In the world today there is a massive dependency on fossil fuels as they are currently used to provide around 80% of the world’s energy. This is hugely detrimental to the environment and is a major contributory factor in climate change. Biofuel is a renewable energy source that is already being used to lessen some of the fossil fuel dependency. 2nd generation biofuels, by using non-food parts of plants, circumvent the food vs fuel argument, and by using farming waste or surplus can also avoid changing land use problems. Additionally liquid biofuels can use existing infrastructure for storage and delivery, and also fit into current lifestyles. Cost-effective 2nd generation biofuel production is directly affected by the presence of the polymer lignin in plant biomass, as it has been shown to impede enzymatic sugar release (saccharification) that is used for biofuel production. The work undertaken in this project developed a high-throughput methodology for the assessment of straw lignin content and composition across a large population of elite varieties in the economically important cereal crop, barley. Saccharification yield was also measured across the same population along with a number of other agronomically important traits, such as thousand-grain weight, biomass, mechanical stem properties and height. The data provided by these measurements allowed correlations between traits to be identified and their strength gauged. Genome wide association studies (GWAS) were also carried out and identified influential regions of the genome for each trait. The results revealed varying levels of association between measured traits and lignin content and monomeric constituents. Importantly a negative connection was shown between lignin content and saccharification yield, with lignin content being responsible for approximately 1/5th of the variation seen. Interestingly there was no correlation between lignin content and mechanical stem properties, an important factor in the agronomically important trait, lodging. GWAS results revealed a number of genomic regions that were influential across several traits indicating regions that would be difficult to separate through breeding due to their close proximities. However, unique QTL were identified for saccharification yield and lignin content providing candidates for breeding or genetic manipulation to improve the crop for biofuel production.

Major changes in expression occur in Arabidopsis in response to cold. It is now clear that genome-wide changes in alternative splicing (AS) also occur in Arabidopsis during the cold-response and many of the genes which undergo cold-induced AS have been linked to roles in either the regulation of the cold-response or regulation of AS. Mutations in splicing factors in Arabidopsis, such as STA1 and SRL1 have been shown to lead to both changes in AS and changes in cold-sensitivity/tolerance, suggesting an important the role for AS in regulating the cold-response. Less is known about the effect of cold on AS in barley or how AS impacts the barley cold-response. There are only a few studied examples of cold-induced AS changes in barley genes, although this is rapidly changing due to both the publication of the barley genome and next generation sequencing of the transcriptome. To investigate AS in the barley cold response, 11 barley genes with cold-induced AS changes were identified and the AS change was analyzed in detail. The barley genes FRY2 and SUA change AS after 30 minutes exposure to cold and are both genes have been linked to roles in regulating AS, indicating a complex role for AS in the earliest stages in the cold-response. The Serine Arginine (SR)-Rich protein genes have been shown to change AS and affect AS under stress conditions in Arabidopsis, rice and other plant species but little is known about the SR protein genes in barley or how the genes change splicing/expression in response to cold. The 16 members of the barley SR protein gene family were identified and analyzed for cold-induced expression changes using available microarray and RNA-Seq data. The HvRS41 gene showed a >2 fold increase in expression after 3h exposure to 6°C in a cold-based microarray experiment. A cold-based microarray experiment in Arabidopsis showed a similar cold-induced expression of the AtRS40 gene, a RS-type SR protein gene with high homology to HvRS41. The cold-induced expression of HvRS41 and AtRS40 indicate a role for the RS-type SR protein genes in the cold response. The RS-type SR proteins form a splicing complex with FRY2 which could potentially be regulated through both AS and expression change during the cold-response. The Barley SR Proteins were dived into six sub-groups previously established for plant SR protein genes. Five out of the six sub-groups of the barley SR protein genes contained AS which could be validated through RT-PCR based methods. The SR-type SR protein genes contained was shown to contain three genes (HvSR34, HvSR30a and HvSR34) within barley. All 3 barley SR-type protein genes showed AS change in response to low temperatures, indicating a role for the barley SR-type SR protein genes in regulating AS during the cold-response. The role for SR-type SR protein genes in regulating AS was tested through a creation of a barley transgenic line over-expressing gene the HvSR34. The HvSR34 overexpression lines are in the process of being tested for changes in AS and cold tolerance.

Barley is one of the important crops in many of the irrigated regions of the world. It is an important crop in Iran for both human consumption and livestock feed. In the United States and Europe it is used for livestock feed and beverages. The income from this crop is moderate in comparison to that from other major agricultural products. Barley production has a definite place in the economy of the region, because it requires only a small amount of care. It can be readily grown on poor lands. One of the factors contributing to the increased acreage of barley in many countries is the tolerance and adaptability of this plant to salt and poor or second class land. Yields have been increased materially in recent years with the development of new varieties superior to the standard varieties previously used. Better cultural practices are improving yields, but much remains to be done in this phase of the work.

