DAQ00152 Herbicide Tolerance Screening in Northern Region (Phase IV)

The previous projects (phase I - III) highlighted that northern region wheat and barley cultivars differ considerably in their sensitivity to herbicides. The new project will focus on increased screening of advanced breeding lines and new cultivars lines to commonly used herbicides, for barley, chickpea and wheat. Studies on impact of environment on herbicide x genotype responses will also be undertaken with the national team. The new information will be added to the existing information package on herbicide tolerance. Thus, adverse impacts of herbicides on productivity in northern region will be reduced, as growers and agronomists will select safer herbicides for their sown variety, or select more tolerant varieties for their important herbicides.

Herbicide tolerance of barley and wheat cultivars in NR (phase III)

During the past 10 years, this project tested 23 barley and 51 wheat varieties with 19 and 34 registered herbicides, respectively. It concentrated on new varieties and herbicides. The research highlighted that Northern Region (NR) wheat and barley varieties differed considerably in their sensitivity to these herbicides. Overall, 9 per cent of wheat variety x herbicide combinations and 6 per cent of barley variety x herbicide combinations had significant yield losses (3 to 38%) from herbicides at recommended rates and crop stages. In addition, 21 to 23 per cent had significant yield losses from herbicides at double rates, indicating a narrow margin of crop safety.

Student understandings of numeracy problems: Semantic alignment and analogical reasoning

Despite compulsory mathematics throughout primary and junior secondary schooling, many schools across Australia continue in their struggle to achieve satisfactory numeracy levels. Numeracy is not a distinct subject in school curriculum, and in fact appears as a general capability in the Australian Curriculum, wherein all teachers across all curriculum areas are responsible for numeracy. This general capability approach confuses what numeracy should look like, especially when compared to the structure of numeracy as defined on standardised national tests. In seeking to define numeracy, schools tend to look at past NAPLAN papers, and in doing so, we do not find examples drawn from the various aspects of school curriculum. What we find are more traditional forms of mathematical worded problems.

Identifying the Function of Sorghum's Drought Tolerance Stay-Green QTL

The goal of this research is to understand the function of allelic variation of genes underpinning the stay-green drought adaptation trait in sorghum in order to enhance yield in water-limited environments. Stay-green, a delayed leaf senescence phenotype in sorghum, is primarily an emergent consequence of the improved balance between the supply and demand of water. Positional and functional fine-mapping of candidate genes associated with stay-green in sorghum is the focus of an international research partnership between Australian (UQ/DAFFQ) and US (Texas A&M University) scientists. Stay-green was initially mapped to four chromosomal regions (Stg1, Stg2, Stg3, and Stg4) by a number of research groups in the US and Australia. Physiological dissection of near-isolines containing single introgressions of Stg QTL (Stg1-4) indicate that these QTL reduce water demand before flowering by constricting the size of the canopy, thereby increasing water availability during grain filling and, ultimately, grain yield. Stg and root angle QTL are also co-located and, together with crop water use data, suggest the role of roots in the stay-green phenomenon. Candidate genes have been identified in Stg1-4, including genes from the PIN family of auxin efflux carriers in Stg1 and Stg2, with 10 of 11 PIN genes in sorghum co-locating with Stg QTL. Modified gene expression in some of these PIN candidates in the stay-green compared with the senescent types has been found in preliminary RNA expression profiling studies. Further proof-of-function studies are underway, including comparative genomics, SNP analysis to assess diversity at candidate genes, reverse genetics and transformation.

Evaluation of potential candidate genes involved in salinity tolerance in striped catfish (Pangasianodon hypophthalmus) using an RNA-Seq approach

