Academic literature on the topic 'Fields of Research – 270000 Biological Sciences – 270200 Genetics'

Barley (Hordeum vulgare L. subsp. vulgare) is an important crop and ranks fourth in overall production of the major cereal crops in the world. Like other cereal crops, barley suffers from a narrowing of its genetic base and susceptibility to diseases, pests and environmental stresses. H. bulbosum is a possible source of desirable genes for introgressing into barley to restore genetic diversity and improve current cultivars. Sexual hybridisation between barley and H. bulbosum is the main method for interspecific gene transfer in barley breeding but there are several barriers to overcome. Two of these are reduced recombination and the ability to identify recombinants quickly and efficiently. The aim in this thesis was to gain a better understanding of meiotic chromosomal behaviour in the two species and their hybrids and to improve the characterisation of recombinants from the hybrids. To study the events during meiosis, synaptonemal complex (SC) analysis was carried out on the two species and two H. vulgare - H. bulbosum hybrids. The results indicated that there were interspecific and intraspecific variations in SC length. Mean SC length was positively correlated with recombination frequency but not related to genome size. This suggests that the ratios of mean SC length to genome size (SC/DNA) show divergence among these Hordeum examples. An hypothesis based on the conformation of chromatin associated with axial element, which is dependent on SC/DNA ratio, was presented to explain the relationship between SC length and recombination frequency. Chromosome pairing in the two hybrids was determined by observation at pachytene and metaphase I (MI). Mean percentages of synapses were similar but there were different frequencies of MI pairing between these two hybrids, indicating that different mechanisms may regulate synapsis and MI pairing in the hybrids. To investigate meiotic recombination, genomic in situ hybridisation (GISH) was performed on the two hybrids at MI and anaphase I (AI). It was observed that intergenomic pairing and recombination events occur in distal chromosome segments. A great discrepancy between mean pairing and recombination frequencies was observed in both hybrids and several possible reasons for this discrepancy were discussed. Hybrid 102C2 with high MI pairing had a significantly higher recombination frequency than the low pairing 103K5, suggesting that high MI pairing appears to be associated with high recombination in the hybrids. An interesting finding is that the ratio of recombination to MI pairing in 103K5 (l:8.9) is twice as high compared with 102C2 (l:17). However, the mechanism for this difference in the ratio between the two hybrids remains unknown. Sequential fluorescence in situ hybridisation (FISH) and GISH were used successfully to localise the introgressions in selfed progeny from a tetraploid hybrid derived from chromosome-doubled 102C2 (102C2/colch). This procedure is fast, cheap and can efficiently detect and locate introgressions. Several disease-resistant recombinants were analysed in more details and leaf rust and powdery mildew resistance was associated with distal introgressions on chromosomes 2HS and 2HL (leaf rust) and 2HS (powdery mildew). It is possible that the leaf rust and powdery mildew resistances were closely linked in the distal region of 2HS. A considerable variation in introgression size was observed at similar chromosomal sites among the different recombinants, which will provide useful information for map-based cloning of genes.

