frequently isolated in these states. F. culmorum accounted for more than 70% of isolates from the Victorian high-rainfall (> 500 mm) region and the South-East region of South Australia. F culmorum comprised 18% of isolates from the Victorian medium-rainfall (350–500 mm) region, and 7% of isolates from each of the Victorian low-rainfall region and the Mid-North region of South Australia. F avenaceum, F crookwellense and F. graminearum were isolated very infrequently. The proportion of F. culmorum among isolates of Fusarium from districts in Victoria and South Australia was strongly correlated with climatic conditions around the end of the growing season, especially with rainfall in November.

Mapping of the root lesion nematode (Pratylenchus neglectus) resistance gene Rlnn1 in wheat

Abstract.The root lesion nematode, Pratylenchus neglectus, is an economically damaging pathogen of wheat and other crops. The development of P. neglectus-resistant wheat cultivars would be greatly accelerated through the use of molecular markers, as resistance phenotyping is extremely time-consuming. A greenhouse bioassay was developed to identify resistance phenotypes of doubled-haploid populations. Bulked-segregant analysis was used to identify AFLP markers linked to P. neglectus resistance in the wheat cultivar Excalibur. One resistance-linked AFLP marker was mapped close to chromosome 7A RFLP markers in a densely-mapped Cranbrook/Halberd population. One of the chromosome 7A RFLP probes, cdo347, was genotyped in the Tammin/Excalibur population segregating for response to root lesion nematode and showed 8% recombination with the P. neglectus resistance gene Rlnn1. The marker Xcdo347-7A was validated on Excalibur-and Krichauff-derived DH populations segregating for Rlnn1 and showed 14% and 10% recombination, respectively, with Rlnn1 in these populations.

The application of species-specific assays based on the polymerase chain reaction to analyse Fusarium crown rot of durum wheat

Crown rot of wheat in Australia is caused by species of Fusarium, particularly F pseudograminearum, formerly known as F. graminearum Group 1. Rapid assays are required to identify the species responsible for disease symptoms, especially those with similar morphology. Previously developed assays based on the polymerase chain reaction (PCR) were able to identify F. pseudograminearum, F. graminearum, F. culmorum and F. crookwellense isolates, but not F. acuminatum. To design novel F. acuminatum and F. pseudograminearum species-specific primer sets, randomly amplified polymorphic DNA profiles were amplified that differentiated F. acuminatum and F. pseudograminearum from the other species and polymorphic bands were cloned and sequenced. The specificity of the PCR assays was verified on 79 isolates from 12 different Fusarium species. For two isolates an apparent misidentification occurred using the F. pseudograminearum PCR assay. These isolates were fingerprinted using Amplified Fragment Length Polymorphism analysis, which showed that they had genotypes more similar to F. graminearum than F. pseudograminearum. The PCR-based assays were validated using seedlings infected with single or multiple isolates. A method was also devised to rapidly identify Fusarium species associated with crown rot symptoms on mature wheat stems by culturing the fungi and extracting DNA directly from infected tissue. This assay can be used for routine diagnosis and for epidemiological studies of this disease.

Resistance to crown rot in wheat identified through an improved method for screening adult plants

An improved method has been devised for the screening of cereals with crown rot caused by Fusarium species. By growing plants in open-ended tubes set into galvanised baskets which are then placed on sand in outdoor terraces, it is possible to screen large numbers of plants, detecting adult plant resistance, using precise quantities of a single pathogen species. This ‘Terrace’ system has been used to seek alternative sources of resistance to crown rot in tetraploid and hexaploid wheats and to demonstrate that resistance in check cultivars is effective against both F. pseudograminearum and F. culmorum. Adult plant resistance in the cv. Kukri has been investigated using the Terraces and bulked segregant analysis of doubled haploid lines, and a resistance locus with polymorphic markers has been identified on chromosome 4B near the dwarfing gene Rht1.

Identification and mapping of a gene conferring resistance to the spot form of net blotch (Pyrenophora teres f maculata) in barley

Abstract Spot form of net blotch (SFNB) (Pyrenophora teres f maculata) is an economically damaging foliar disease of barley in many of the world’s cereal growing areas. The development of SFNB-resistant cultivars may be accelerated through the use of molecular markers. A screen for SFNB resistance in 96 lines identified four new sources of resistance, including a feed variety, ‘Galleon’, for which a fully mapped doubled haploid population was available. Segregation data indicated SFNB resistance was conferred by a single gene in the ‘Galleon’בHaruna Nijo’ cross, positioned on the long arm of chromosome 7H. This gene is designated Rpt4 and is flanked by the RFLP loci Xpsr117(D) and Xcdo673 at distances of 6.9 cM and 25.9 cM, respectively. The marker Xpsr117(D) was validated using another population segregating for Rpt4, correctly predicting SFNB resistance with more than 90% accuracy.