Increasing salinity levels in freshwater and coastal environments caused by sea level rise linked to climate change is now recognized to be a major factor that can impact fish growth negatively, especially for freshwater teleost species. Striped catfish (Pangasianodon hypophthalmus) is an important freshwater teleost that is now widely farmed across the Mekong River Delta in Vietnam. Understanding the basis for tolerance and adaptation to raised environmental salinity conditions can assist the regional culture industry to mitigate predicted impacts of climate change across this region. Attempt of next generation sequencing using the ion proton platform results in more than 174 million raw reads from three tissue libraries (gill, kidney and intestine). Reads were filtered and de novo assembled using a variety of assemblers and then clustered together to generate a combined reference transcriptome. Downstream analysis resulted in a final reference transcriptome that contained 60,585 transcripts with an N50 of 683 bp. This resource was further annotated using a variety of bioinformatics databases, followed by differential gene expression analysis that resulted in 3062 transcripts that were differentially expressed in catfish samples raised under two experimental conditions (0 and 15 ppt). A number of transcripts with a potential role in salinity tolerance were then classified into six different functional gene categories based on their gene ontology assignments. These included; energy metabolism, ion transportation, detoxification, signal transduction, structural organization and detoxification. Finally, we combined the data on functional salinity tolerance genes into a hypothetical schematic model that attempted to describe potential relationships and interactions among target genes to explain the molecular pathways that control adaptive salinity responses in P. hypophthalmus. Our results indicate that P. hypophthalmus exhibit predictable plastic regulatory responses to elevated salinity by means of characteristic gene expression patterns, providing numerous candidate genes for future investigations.

Field evaluation of tolerance to Tobacco streak virus in sunflower germplasm, and observations of seasonal disease spread

Strong statistical evidence was found for differences in tolerance to natural infections of Tobacco streak virus (TSV) in sunflower hybrids. Data from 470 plots involving 23 different sunflower hybrids tested in multiple trials over 5 years in Australia were analysed. Using a Bayesian Hierarchical Logistic Regression model for analysis provided: (i) a rigorous method for investigating the relative effects of hybrid, seasonal rainfall and proximity to inoculum source on the incidence of severe TSV disease; (ii) a natural method for estimating the probability distributions of disease incidence in different hybrids under historical rainfall conditions; and (iii) a method for undertaking all pairwise comparisons of disease incidence between hybrids whilst controlling the familywise error rate without any drastic reduction in statistical power. The tolerance identified in field trials was effective against the main TSV strain associated with disease outbreaks, TSV-parthenium. Glasshouse tests indicate this tolerance to also be effective against the other TSV strain found in central Queensland, TSV-crownbeard. The use of tolerant germplasm is critical to minimise the risk of TSV epidemics in sunflower in this region. We found strong statistical evidence that rainfall during the early growing months of March and April had a negative effect on the incidence of severe infection with greatly reduced disease incidence in years that had high rainfall during this period.

Constructive alignment: A journey for new eyes

The needs of students engaged in enterprise education programs are of ever growing importance. This paper considers the pedagogical challenges that confront the designers of such-programs. It is argued that it is the designer's mindset that will most likely determine the program's outcomes. That, regardless of where such programs reside, their development should be guided by a learner-centred approach. The recently developed hic et nunc framework, provides an example of such a student-centred approach. The process through which student learning outcomes occur is argued to be essentially Darwinian in nature. Taking into account both knowledge and skills, it is also argued that assessment of desirable learning outcomes should occur in visible interaction spaces. That the failure to eliminate invisible interaction spaces from such programs is an invitation for criticism from those who favour a more traditional lecturer-centred approach to teaching and learning.

Computational Techniques for Haplotype Inference and for Local Alignment Significance