Individuals of the cnidarian genus Physalia are a common sight at New Zealand beaches and are the primary cause of jellyfish stings to beachgoers each year. The identity of the species and the environmental factors that determine its presence are unknown. Lack of knowledge of many marine species is not unusual, as pelagic invertebrates often lack detailed taxonomic descriptions as well as information about their dispersal mechanisms such that meaningful patterns of distribution and dispersal are almost impossible to determine. Molecular systematics has proven to be a powerful tool for species identification and for determining geographical distributions. However, other techniques are needed to indicate the causal mechanisms that may result in a particular species distribution. The aim of this study was to apply molecular techniques to the cnidarian genus Physalia to establish which species occur in coastal New Zealand, and to apply models to attempt to forecast its occurrence and infer some mechanisms of dispersal. Physalia specimens were collected from New Zealand, Australia and Hawaii and sequenced for Cytochrome c oxidase I (COI) and the Internal transcribed spacer 1 (ITS1). Three clans were found: a Pacific-wide clan, an Australasian clan and New Zealand endemic clan with a distribution confined to the Bay of Plenty and the East Coast of the North Island. Forecasting Physalia occurrence directly from presence data using artificial neural networks (ANN) proved unsuccessful and it was necessary to pre-process the presence data using a variable sliding window to reduce noise and improve accuracy. This modelling approach outperformed the time lagged based networks giving improved forecasts in both regions that were assessed. The ANN models were able to indicated significant trends in the data but would require more data at higher resolution to give more accurate forecasts of Physalia occurrence suitable for decision making on New Zealand beaches. To determine possible causal mechanisms of recorded occurrences and to identify possible origins of Physalia the presence and absence of Physalia on swimming beaches throughout the summer season was modelled using ANN and Naϊve Bayesian Classifier (NBC). Both models were trained on the same data consisting of oceanographic variables. The modelling carried out in this study detected two dynamic systems, which matched the distribution of the molecular clans. One system was centralised in the Bay of Plenty matching the New Zealand endemic clan. The other involved a dynamic system that encompassed four other regions on both coasts of the country that matched the distribution of the other clans. By combining the results it was possible to propose a framework for Physalia distribution including a mechanism that has driven clan divergence. Moreover, potential blooming areas that are notoriously hard to establish for jellyfish were hypothesised for further study and/or validation.

Content removed from thesis due to copyright restrictions: Eaton, C. J., I. Jourdain, et al. (2008). "Functional analysis of a fungal endophyte stress-activated MAP kinase." Current Genetics 53(3): 163-174. Scott, B. and C. J. Eaton (2008). "Role of reactive oxygen species in fungal cellular differentiations." Current Opinion in Microbiology 11(6): 488-493.
In the mutually beneficial association between the fungal endophyte Epichloë festucae and perennial ryegrass, fungal growth is highly regulated and coordinated with that of the host. This implies there must be signalling between the fungus and its host to maintain this close association. Recent work has shown a novel role for reactive oxygen species (ROS) in this symbiotic maintenance, with multiple components of the superoxideproducing NADPH oxidase (Nox) complex being essential for normal association. However, the mechanism by which the Nox complex is regulated is unclear. To identify potential regulators of the E. festucae Nox complex, comparisons were made with well-characterised mammalian systems. This search identified three candidate regulators: a stress activated MAP kinase, sakA, and the p21-activated kinases, pakA and pakB. To investigate if these genes were involved in symbiotic maintenance, replacement mutants were generated by homologous recombination. In culture analysis revealed that the ?sakA mutant was hypersensitive to a range of stresses, whereas the pak mutants were hypersensitive to cell wall stress-inducing agents and displayed altered growth and morphology. Examination of perennial ryegrass infected with these mutants revealed drastically altered plant interaction phenotypes for the ?sakA and ?pakA mutants in comparison to the wild-type strain. ?sakA-infected plants were stunted and displayed striking changes in development, with the base of tillers showing loss of anthocyanin pigmentation and disorganisation of host cells below the meristem, resulting in swollen bases. Plants infected with the ?pakA mutant were severely stunted, had no more than two tillers and senesced soon after planting. In contrast, plants infected with the ?pakB mutant were similar to wild-type, with only slight deregulation of growth in planta. Examination of ROS in culture revealed that ?sakA and ?pakA displayed elevated levels of both superoxide and hydrogen peroxide. ROS levels were also elevated around ?sakA hyphae in planta. These results support roles for SakA and PakA in Nox regulation. This work highlights the fine balance between mutualism and antagonism, and provides insight into the molecular basis for mutualism.