In plants, P450 enzymes encoded by P450 genes play a central role in numerous biosynthetic pathways, such as the production of secondary metabolites, stress responses and disease resistance. This thesis reports upon the utilization of molecular biology techniques to study P450 gene(s) in barley (Hordeum vulgare L.).Using several combinations of degenerate primers, a large number of barley P450 gene fragments were cloned and sequenced from two commercial varieties, Chebec and Harrington. Among 247 isolated sequences, twenty six percent were homologous to genes of known function. The abundance of these sequences differed between the two cultivars. Variations in the motif sequences of the cloned genes were also found between these two cultivars. In addition to the cloned fragments, twenty-two putative barley P450 encoding genes were identified from 24,000 cereal sequences in the International Triticeae EST Cooperative (ITEC) database by homology search. Among these Expressed Sequence Tag (EST) sequences, a full-length P450 sequence was selected for further investigation in this study.This novel P450 gene, CYP72A39, was expressed at a very early vegetative stage, but no expression was detected at the reproductive stage. Comparison of expression profiles of this gene and "digital expression" databases confirmed that this gene was homologous to several cereal EST clones with tissue-specific transcripts responding to various environmental stimuli, such as stresses and disease. Among these, many transcripts in barley were obtained from stressed tissues at the vegetative stage, and two transcripts in wheat (Triticum aestivum L.) were expressed after being challenged by barley powdery mildew pathogen (Blumeria graminis f. sp. hordei L.). This suggested that CYP72A39 may play a defence role in the barley seedling.The secondary structure of CYP72A39 was investigated in a comparative analysis using a computational approach. However, attempts to express CYP72A39 in a heterologous system and functional assays of the native protein in barley did not give decisive results, due to the disordered structure of the native protein and limitations of the current method. Screening the 3' UnTranslated Region (3'UTR) of this gene in 158 genotypes of domesticated, landrace and wild barley revealed two haplotypes, which differed by a 12 base indel positioned between two transversions. The presence of both haplotypes in wild and cultivated barley suggests this polymorphism predates the domestication of barley. This indel was mapped to the long arm of chromosome 6H, less than 10 centi-Morgans (cM) from the gene encoding resistance to powdery mildew in barley (B. graminis). A comparison between haplotype diversity and powdery mildew resistance data for over 102 genotypes showed a weak link between the 'long' haplotype and resistance, while the 'short' haplotype was associated with susceptibility. There was no evidence for a strong correlation between haplotype and quality type (malt or feed); however, more malting varieties had the long haplotype, suggesting a possible association with some attributes in modern malting barley. There was no evidence for association with other characteristics such as geographic origin, growth habit or row number. Phylogenetic relationship of the CYP72A39 to other CYP72 members was also investigated.

Near infrared spectroscopy (NIS) has been used routinely for many years for the measurement of grain protein and moisture in plant breeding programmes. Investigation as to the applicability of NIS to the identification from a barley breeding programme of the progeny with high malting quality potential was carried out over several harvests. The project concentrated on the determination of correlations between Hot Water Extract, Total Soluble Nitrogen, and Free Alpha Amino Nitrogen contents of worts (the extract of malt used to make beer) and NIR transmission data using the multivariate method of partial least squares regression. The correlation coefficients, for both calibration and prediction data sets, were significant and the standard error of prediction was similar to that obtained in the standard methods in the first year, but were unsatisfactory in the second. The instrument chosen for the study gave satisfactory correlations for the purpose of selection in the intermediate generations of the breeding programme with errors similar to the analytical methods, as long as a very wide range of calibration samples were collected from more than one harvest. It was shown that the use of an NIR spectrophotometer as a selection tool for malting quality within a barley breeding programme would not be sufficient to justify investment in this type of instrument for this purpose alone. / Master of Science (Hons)

Bibliography: leaves 114-141. / vi, 141, [42] leaves, [19] leaves of plates : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Using RFLP, STS-PCR and isoenzyme techniques, maps the structural genes of hydrolytic enzymes important in seed germination processes, and determines the contribution of each gene to the activity of the enzyme. / Thesis (Ph.D.)--University of Adelaide, Dept. of Plant Science, 1998