Quantitative trait loci for grain fructan concentration in wheat (Triticum aestivum L.)

Fructans (fructo-oligosaccharides) are prebiotics that are thought to selectively promote the growth of colonic bifidobacteria, thereby improving human gut health. Fructans are present in the grain of wheat, a staple food crop. In the research reported here, we aimed to detect and map loci affecting grain fructan concentration in wheat using a doubled-haploid population derived from a cross between a high-fructan breeding line, Berkut, and a low-fructan cultivar, Krichauff. Fructan concentration was measured in grain samples grown at two locations in Australia and one in Kazakhstan. Fructan concentration varied widely within the population, ranging from 0.6 to 2.6% of grain dry weight, and was quite repeatable, with broad-sense heritability estimated as 0.71. With a linkage map of 528 molecular markers, quantitative trait loci (QTLs) were detected on chromosomes 2B, 3B, 5A, 6D and 7A. Of these, the QTLs on chromosomes 6D and 7A had the largest effects, explaining 17 and 27% of the total phenotypic variance, respectively, both with the favourable (high-fructan concentration) alleles contributed from Berkut. These chromosome regions had similar effects in another mapping population, Sokoll/Krichauff, with the favourable alleles contributed from Sokoll. It is concluded that grain fructan concentration of wheat can be improved by breeding and that molecular markers could be used to select effectively for favourable alleles in two regions of the wheat genome.

Development and use of an assay based on the polymerase chain reaction that differentiates the pathogens causing spot form and net form of net blotch of barley

Two forms of barley net blotch are caused by different formae of the fungus Pyrenophora teres and both are economically important pathogens. The spot form of the net blotch fungus (P. teres f. maculata) and the net form of the net blotch fungus (P. teres f. teres) cause the lesion types indicated by their disease names, although symptom overlap and similar spore morphology can make identification difficult. Randomly amplified polymorphic DNA fragments differentiated the two forms of Pyrenophora. Polymorphic bands were cloned and sequenced to develop specific primer sets. A simple assay based on the polymerase chain reaction was developed and can identify the Pyrenophora formae causing disease symptoms directly from infected plant tissues in a single multiplex reaction. The assay was validated using amplified fragment length polymorphism genotyping of isolates and was shown to be more accurate than reliance on symptom expression. This assay can now be used for routine diagnosis, epidemiological studies and resistance breeding, where correct identification of each pathogen is critical.

Proteinaceous Metabolites from Pyrenophora teres Contribute to Symptom Development of Barley Net Blotch

ABSTRACT Pyrenophora teres, the causal agent of net blotch of barley (Hordeum vulgare L.), induces a combination of necrosis and extensive chlorosis in susceptible barley cultivars. Cell-free filtrates from both net and spot forms of P. teres; P. teres f. sp. teres, and P. teres f. sp. maculata were found to contain phytotoxic low molecular weight compounds (LMWCs) and proteinaceous metabolites which appear to be responsible for different components of the symptoms induced by the two forms of the pathogen in a susceptible cultivar of barley (cv. Sloop). Proteins induced only brown necrotic spots or lesions similar to those induced by the pathogens 72 h after inoculation. In contrast, LMWCs induced general chlorosis seen 240 h after inoculation but not the localized necrosis. Neither hydrolyzed or heat- or protease-treated proteinaceous metabolites induced the symptoms. This is the first report of the involvement of proteins produced by P. teres in symptom development during net blotch disease of barley.

Molecular variation in Rhynchosporium secalis isolates obtained from hotspots

A unique sampling strategy was used to search for genetic similarity among isolates of Rhynchosporium secalis, the causal agent of leaf scald of barley. A field of barley cv. Skiff with discrete ‘hotspots’ of leaf scald was identified and infected leaves were collected for this study. The isolates were genotyped using amplified fragment length polymorphism (AFLP) analysis. In contrast to previous reports of high diversity of R. secalis isolates in areas of only 1 m2, we found close genetic similarity between isolates from the same hotspot as determined by AFLP marker alleles, with a higher level of variation between isolates from different hotspots. A UPGMA phenogram showed that most isolates clustered with members of the same hotspot. This pattern is consistent with the inoculum source for the hotspots being airborne ascospores or wind-dispersed conidia from a local founder population, borne in splash-created aerosols. AFLP variation within hotspots suggests a high rate of mutation within a few cycles of fungal infection. A collection of isolates from cv. Skiff plots in a straw-inoculated disease nursery 60 km from the hotspot field was fingerprinted with AFLPs for comparison and was found to have much greater genotypic diversity.