Survival in the face of unpredictable environments is a challenge faced by all organisms. One solution is the evolution of mechanisms that cause stochastic switching between phenotypic states. Despite the wide range of switching strategies found in nature, their evolutionary origins and adaptive significance remain poorly understood. Recently in the Rainey laboratory, a long-term evolution experiment performed with populations of the bacterium Pseudomonas fluorescens SBW25 saw the de novo evolution of a phenotypic switching strategy. This provided an unprecedented opportunity to gain insight into the evolution and maintenance of switching strategies. The derived ‘switcher’ genotype was detected through colony level phenotypic dimorphism. Further microscopic examination revealed the cellular basis of phenotypic switching as the bistable (ON/OFF) expression of a capsule. Transposon mutagenesis demonstrated that the structural basis of the capsule was a colanic acid-like polymer encoded by the Pflu3656-wzb locus. Subsequently, whole genome re-sequencing enabled elucidation of the series of mutational events underlying the evolution of capsule bistability: nine mutations were identified in the switcher. Present in both forms of the switcher, the final mutation – a point mutation in a central metabolic pathway – was shown to be the sole mechanistic cause of capsule switching; it ‘set the stage’ for a series of molecular events directly responsible for bistability. Two models were proposed to explain capsule switching at the molecular level: the genetic amplification-reduction model, and the epigenetic feedback model. Collective results of biochemical and genetic assays proved consistent with the epigenetic model, whereby a decrease in flux through the pyrimidine biosynthetic pathway activates an already-present feedback loop. Subsequent analysis of a second switcher (evolved independently of and in parallel with the first) revealed a radically different genetic route leading to phenotypically and mechanistically similar capsule switching. In addition to providing the first empirical insight into the evolutionary bases of switching strategies, the work presented in this thesis demonstrates the power of natural selection – operating on even the simplest of organisms – to forge adaptive solutions to evolutionary challenges; in a single evolutionary step, selection took advantage of inherent intracellular stochasticity to generate an extraordinarily flexible phenotype.

Butyrivibrio proteoclasticus B316T is the most recently described species of the Butyrivibrio / Pseudobutyrivibrio assemblage and now the first to have its genome sequenced. The genome of this organism was found to be spread across four replicons: a 3.5 Mb major chromosome and three additional large replicons: 186, 302 and 361 Kb in size. This thesis describes the sequencing, analysis, annotation and initial characterisation of all three B. proteoclasticus auxiliary replicons. Most significantly, these analyses revealed that the 302-Kb replicon is a second chromosome. This small chromosome, named BPc2, encodes essential systems for the uptake and/or biosynthesis of biotin and nicotinamide adenine mononucleotide, as well as the enzymes required for utilisation of fumarate as the terminal electron acceptor during anaerobic respiration, none of which are found on the main chromosome. In addition, BPc2 contains two complete rRNA operons, a large number of enzymes involved in the metabolism of carbohydrates, nitrogen and fatty acids. In contrast to BPc2, both megaplasmids appear largely cryptic, collectively encoding 421 genes not previously described in public databases. Nevertheless, only the 186-Kb, but not 361-Kb megaplasmid, could be cured from Butyrivibrio proteoclasticus B316T. The largest megaplasmid has a copy number of 5, while all other replicons are present at a copy number of 1. %GC content and codon usage analyses strongly suggests that all three auxiliary replicons have co-resided with the major chromosome for a significant evolutionary period. Moreover, the replication machineries of these three replicons are conserved. Interestingly, a survey of a number of Butyrivibrio / Pseudobutyrivibrio species revealed that the megaplasmids are widespread in this assemblage, however these other large plasmids do not show concordance with their 16S rRNA phylogeny and appear distinct to those of B. proteoclasticus B316T. A microarray analysis of gene expression in a co-culture experiment between B. proteoclasticus and the important ruminal methanogen, Methanobrevibacter ruminantium M1, revealed a potentially mutualistic interspecies interaction. In this relationship M. ruminantium appears to provide B. proteoclasticus with glutamate, essential to the final step of NAD+ biosynthesis, while B. proteoclasticus appears to provide M. ruminantium with formate, hydrogen and carbon dioxide, each important substrate for methanogenesis.