<p>Plant resistance hypothesis says that under a period of time when a plant is exposed to powerful herbivore attack it will prioritise defence as a major metabolic function. In theory, induced plant defence (resistance) will provide opportunities for this organism to “invest” in other functions, in example growth when attackers are absent.</p><p>One of the compounds taking part in plant defence is callose. This β-1,3-glucan is synthesised by callose synthase and broken down by β-1,3-glucanase. Deposition of callose occurs as a reaction to aphid attack an varies, depending on cultivars, and aphid species. In this experiment barley (Hordeum vulgare) cultivar Clipper is being infested with two types of aphids: Russian wheat aphid (RWA, Diuraphis noxia) and bird cherry-oat aphid (BCA, Rhopalosiphium padi) over a time period. Infestation by those two insects results in different callose formation and deposition level.</p><p>Six sequences encoding for putative callose synthase genes and nine sequences encoding for β-1,3-glucanase were examined by RT-PCR and Real – Time PCR methods for different expression patterns.</p><p>The results did not show any significant regulation of gene expression during RWA and BCA attack for any of these genes. Thus the pathway regulating aphid – induced callose deposition in barley reminds unresolved.</p>

Understanding complex traits is a fundamental challenge in plant genetics and a prerequisite for molecular breeding. Tools for trait dissection are markers, maps, and quantitative trait locus (QTL) analysis. Marker-assisted selection (MAS) is an application that integrates these tools. In this thesis research, a new sequence-based marker was evaluated, maps were constructed and used, and QTLs were detected using two types of populations. Marker-assisted selection was used to develop a novel class of barley. Restriction-site Associated DNA (RAD), a sequence based-marker technology, allows for simultaneous high-density single nucleotide polymorphism (SNP) discovery and genotyping. We assessed the value of RAD markers for linkage map construction using the Oregon Wolfe Barley (OWB) mapping population. We compared a RAD-based map to a map generated using Illumina GoldenGate Assay (EST-based SNPs). The RAD markers generated a high quality map with complete genome coverage. We then used the RAD map to locate QTL for agronomic fitness traits. A paper describing this research was published (Chutimanitsakun et al., 2011). Marker-assisted selection was used to rapidly develop fall-sown barley germplasm for human food uses. The target traits were high grain β-glucan, vernalization sensitivity (VS) and low temperature tolerance (LTT). The target loci were WX and VRN-H2. Marker-assisted selection was effective in fixing target alleles at both loci and waxy starch led to increase in grain β-glucan. Unexpected segregation at VRN-H1 and VRN-H3, revealed by genome-wide association mapping (GW-AM), led to unanticipated phenotypic variation in VS and LTT. We found that GW-AM is an efficient and powerful method for identifying the genome coordinates of genes determining target traits. Precise information is obtained with perfect markers; additional research may be needed when multiple alleles are segregating at target loci and significant associations are with markers in linkage disequilibrium (LD) with the target loci. A paper describing this research will be submitted for publication. / Graduation date: 2012

Characterization of the determinants of economically important phenotypes showing complex inheritance should lead to more effective use of genetic resources. This study was conducted to determine the number, genome location and effects of QTLs determining malting quality and agronomic traits in the two North American barley quality standards. Using a doubled haploid population of 140 lines from the cross of Harrington x Morex, agronomic phenotype and malting quality data sets from nine and eight environments, respectively, and a 107-marker linkage map, QTL analyses were performed using simple interval mapping and simplified composite interval mapping procedures. Thirty five QTLs were associated either across environments or in individual environments, with five grain and agronomic traits (yield, kernel plumpness, test weight, heading date and plant height). Thirteen QTLs were associated with five malting quality traits (grain protein percentage, soluble/total protein ratio, ��-amylase activity, diastatic power and malt extract percentage). QTLs for multiple traits were coincident. The loci controlling inflorescence type [vrsl on chromosome 2 (2H) and int-c on chromosome 4 (4H)] were coincident with QTLs affecting all traits except heading date and malt extract percentage. The largest effect QTLs -for yield, kernel plumpness test weight, plant height grain protein percentage, S/T ratio, and diastatic power- were coincident with the vrsl locus. QTL analyses were conducted separately for each sub-population (six-rowed and two-rowed). Ten new QTLs were detected in the sub-populations. There were significant interactions between the vrsl and int-c loci for plant height, grain protein percentage, and SIT protein ratio. Positive transgressive segregants were found for all agronomic traits. They were more prevalent in the six-rowed sub-population, indicating that more favorable alleles were fixed in the two-rowed parent. Results suggest that this mating of two parents representing different germplasm groups caused a disruption in the balance of traits involved in malting quality, which resulted in no progeny carrying all favorable alleles and therefore surpassing the quality of either parent. This study describes some of the genetic determinants of agronomic and malting quality traits in a two-rowed x six-rowed cross and it is a first step toward the further characterization and manipulation of these determinants. / Graduation date: 2001

Graduation date: 1994