The Russian wheat aphid (RWA) (Diuraphis noxia Kurdjumov) is a major pest of cultivated small grains. It is particularly devastating because of is high reproductive rate which results in the growth of large populations which become damaging to its host plants. Development of resistant barley (Hordeum vulgare L.) cultivars is complicated as resistance is polygenic. As a result, the industry remains at risk now that the RWA has spread throughout South Africa. It has, as recently as, 2013, been identified in the SW Cape, which was previously geographically isolated. This is South Africa‟s principle barley growing region. Now a potentially huge problem exists. Therefore, it is imperative that an alternative to pesticide use is found. Testing potential innate resistance in barley cultivars is thus, critical. In this thesis, I present data on four barely cultivars where I have examined their resistance/ lack of resistance to three known RWA biotypes, RWASA1, RWASA2 and RWASA3. The barley varieties used were two economically important South African malt barley cultivars (S5 and SSG 564) along with two potentially RWA resistant Afghan accessions (CIho 4125 and CIho 4159). The RWA biotype population growth rates on each of the plants were determined over a 14 day period. The aim was to establish baseline data of the effects of RWA population growth on the host plants under ambient CO2 (380 – 400 ppm) conditions. The extent of RWA feeding damage was investigated at the cell level by examining saliva deposition and cell disruption using Transmission Electron Microscopy; at the tissue/vascular level using fluorescence microscopy, to determine the extent of callose formation; at a whole leaf level by recording percent chlorosis and leaf roll; and finally, at a whole plant level by measuring biomass loss.The experiments were repeated under elevated CO2 (450 ppm) to model any changes in RWA/plant interaction with respect to future climate change. The effects of an elevated CO2 environment and RWA feeding on host plant foliar N and C:N ratio were compared to ambient CO2 conditions, to provide a clearer picture of the potential nutrient drain that a feeding RWA colony exacts on its host. Of the varieties tested, the CIho accessions performed better than the two SA barley cultivars as the CIho accessions appeared to express a mild antibiosis resistance response as RWA populations, particularly those of RWASA1, were smaller than those observed on either S5 or SSG 564. In addition, less damage was evident in the two CIho accessions due to RWA feeding. II RWASA2 was the most virulent of the three RWA biotypes tested, followed by RWASA3 while RWASA1 was the least virulent. Under elevated CO2 conditions, RWA feeding damage was exacerbated but the trend of biotype virulence remained the same. Higher aphid population sizes were recorded under elevated CO2, meant that even the more resistant CIho accessions were overcome by the increased demand made by the larger aphid colonies on the host plants. The % foliar N data showed that under elevated CO2 aphid-free control plants had increased N levels in their leaves. Increased “food” supply (as shown by the increased N levels) therefore allowed significantly larger aphid populations to develop on the plants exposed to elevated CO2, due to improved nutrient status of the phloem sap taken up by RWA. The knock-on effect of a higher aphid population was increased cell disruption as a result of extensive probing, extensive formations of wound callose, with the result that phloem damage impeded nutrient flow through the vascular tissues which contributed to chlorosis and (eventually plant) death. The major conclusion from this study is that even a mild CO2 elevation resulted in an increase aphid population which may pose a severe and very real threat to a barley crop. Therefore, without effort to identify and deploy resistant barley cultivars, it could well be possible that future barley cultivation in South Africa may no longer be viable.

