K. J. Williams

A QTL located on chromosome 4A associated with dormancy in white- and red-grained wheats of diverse origin

Improved resistance to preharvest sprouting in modern bread wheat (Triticum aestivum. L.) can be achieved via the introgression of grain dormancy and would reduce both the incidence and severity of damage due to unfavourable weather at harvest. The dormancy phenotype is strongly influenced by environmental factors making selection difficult and time consuming and this trait an obvious candidate for marker assisted selection. A highly significant Quantitative Trait Locus (QTL) associated with grain dormancy and located on chromosome 4A was identified in three bread wheat genotypes, two white- and one red-grained, of diverse origin. Flanking SSR markers on either side of the putative dormancy gene were identified and validated in an additional population involving one of the dormant genotypes. Genotypes containing the 4A QTL varied in dormancy phenotype from dormant to intermediate dormant. Based on a comparison between dormant red- and white-grained genotypes, together with a white-grained mutant derived from the red-grained genotype, it is concluded that the 4A QTL is a critical component of dormancy; associated with at least an intermediate dormancy on its own and a dormant phenotype when combined with the R gene in the red-grained genotype and as yet unidentified gene(s) in the white-grained genotypes. These additional genes appeared to be different in AUS1408 and SW95-50213.

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.

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.

Mapping of a novel QTL for resistance to cereal cyst nematode in wheat

Cereal cyst nematode (CCN; Heterodera avenae Woll.) is a root pathogen of cereals that can cause severe yield losses in intolerant wheat cultivars. Loci for resistance to CCN, measured by a seedling bioassay, were identified by creating a genetic map based on a Trident/Molineux doubled haploid population of 182 lines. A novel locus accounting for up to 14% of the resistance to CCN was mapped to chromosome 1B of Molineux by association with microsatellite marker loci Xwmc719 and Xgwm140. This locus acts additively with the previously identified CCN resistance loci identified on chromosomes 6B (Cre8) and 2A (Cre5 on the VPM1 segment) in this population to explain 44% of the genetic variance for this major wheat pathogen.

Expression profiling and mapping of defence response genes associated with the barley–Pyrenophora teres incompatible interaction

Barley net- and spot-form of net blotch disease are caused by two formae of the hemibiotrophic fungus Pyrenophora teres (P. t. f. teres and P. t. f. maculata). In the present study, suppression subtractive hybridization (SSH) was used in combination with quantitative real-time reverse transcriptase PCR to identify and profile the expression of defence response (DR) genes in the early stages of both barley-P. teres incompatible and compatible interactions. From a pool of 307 unique gene transcripts identified by SSH, 45 candidate DR genes were selected for temporal expression profiling in infected leaf epidermis. Differential expression profiles were observed for 28 of the selected candidates, which were grouped into clusters depending on their expression profiles within the first 48 h after inoculation. The expression profiles characteristic of each gene cluster were very similar in both barley-P. t. f. teres and barley-P. t. f. maculata interactions, indicating that resistance to both pathogens could be mediated by induction of the same group of DR genes. Chromosomal map locations for 21 DR genes were identified using four doubled-haploid mapping populations. The mapped DR genes were distributed across all seven barley chromosomes, with at least one gene mapping to within 15 cM of another on chromosomes 1H, 2H, 5H and 7H. Additionally, some DR genes appeared to co-localize with loci harbouring known resistance genes or quantitative trait loci for net blotch resistance on chromosomes 6H and 7H, as well as loci associated with resistance to other barley diseases. The DR genes are discussed with respect to their map locations and potential functional role in contributing to net blotch disease resistance.

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.

Mapping of a QTL contributing to cereal cyst nematode tolerance and resistance in wheat

Cereal cyst nematode (CCN; Heterodera avenae Woll.) is a root pathogen of cereals that can cause severe yield losses in intolerant wheat cultivars. Tolerance to CCN, measured as early vigour in CCN-infested plots, was mapped in a Trident/Molineux doubled-haploid (DH) population. A locus accounting for a significant proportion of the tolerance to CCN was mapped to chromosome 6B of Molineux by association with RFLP loci Xcdo347-6B and Xbcd1 and also by nullisomic/tetrasomic substitution line analysis, and has been designated Cre8. The linkage of CCN tolerance with Xcdo347-6B was validated using a Barunga/Suneca DH population. The Cre8 locus also contributed to CCN resistance in the Trident/Molineux population. The RFLP locus Xbcd175, which is diagnostic for the Aegilops ventricosa segment VPM1 of Trident, explained up to 18% of the variation for early vigour in CCN-infested soils in the Trident/Molineux population. However, the Trident/Molineux population also segregated for early vigour in the absence of CCN, with Xbcd175 explaining up to 7% of the variation for this trait. The VPM1 segment of Trident therefore provides early vigour that may contribute to CCN tolerance in this cultivar.