The sap beetle genus Carpophilus Stephens (Coleoptera: Nitidulidae) is a large genus consisting of over 200 species and are found worldwide. Several species are important pests of crops and stored products, and are frequently intercepted as part of biosecurity operations. The genus is poorly known taxonomically, and there are several species groups that are challenging to identify by morphological methods. In particular, two species found across the Pacific, C. maculatus Murray and C. oculatus Murray are frequently confused with each other. These two species are similar in size and colour, but differ primarily by the shape of the colour pattern on their elytra. However, this colour pattern is highly variable within both species, leading to ambiguity in the indentification of these species. Within C. oculatus, three subspecies have been described based on differences in the male genitalia and pronotal punctation: C. o. oculatus and C. o. gilloglyi Dobson are distributed widely across the Pacific, while C. o. cheesmani Dobson is known only from Vanuatu. A search of literature records and specimen collections revealed 32 species of Carpophilus recorded from the Pacific region. In addition there remain several unidentified specimens representing at least four species, two of which will be described subsequent to this research. A number of species recorded in the literature may have been misidentified, and these require further field collections and inspection of museum specimens to confirm their presence in the Pacific. To test the validity of the subspecies of C. oculatus, and its distinctiveness from C. maculatus, a phylogeny of available specimens of Carpophilus was inferred from one mitochondrial gene (cytochrome c oxidase subunit I (COI)), and two nuclear genes (28S ribsomal RNA (28S) and the internal transcribed spacer 2 (ITS2)). These data show large genetic distances between the three subspecies of C. oculatus of 7-12%. Given these distances are similar to those between other species in the genus, this indicates these subspecies may be elevated to full species. The data also consistently support a monophyletic relationship between C. o. oculatus and C. o. gilloglyi. Nuclear genes also support C. o. cheesmani as part of a clade with the other subspecies, but these relationships are unresolved in COI. Carpophilus maculatus was not supported as being the sister taxon of the C. o. oculatus and C. o. gilloglyi clade. Other relationships within Carpophilus were unresolved, possibly due to a combination of incomplete taxon sampling, and saturation of substitutions within the COI gene. Phylogeographic analysis of specimens collected from several localities within the range of C. oculatus showed that, with only one exception, there were no shared haplotypes between archipelagoes. This result suggests it may be possible to determine the provenence of intercepted specimens, providing further information regarding potential invasion pathways. A degree of geographic structuring was also present within C. o. gilloglyi, being separated into a western clade found in Fiji and Rotuma and an eastern clade distributed from the Kermadec Islands and Tonga to French Polynesia. This separation was most profound in COI data, with a mean pairwise distance between the clades of 7%. ITS2 data also demonstrates a degree of differentiation between the two clades, based on differences in the insertions and deletions between the clades. The variability in the shape and colour of the elytral pattern of C. oculatus was also investigated. Colour was quantified using a method based on Red-Green-Blue (RGB) colour values derived from digital photographs, while an outline analysis of the elytral pattern was conducted using elliptic Fourier analysis (EFA). Principal Components Analysis of the RGB values and EFA coefficients showed no clear separation between subspecies, nor were any trends correlated with host fruit or collection localities. Variation at all levels and all measures studied in this thesis show that this geographic region and this genus of beetles offer intruiging insights into speciation, biogeography and biological invasions. There is much scope for further research on the causes and consequences of this variation and the lives of these interesting insects.

The fungal endophyte Epichloë festucae forms a largely mutualistic association with the ryegrass species Lolium perenne. E. festucae produces a range of bio-protective alkaloids that protect the host grass from herbivory by both mammals and insects. One such alkaloid, Lolitrem B, is a potent mycotoxin and the causative agent of ryegrass staggers in livestock. Ten genes required for biosynthesis of lolitrem B are encoded in the ltm gene cluster. The ltm genes are expressed in a plant-specific manner, with high levels of expression in planta and very low levels of expression in culture. The mechanism regulating ltm gene expression is unknown but it is predicted to involve signalling from the host plant. The ltmM gene was chosen for use in the investigation of ltm gene regulation because the flanking regions do not contain retrotransposon sequence, which surrounds much of the ltm gene cluster. To identify fungal genes involved in the plant-induced expression of ltmM, a mutagenesis and screening system was developed using a PltmM-gusA ‘knock-in’ construct to detect expression from the ltmM promoter. Agrobacterium tumefaciens-mediated T-DNA mutagenesis was used to create a set of mutants with random insertions in the genome. Mutants were then screened for altered PltmM-gusA expression, both in culture and in planta. Three mutants were identified with increased PltmM-gusA expression in culture, however, no mutants were identified with loss of PltmM-gusA expression in planta. This indicates that a mechanism of repression is involved in the plant-induced expression of ltmM, either directly or indirectly. TM mutants of interest were also observed for altered symbiosis phenotypes. Mutants were identified with reduced colonisation rates and altered hyphal growth in planta. Integration sites were identified for two colonisation mutants and the disrupted genes are predicted to be the CTP:cholinephosphate cytidylyltransferase (CCT) gene PCT1 and the mitogen-activated protein kinsase kinase (MAPKK) gene mkk2.