This thesis which consists of an introduction and four peer-reviewed original publications studies the problems of haplotype inference (haplotyping) and local alignment significance. The problems studied here belong to the broad area of bioinformatics and computational biology. The presented solutions are computationally fast and accurate, which makes them practical in high-throughput sequence data analysis. Haplotype inference is a computational problem where the goal is to estimate haplotypes from a sample of genotypes as accurately as possible. This problem is important as the direct measurement of haplotypes is difficult, whereas the genotypes are easier to quantify. Haplotypes are the key-players when studying for example the genetic causes of diseases. In this thesis, three methods are presented for the haplotype inference problem referred to as HaploParser, HIT, and BACH. HaploParser is based on a combinatorial mosaic model and hierarchical parsing that together mimic recombinations and point-mutations in a biologically plausible way. In this mosaic model, the current population is assumed to be evolved from a small founder population. Thus, the haplotypes of the current population are recombinations of the (implicit) founder haplotypes with some point--mutations. HIT (Haplotype Inference Technique) uses a hidden Markov model for haplotypes and efficient algorithms are presented to learn this model from genotype data. The model structure of HIT is analogous to the mosaic model of HaploParser with founder haplotypes. Therefore, it can be seen as a probabilistic model of recombinations and point-mutations. BACH (Bayesian Context-based Haplotyping) utilizes a context tree weighting algorithm to efficiently sum over all variable-length Markov chains to evaluate the posterior probability of a haplotype configuration. Algorithms are presented that find haplotype configurations with high posterior probability. BACH is the most accurate method presented in this thesis and has comparable performance to the best available software for haplotype inference. Local alignment significance is a computational problem where one is interested in whether the local similarities in two sequences are due to the fact that the sequences are related or just by chance. Similarity of sequences is measured by their best local alignment score and from that, a p-value is computed. This p-value is the probability of picking two sequences from the null model that have as good or better best local alignment score. Local alignment significance is used routinely for example in homology searches. In this thesis, a general framework is sketched that allows one to compute a tight upper bound for the p-value of a local pairwise alignment score. Unlike the previous methods, the presented framework is not affeced by so-called edge-effects and can handle gaps (deletions and insertions) without troublesome sampling and curve fitting.

Development of salinity tolerance in rice by constitutive-overexpression of genes involved in the regulation of programmed cell death

Environmental factors contribute to over 70% of crop yield losses worldwide. Of these drought and salinity are the most significant causes of crop yield reduction. Rice is an important staple crop that feeds more than half of the world’s population. However among the agronomically important cereals rice is the most sensitive to salinity. In the present study we show that exogenous expression of anti-apoptotic genes from diverse origins, AtBAG4 (Arabidopsis), Hsp70 (Citrus tristeza virus) and p35 (Baculovirus), significantly improves salinity tolerance in rice at the whole plant level. Physiological, biochemical and agronomical analyses of transgenic rice expressing each of the anti-apoptotic genes subjected to salinity treatment demonstrated traits associated with tolerant varieties including, improved photosynthesis, membrane integrity, ion and ROS maintenance systems, growth rate, and yield components. Moreover, FTIR analysis showed that the chemical composition of salinity-treated transgenic plants is reminiscent of non-treated, unstressed controls. In contrast, wild type and vector control plants displayed hallmark features of stress, including pectin degradation upon subjection to salinity treatment. Interestingly, despite their diverse origins, transgenic plants expressing the anti-apoptotic genes assessed in this study displayed similar physiological and biochemical characteristics during salinity treatment thus providing further evidence that cell death pathways are conserved across broad evolutionary kingdoms. Our results reveal that anti-apoptotic genes facilitate maintenance of metabolic activity at the whole plant level to create favorable conditions for cellular survival. It is these conditions that are crucial and conducive to the plants ability to tolerate/adapt to extreme environments.

Tripogon loliiformis elicits a rapid physiological and structural response to dehydration for desiccation tolerance

Resurrection plants can withstand extreme dehydration to an air-dry state and then recover upon receiving water. Tripogon loliiformis (F.Muell.) C.E.Hubb. is a largely uncharacterised native Australian desiccation-tolerant grass that resurrects from the desiccated state within 72 h. Using a combination of structural and physiological techniques the structural and physiological features that enable T. loliiformis to tolerate desiccation were investigated. These features include: - (i) a myriad of structural changes such as leaf folding, cell wall folding and vacuole fragmentation that mitigate desiccation stress; - (ii) potential role of sclerenchymatous tissue within leaf folding and radiation protection; - (iii) retention of ~70% chlorophyll in the desiccated state; - (iv) early response of photosynthesis to dehydration by 50% reduction and ceasing completely at 80 and 70% relative water content, respectively; - (v) a sharp increase in electrolyte leakage during dehydration, and; - (vi) confirmation of membrane integrity throughout desiccation and rehydration. Taken together, these results demonstrate that T. loliiformis implements a range of structural and physiological mechanisms that minimise mechanical, oxidative and irradiation stress. These results provide powerful insights into tolerance mechanisms for potential utilisation in the enhancement of stress-tolerance in crop plants.