Thirty-two resistant varieties of winter type barley from the United States Department of Agriculture's World Collection were chosen for a study of the inheritance of scald resistance. They were crossed with Wong (0.1. 6728), a highly susceptible variety and with Hudson (0.I. 8067), a highly resistant variety. The crosses with Wong were made for the purpose of determining the number of pairs of genes for resistance in each of the resistant varieties. The crosses with Hudson were made for the purpose of determining whether any of the resistant varieties has genes for scald resistance at loci other than in Hudson. The F2 seedling populations from these crosses were studied in the greenhouse at Blacksburg in the early spring of 1960 and the winter of 1961. / Master of Science

Barley yellow dwarf virus (BYDV), an aphid transmitted luteovirus, is the most widespread and economically damaging virus of cereal crops. The work in this thesis aims to characterise the basis of the naturally occurring resistance to BYDV in cereals in three ways: Firstly, by facilitating the isolation of the Yd2 gene for BYDV resistance from barley by a map-based approach. Secondly, by determining if a BYDV resistance gene in rice is orthologous to Yd2. Thirdly, by establishing if other BYDV resistance genes in non- Ethiopian barleys are allelic to Yd2. It is hoped that the information generated in this study will ultimately assist in the production of BYDV resistant cereal cultivars. A detailed genetic map of the Yd2 region of barley chromosome 3 was constructed, containing 19 RFLP loci, the centromere and the Yd2 gene. Yd2 mapped on the long arm, 0.5 cM from the centromere, and in the mapping population of 106 F2 individuals, perfectly cosegregated with the RFLP loci XYlp, and Xwg889. This map represents the first stage in a project to isolate the Yd2 gene by a map-based approach. The isolation of Yd2 could help to elucidate the molecular mechanism of the Yd2-mediated BYDV resistance, and may allow the production of BYDV resistant cereals by genetic transformation. The RFLP markers mapped closest to Yd2 could also be useful in barley breeding, by enabling selection for both the presence of Yd2 and the absence of agronomically undesirable traits known to be closely linked to Yd2. Genetically Directed Representational Difference Analysis (GDRDA) is a technique based on subtractive hybridisation, which can be used to identify RFLP markers closely linked to a gene of interest. Two GDRDA experiments were performed with the intention of generating additional RFLP markers close to Yd2. However, the first experiment yielded RFLP probes that were not derived from the barley genome, while the second experiment yielded probes that detected repetitive sequences. It was concluded that GDRDA is of limited use in generating further markers close to Yd2. To isolate the Yd2 gene by a map-based approach, a much larger mapping population will need to be analysed to genetically resolve markers tightly linked to Yd2. If the two morphological markers uzu dwarf and white stripe,,j flank Yd2, then they could assist in this task by enabling the visual identification of F2 seedlings resulting from recombination close to Yd2. However, in this study, both morphological markers were found to be located distal to Yd2. Therefore, these two morphological markers can not be used together to facilitate high resolution genetic mapping of the Yd2 locus. It may be possible to use large-insert genomic DNA clones from the relatively small genome of rice to generate further RFLP markers close to the Yd2 gene in barley, provided that the order of orthologous sequences in barley and rice is conserved close to the Yd2 locus. To assess the feasibility of this approach, RFLP probes used to identify loci close to Yd2 were mapped in rice using a segregating rice F2 population. Five of the RFLP loci mapped together and in the same order as RFLP loci mapped close to Yd2 in barley using the same probes. By comparing the location of RFLPs mapped by other researchers in rice using probes mapped close to Yd2, the region of conserved linkage between rice and the Yd2 region was tentatively identified as the central portion of rice chromosome 1. The collinearity shown by orthologous sequences in barley and rice indicated that it may indeed be possible to use rice to assist in generating RFLP markers close to Yd2. Of all the cereals, rice is the most amenable to map-based gene isolation, due to its small genome, well developed physical and genetic maps, and its ability to be genetically transformed with high efficiency. If a BYDV resistance gene that is orthologous to Yd2 could be identified in rice, this gene could be isolated with relative ease, and then used to identify barley cDNA clones corresponding to Yd2 gene by virtue of