This thesis is presented as a collection of research papers synthesising knowledge gained during the period of candidacy. Its underlying focus is the examination of evolution from a variety of perspectives for terrestrial arthropods (springtails) in an Antarctic setting. These perspectives include investigation of the ways in which springtail populations respond both physiologically and genetically to environmental variability over historical and contemporary time-scales. While the physiological and genetic may seem two worlds apart, this thesis recognises that, in reality the two are inextricably linked. Thus, when genetic differentiation between populations of the same species can be demonstrated, physiological differentiation of these populations may also be predicted (and vice versa). Therefore, across several locations and springtail species, physiological and genetic parameters of individuals and populations are examined both separately and, where possible, in concert. The physiological aspect of this thesis focuses on the springtail Gomphiocephalus hodgsoni from continental Antarctica. In addition to providing the first metabolic rate data for a continental Antarctic springtail, seasonal variation in metabolic rates is examined across multiple temporal and spatial scales to evaluate the ways in which individuals and populations respond to environmental variability. Metabolic activity in this species is intricately linked to a variety of factors, both intrinsic and extrinsic. These include biological function, temperature profiles in the local microclimate, and body mass and genetic differences among populations. In the genetically-focused aspect of this thesis, population genetic patterns of G. hodgsoni from several continental locations and Cryptopygus antarcticus antarcticus from locations across the Antarctica Peninsula are compared. Here, the importance of differing evolutionary histories in influencing patterns of contemporary genetic population structure is highlighted. While both species have been similarly affected genetically by Pleistocene (2 Ma – present) glacial cycling, it is clear that differences in timing of colonisation events and subsequent population expansions have left distinct genetic signatures in each species. In a separate molecular study, phylogenetic analyses are employed to study members of the circum-Antarctic springtail family Isotomidae. Thesis Abstract The genetic ancestry among these closely related species is shown to reflect a diverse evolutionary origin in the Miocene (23 – 5 Ma), subsequent to which both vicariant and dispersal processes have been important. Phylogenetic re-constructions tease out the relationships among sister species, and the identification of several genetically distant lineages suggests that a revision of current species designations is required. Finally, two studies that integrate the physiological and molecular genetic are presented. First, metabolic rate variation across several locations on sub-Antarctic Marion Island in the springtail Cryptopygus antarcticus travei is examined. This variation is related to the genetic structure of populations to show that historical and contemporary environmental characteristics have left their trace in the expression of both genetic and physiological variability of these populations. Second, the perceived association between metabolic rate and genetic (mutation) rate is investigated more closely - a sophisticated Bayesian correlation analysis detects that there is an indirect relationship between metabolic rate and underlying species phylogeny in C. a. travei. Thus, the physiological and molecular genetic elements of this thesis test or advance important hypotheses within their own fields, and the integrated approach applied is a new step in interpreting evidence of physiological adaptation in Antarctic species. In its multi-faceted approach to evolutionary studies, this thesis enhances understanding of the current picture of springtail evolution in polar environments.