Recognition and alignment of homologous DNA sequences between minichromosomes and single-stranded DNA promoted by RecA protein

The incorporation of DNA into nucleosomes and higher-order forms of chromatin in vivo creates difficulties with respect to its accessibility for cellular functions such as transcription, replication, repair and recombination. To understand the role of chromatin structure in the process of homologous recombination, we have studied the interaction of nucleoprotein filaments, comprised of RecA protein and ssDNA, with minichromosomes. Using this paradigm, we have addressed how chromatin structure affects the search for homologous DNA sequences, and attempted to distinguish between two mutually exclusive models of DNA-DNA pairing mechanisms. Paradoxically, we found that the search for homologous sequences, as monitored by unwinding of homologous or heterologous duplex DNA, was facilitated by nucleosomes, with no discernible effect on homologous pairing. More importantly, unwinding of minichromosomes required the interaction of nucleoprotein filaments and led to the accumulation of circular duplex DNA sensitive to nuclease P1. Competition experiments indicated that chromatin templates and naked DNA served as equally efficient targets for homologous pairing. These and other findings suggest that nucleosomes do not impede but rather facilitate the search for homologous sequences and establish, in accordance with one proposed model, that unwinding of duplex DNA precedes alignment of homologous sequences at the level of chromatin. The potential application of this model to investigate the role of chromosomal proteins in the alignment of homologous sequences in the context of cellular recombination is considered.

Tailoring texture of γ-Y2Si2O7 by strong magnetic field alignment and two-step sintering

In this study, a well-dispersed γ-Y2Si2O 7 ethanol-based suspension with 30 vol% solid loading was prepared by adding 1 dwb% polyethylene imine dispersant, which allows feeble magnetic γ-Y2Si2O7 particles with anisotropic magnetic susceptibility to rotate in a 12 T strong magnetic field during slip casting, resulting in the development of a strong texture in green bodies. Pressureless sintering gives rise to more pronounced grain growth in the textured sample than in the untextured sample prepared without the magnetic field due to the rapid migration of the grain boundaries of the well-oriented grains, which was revealed by constant-heating-rate sintering kinetics. It was found that the use of two-step sintering is very efficient not only for inhibiting the grain growth but also for enhancing the texture. This implies that controlled grain growth is crucial for enhancing texture development in γ-Y2Si2O7.

Mechanical properties and damage tolerance of Y2SiO5

Y2SiO5 has potential applications as functional-structural ceramic and environmental/thermal barrier coating material. As an important grain-boundary phase in the sintered Si3N4, it also influences the mechanical and dielectric performances of the host material. In this paper, we present the mechanical properties of Y2SiO5 including elastic moduli, hardness, strength and fracture toughness, and try to understand the mechanical features from the viewpoint of crystal structure. Y2SiO5 has low shear modulus, low hardness, as well as high capacity for dispersing mechanical damage energy and for resisting crack penetration. Particularly, it can be machined by cemented carbides tools. The crystal structure characteristics of Y2SiO5 suggest the low-energy weakly bonded atomic planes crossed only by the easily breaking Y-O bonds as well as the rotatable rigid SiO4 tetrahedra are the origins of low shear deformation, good damage tolerance and good machinability of this material. TEM observations also demonstrate that the mechanical damage energy was dispersed in the form of the micro-cleavages, stacking faults and twins along these weakly bonded atomic planes, which allows the "microscale-plasticity" for Y2SiO5.