the sequence homology expected between these genes. To test if a BYDV resistance gene from an Italian rice line is orthologous to Yd2, recombinant-inbred rice lines previously characterised for this gene were analysed using probes mapped close to Yd2 in barley. No genetic linkage was detected between the RFLP loci and the BYDV resistance gene, indicating that the gene is unlikely to be orthologous to Yd2. BYDV resistance alleles at the Yd2 locus which are of a non-Ethiopian origin may show interesting differences to Ethiopian Yd2 resistance alleles. To identify barleys which may contain resistance alleles of Yd2, ten BYDV resistant barleys not known to contain Yd2 were assessed for their resistance to the PAVadel isolate of BYDV in the glasshouse. CI 1179, Rojo, Perry, Hannchen, Post and CI 4228 were found to be the most resistant under these conditions, and were analysed further. If the resistance from these barleys is controlled by alleles of Yd2, RFLP markers close to Yd2 will be expected to cosegregate with the resistance in F2 families derived from crosses between these resistant barleys and the BYDV susceptible barleys Atlas and Proctor. RFLPs suitable for use in these allelism tests were identified using probes mapped close to Yd2. However, time did not permit the analysis of these F2 populations. / Thesis (Ph.D.) -- University of Adelaide, Dept. of Plant Science, 1996

Errata attached to inside front cover. / Bibliography: leaves [156-188] / vi, 155, [33] leaves, [48] leaves of plates : ill. (some col.) ; 30cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Plant Science, 2001

Includes bibliographical references. The thesis aims to characterise the basis of naturally occuring resistance to BYDV in cereals in three ways: i. A map-based approach to the isolation of the Yd2 gene for BYDV resistance from barley. -- ii. Determining if a BYDV resistance gene in rice is orthologous to Yd2. -- iii. Establishing if other BYDV resistance genes in non-Ethiopian barleys are allelic to Yd2.

Errata sheet pasted on front end-paper. / Includes bibliographical references. / v, 125, [99] leaves, [3] leaves of plates : ill. (chiefly col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Plant Science, (1995?)

The occurrence of Boron (B) toxicity in Australian soils is recognised as a limiting factor for cereal productivity. A number of loci conferring tolerance to B toxicity have been identified in barley and chromosomally mapped. However, a lack of knowledge relating to the physiological and molecular events that occur under B toxicity and the molecular basis for B stress tolerance has been a bottleneck in harnessing available genetic diversity in barley and wheat. The recent advances in functional genomics provided an opportunity to examine B stress in barley in more detail. The aim of this project was to analyse genes differentially expressed under B stress in tolerant and intolerant barley to identify candidate genes involved in B toxicity tolerance. Two experimental approaches, Suppression Subtractive Hybridization (SSH) and microarray were adopted. Firstly, SSH was performed to examine gene expression in roots of selected tolerant and intolerant doubled haploid lines from a Clipper (B intolerant) X Sahara 3771 (B tolerant) mapping population, grown under moderate B stress. The SSH experiment aimed to investigate the early transcriptional response of B tolerant barley lines to B stress in order to identify the basis for B toxicity tolerance in roots. Differential screening of the subtracted library generated from B treated plants identified a total of 111 non-redundant clones up-regulated in bulked tolerant lines. On the other hand 94 clones were differentially expressed under non-treated conditions. Among the clones identified from subtracted library generated from B treated plants, metabolism was the largest functional category, representing 21% of the clones. The largest functional category in the subtracted library generated from non treated plants was cellular transport, representing 19% of the clones. Based on sequence similarity, about 170 transcripts identified in this experiment were assigned to chromosomal segments (bins) on the three homoeologous genomes of bread wheat. In total, 36 clones from the subtracted library generated from B treated plants were analysed as candidates. Nine were genetically mapped within the region of B tolerance QTL on three chromosomes (2H, 4H and 6H). The genes mapped to 4H and 6H QTL have the highest association with these loci in the Clipper X Sahara 3771 doubled haploid mapping population. A 4H B tolerance QTL candidate gene was identified as a B transporter gene with similarity to the Arabidopsis BOR1 gene. Genes identified to be differentially expressed in the tolerant lines from SSH suggest activation