Two main themes to the avian research presented in this thesis are, 1. Deep resolution of birds generally, and 2. Investigation of specific aspects of the New Zealand avifauna. More specifically, this thesis covers phylogeny, and predictions about palaeognaths, pigeons, pelecaniforms and passerines. Significant progress is made in resolving the basal branches of Neoaves. This thesis examines whether the six-way basal Neoavian split of Cracraft (2001) is, in principle, resolvable. New mitochondrial genomes are added to improve taxon sampling, break up long branches, and allow testing of the prior assumptions of six Neoavian groups. This research shows the six-way split is resolvable, although more work is required for specific details. From a life-history perspective, it is interesting that the two bird-of-prey groups (falcons and buzzards) are very divergent, and may not be sister groups. Molecular dating supports major diversification of at least 12 Neoavian lineages in the Late Cretaceous. Additionally, novel avian mitochondrial gene orders are investigated and a hypothesis put forward suggesting gene conversion and stable intermediate forms allows an apparently rare event (gene rearrangement) to occur multiple times within Neoaves. One of Cracraft’s six groups, informally called the ‘Conglomerati’, is particularly difficult to resolve. The pigeons (Columbiformes) lie within the ‘Conglomerati’, and this chapter examines two aspects along the continuum of pigeon evolution. Firstly the large South Pacific fruit pigeon radiation is examined with mid-length mitochondrial sequences. This clade contains a third of all pigeon species, and has been very successful in island colonisation throughout South East Asia and the Pacific. Secondly, candidates for the closest relative of pigeons are tested using analysis of whole mitochondrial genomes. Highest support was found for the grouping of sandgrouse and pigeon, although they are clearly very divergent. Also within the ‘Conglomerati’ is the traditional order Pelecaniformes, and their close allies the Ciconiiformes. These orders (the P&C) are part of an adaptive radiation of seabird water-carnivores, including loons, penguins, petrels and albatrosses. This group is separate from the large shorebird water-carnivore group; although both appear to have begun radiating abut 70 million years ago. The tropicbird represents a separate, convergent life history and is not part of the Pelecaniformes, nor within the larger seabird water-carnivore group. Resolution of the basal phylogeny of oscine passerines is important for interpreting the radiation of this group out of the Australasian region. Many endemic New Zealand oscine passerines belong to ‘basal corvid’ lineages, but have not previously been investigated with mitochondrial DNA. This chapter shows that many ‘basal corvid’ lineages are actually ‘basal passerine’ lineages, and there is a discrepancy between nuclear Rag-1 phylogenies (the most commonly used gene in passerine phylogenetics) and other phylogenies, including mitochondrial, that requires further investigation. Taken as a whole, this thesis adds significantly to our understanding of the evolution of birds, and provides a foundation for future research, not only of phylogenetic relationships, but also of avian life history, long-term niche stability and macroevolution.

Dothistromin (DOTH) is a secondary metabolite produced by the fungal peanut pathogen Passalora arachidicola and pine needle pathogen Dothistroma septosporum. The chemical structure of DOTH is similar to a precursor of aflatoxin (AF) and sterigmatocystin (ST), which are secondary metabolites produced by Aspergillus species. A size fractionated genomic library was made and 11 putative DOTH genes were identified in P. arachidicola. The DOTH genes in P. arachidicola were compared to DOTH genes in D. septosporum as well as to AF and ST genes in Aspergillus species. The DOTH gene products in P. arachidicola showed 73 - 96% amino acid identity to DOTH genes in D. septosporum and 50 - 69% amino acid identity to AF/ST genes in Aspergillus. The DOTH biosynthesis genes in P. arachidicola had similar gene organization and direction of transcription to DOTH biosynthesis genes in D. septosporum and is similar in that 11 putative DOTH genes are separated into three mini-clusters. This differs from the AF/ST clusters in which 25 AF/ST genes are tightly clustered in a 70 kb region. Identification of transcription factor binding sites upstream of DOTH genes in P. arachidicola and D. septosporum suggested similar co-regulation of DOTH gene expression in P. arachidicola and D. septosporum. Tandem and inverted repeat sequences were identified in intergenic regions in the P. arachidicola DOTH gene cluster, but the distribution of those repeats appears to be random. This suggests that the fragmentation of the DOTH biosynthesis gene cluster is not due to retrotransposon activity or recombination between repeat sequences. The DOTH biosynthesis gene clusters in P. arachidicola and D. septosporum could be ancestral to AF/ST biosynthesis clusters in Aspergillus species.