Cow's milk allergy and the development of tolerance

Aims: To gain insight on the immunological processes behind cow’s milk allergy (CMA) and the development of oral tolerance. To furthermore investigate the associations of HLA II and filaggrin genotypes with humoral responses to early oral antigens. Methods: The study population was from a cohort of 6209 healthy, full-term infants who in a double-blind randomized trial received supplementary feeding at maternity hospitals (mean duration 4 days): cow’s milk (CM) formula, extensively hydrolyzed whey formula or donor breast milk. Infants who developed CM associated symptoms that subsided during elimination diet (n=223) underwent an open oral CM challenge (at mean age 7 months). The challenge was negative in 112, and in 111 it confirmed CMA, which was IgE-mediated in 83. Patients with CMA were followed until recovery, and 94 of them participated in a follow-up study at age 8-9 years. We investigated serum samples at diagnosis (mean age 7 months, n=111), one year later (19 months, n=101) and at follow-up (8.6 years, n=85). At follow-up, also 76 children randomly selected from the original cohort and without CM associated symptoms were included. We measured CM specific IgE levels with UniCAP (Phadia, Uppsala, Sweden), and β-lactoglobulin, α-casein and ovalbumin specific IgA, IgG1, IgG4 and IgG levels with enzyme-linked immunosorbent assay in sera. We applied a microarray based immunoassay to measure the binding of IgE, IgG4 and IgA serum antibodies to sequential epitopes derived from five major CM proteins at the three time points in 11 patients with active IgE-mediated CMA at age 8-9 years and in 12 patients who had recovered from IgE-mediated CMA by age 3 years. We used bioinformatic methods to analyze the microarray data. We studied T cell expression profile in peripheral blood mononuclear cell (PBMC) samples from 57 children aged 5-12 years (median 8.3): 16 with active CMA, 20 who had recovered from CMA by age 3 years, 21 non-atopic control subjects. Following in vitro β-lactoglobulin stimulation, we measured the mRNA expression in PBMCs of 12 T-cell markers (T-bet, GATA-3, IFN-γ, CTLA4, IL-10, IL-16, TGF-β, FOXP3, Nfat-C2, TIM3, TIM4, STIM-1) with quantitative real time polymerase chain reaction, and the protein expression of CD4, CD25, CD127, FoxP3 with flow cytometry. To optimally distinguish the three study groups, we performed artificial neural networks with exhaustive search for all marker combinations. For genetic associations with specific humoral responses, we analyzed 14 HLA class II haplotypes, the PTPN22 1858 SNP (R620W allele) and 5 known filaggrin null mutations from blood samples of 87 patients with CMA and 76 control subjects (age 8.0-9.3 years). Results: High IgG and IgG4 levels to β-lactoglobulin and α-casein were associated with the HLA (DR15)-DQB1*0602 haplotype in patients with CMA, but not in control subjects. Conversely, (DR1/10)-DQB1*0501 was associated with lower IgG and IgG4 levels to these CM antigens, and to ovalbumin, most significantly among control subjects. Infants with IgE-mediated CMA had lower β -lactoglobulin and α-casein specific IgG1, IgG4 and IgG levels (p<0.05) at diagnosis than infants with non-IgE-mediated CMA or control subjects. When CMA persisted beyond age 8 years, CM specific IgE levels were higher at all three time points investigated and IgE epitope binding pattern remained stable (p<0.001) compared with recovery from CMA by age 3 years. Patients with persisting CMA at 8-9 years had lower serum IgA levels to β-lactoglobulin at diagnosis (p=0.01), and lower IgG4 levels to β-lactoglobulin (p=0.04) and α-casein (p=0.05) at follow-up compared with patients who recovered by age 3 years. In early recovery, signal of IgG4 epitope binding increased while that of IgE decreased over time, and binding patterns of IgE and IgG4 overlapped. In T cell expression profile in response to β –lactoglobulin, the combination of markers FoxP3, Nfat-C2, IL-16, GATA-3 distinguished patients with persisting CMA most accurately from patients who had become tolerant and from non-atopic subjects. FoxP3 expression at both RNA and protein level was higher in children with CMA compared with non-atopic children. Conclusions: Genetic factors (the HLA II genotype) are associated with humoral responses to early food allergens. High CM specific IgE levels predict persistence of CMA. Development of tolerance is associated with higher specific IgA and IgG4 levels and lower specific IgE levels, with decreased CM epitope binding by IgE and concurrent increase in corresponding epitope binding by IgG4. Both Th2 and Treg pathways are activated upon CM antigen stimulation in patients with CMA. In the clinical management of CMA, HLA II or filaggrin genotyping are not applicable, whereas the measurement of CM specific antibodies may assist in estimating the prognosis.