of a diverse defence response in the roots of barley plants under B stress. Data from SSH experiment indicate that cell wall-plasma membrane cytoskeleton continuum constitute the first action site against B toxicity and the influence of toxic B on K+ uptake could be the key initiating factor. In the second approach, the Affymetrix 22K Barley1 GeneChip(TM) was used to investigate B stress adaptation processes in barley. Gene expression was profiled in leaves of Sahara 3771 and Clipper plants grown under various B concentrations. The results show that the two genotypes respond differently to B toxicity. The B intolerance of Clipper is expressed through the induction of a high number of probe sets (2310) even at a low B concentration of 100 µM. In contrast, Sahara 3771 responded to a high B concentration (2000 µM) through the induction of only a few hundred (266) probe sets. In Sahara 3771 no change in the expression level of any probe sets was observed at 100 µM B. Altogether 286 probe sets showed differential expression in Sahara 3771 under three levels of B treatment (500, 1000 and 2000 µM). About 30% of these were down-regulated and about 70% were up-regulated in Sahara 3771 in response to B treatment. Most of the probe sets (59%) up-regulated in Sahara 3771 did not respond to B treatment in Clipper. These genes are either salt stress responsive or related to plant defense and thus could play a key role in protecting barley plants from the toxic effects of B. Two differentially expressed probe sets annotated as B transporters were identified between Sahara 3771 and Clipper under control condition. These two B transporter probe sets did not respond to B treatment but showed opposing expression patterns in the two varieties. One of these probe sets (Contig21126_at) is similar to the B transporter gene isolated from the SSH experiment that maps to the 4H tolerance locus. The map location and expression of this B transporter gene suggest that it could be the borate anion efflux transporter predicted by the proposed efflux model of B tolerance in Sahara 3771 barley. The other B transporter gene (Contig14139_at) showed over expression in Clipper under control condition and could be contributing to high B accumulation in Clipper which needs further investigation. Data from both experiments have indicated that B toxicity triggers oxidative stress and that jasmonate-based signaling plays a key role in B toxicity tolerance. SSH data indicate that Sahara 3771 which evolved in the harsh environment of Africa is more efficient in osmoregulation and ROS scavenging than Clipper. This trait is likely to give Sahara 3771 an edge over Clipper in tolerating toxic the effect of B. In addition to the efflux mechanism, which becomes less efficient with increasing B supply, Sahara 3771 appears to apply a number of other mechanisms for alleviating or withstanding toxic B induced stress to sustain growth. Some of these mechanisms are already known to be used by plants to cope with a number of stresses. / Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2008

Fall-sown barley will be increasingly important in the era of climate change due to higher yield potential and efficient use of water resources. Resistance/tolerance to biotic and abiotic stresses will be critical. Low temperature is an abiotic stress of great importance. Resistance to barley stripe rust (incited by Puccinia striifomis f. sp. hordei) and scald (incited by Rhynchosporium secalis) will be important in higher rainfall areas. Simultaneous gene discovery and breeding will accelerate the development of agronomically relevant germplasm. The role of FR-H1 and FR-H2 in low temperature tolerance (LTT) has been well documented. However the question still remains: is LTT due only to FR-H1 and FR-H2 or are there other, undiscovered, determinants of this critical trait? We developed two doubled haploid mapping populations using two lines from the University of Nebraska (NE) with superior cold tolerance and one line from Oregon State University (OR) with good malting quality and disease resistance: NB3437f/OR71 (facultative x facultative) and NB713/OR71 (winter x facultative). Both were genotyped with a custom 384 oligonucleotide pool assay (OPA). QTL analyses were performed for LTT, vernalization sensitivity (VS), and resistance to barley stripe rust and scald. Disease resistance QTL were identified with favorable alleles from both NE and OR germplasm. The role of VRN-H2 in VS was confirmed and a novel alternative winter allele at VRN-H3 was discovered in the Nebraska germplasm. FR-H2 was identified as a determinant of LTT and a new QTL, FR-H3, was discovered on chromosome 1H that accounted for up to 48% of the phenotypic variation in field survival at St. Paul, Minnesota, USA. The discovery of FR-H3 is a significant advancement in barley LTT genetics and will assist in developing the next generation of fall-sown varieties. / Graduation date: 2012

Black point of barley grain refers to a discolouration of the embryo end of the grain. Historically black point has been proposed to be due to fungal colonisation of the grain. However, Koch’s postulates have yet to be satisfied. The discolouration occurs during grain fill in response to high humidity or rainfall during the grain filling period. In wheat, which is also affected by black point, the discolouration has been proposed to be due to the oxidation of phenolic acids within the grain to form discoloured end products. Within this study, two approaches were investigated in order to understand the proteins and genes associated with this disorder. Firstly, a proteomics approach enabled the identification of individual proteins associated with black point. Two-dimensional gel electrophoresis was used to compare the proteome of the husk and whole grain tissue of mature black pointed and healthy grain. Very little watersoluble protein was extracted from the husk tissue. However, a significantly larger amount of protein was extracted using a salt extraction buffer, indicating the husk proteins were mostly cell wall bound. Due to the effect of residual salt and low protein concentrations these proteins were not conducive to analysis using two-dimensional gel electrophoresis. Further experiments using acid hydrolysis of the husk tissue and subsequent amino acid analysis revealed that the proteins were bound to the husk cell walls via covalent bonds. In contrast, large quantities of protein were obtained from the whole grain samples. This allowed statistically significant comparisons to be made between gels from healthy and black pointed grains. Two proteins were identified as being more abundant in black pointed grains. Mass spectrometry identified these as isoforms of barley grain peroxidase 1 (BP1). In addition, three proteins were identified as being more abundant in healthy grain. Mass spectrometry revealed these to be isoforms of the same protein with sequence similarity to a partial EST sequence from barley. Using 3' RACE the entire coding sequence of the gene was isolated which revealed that it encoded a novel putative late embryogenesis abundant (LEA) protein. Northern blot analysis was performed for BP1 and LEA and showed that gene expression differences could not account for the differences seen in protein quantities. Western blot analysis revealed that the LEA protein was biotinylated in vivo which is consistent with similar LEA proteins from other plant species. To further understand the role of these proteins in black point, antibodies were raised against the two proteins. Subsequent immunolocalisation studies indicated BP1 was present throughout all tissues of the grain whilst LEA was most abundant in the embryo and aleurone tissue. The second major area of investigation within this thesis was to further delineate the previously identified quantitative trait loci (QTL) associated with black point in barley. Previous studies have reported QTL for black point and kernel discolouration in both barley and wheat. Comparison of the published QTL revealed a locus on the short arm of chromosome 2H to be of particular interest. To identify genes underlying this QTL the genomes of barley, wheat and rice were compared. An in silico approach showed that the QTL shared macro-synteny with rice chromosomes 4 and 7. From the rice genome sequence, barley ESTs with sequence similarity were selected. In total, 20 ESTs were selected based on two main criteria: their putative role in black point and also being evenly spread across the region of the QTL length. These QTL were mapped within the Alexis x Sloop double haploid population. This approach revealed that there was some conservation of synteny but also identified clear boundaries where synteny between barley and rice had been lost since divergence. Significantly, the additional markers mapped to this region have enabled the initial black point QTL to be reduced from approximately 30cM to 20cM. In conclusion, this study has added significant knowledge our understanding of the genetics of black point in barley through the use of two approaches. The proteomics approach has aided in understanding the biochemical processes occurring within the grain in response to black point. The comparative genetics approach has aided in understanding the genetic control of an important region of the genome influencing black point susceptibility. Combined, these findings will direct future research endeavours aimed at producing black point resistant barley cultivars. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1323053 / Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2008