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    Reciprocally formed Tragopogon allopolyploids and their diploid parents : a comparative study : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Plant Biology, School of Natural Sciences, Massey University, Palmerston North. EMBARGOED to 14 March 2027.
    (Massey University , 2025-02-28) Mukhtar, Usama
    Allopolyploidy has been a significant evolutionary force across the eukaryotic tree of life, particularly in plants. Newly formed polyploids inherit traits from their progenitors but may also show transgressive characters that allow them to inhabit different areas and/or outcompete their parents in similar habitats. In this thesis, multiple approaches were used to study differences between reciprocally formed allopolyploids (Tragopogon miscellus) and their diploid parents (T. dubius and T. pratensis) in the genus Tragopogon. This system was chosen because the parentage of the allopolyploids is known and the polyploids were recently (within the last 100 years) formed. These four species were analysed for: growth parameters under variable temperature and water conditions; physiology and cellular characteristics; and variations in plastid genomes. Both reciprocally formed polyploids were found to have different growth profiles from each other, with short-liguled Tragopogon miscellus being potentially more robust. Leaf physiology revealed T. dubius had low water use efficiency, but a higher transpiration capacity than the other diploid T. pratensis and the polyploids. Comparison of whole plastid genomes revealed variations in both DNA sequence and base modifications, including methylation patterns, among the four species. Collectively, these results help further our understanding of phenotypic and genotypic evolution in young allopolyploids.
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    Gene duplication fate in a genetic pathway context : an examination of the trichome initiation pathway in the allopolyploid genus Pachycladon (Brassicaceae) : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Plant Biology at Massey University, Manawatu, New Zealand
    (Massey University, 2024-10-29) Warusawithana, Thilini
    The evolutionary significance of polyploidy is difficult to overstate and one of the primary foci of modern polyploidy research is on the fates of duplicated genes (homeologs). The impact of gene duplication on biological evolution is strongly related to evolutionary novelty, genome architecture, and even speciation. However, current approaches to study homeolog fates are largely descriptive (e.g., whole genome scans) and lack a critical context: the genetic pathway. As a result, relatively little is known about how homeologs interactively behave within a duplicated genetic pathway to form protein-protein interactions and, how homeolog functional fates are associated with particular gene attributes such as pleiotropy and epistasis. We examined the well-characterized trichome initiation pathway in the allopolyploid Pachycladon (Brassicaceae) in this study to investigate how integrated genetic systems respond to the common phenomenon of duplication. The genus is the product of a hybridization event that took place during the Pleistocene ~0.8-1.6 mya, between two highly diverged lineages, followed by polyploidy and diversification. Considering morphological and phylogenetic diversity, four Pachycladon species, P. enysii, P. fastigiatum, P. novae-zealandiae and P. cheesemanii were selected to study the trichome initiation pathway focusing on the key genes TTG1, GL1, GL3, and EGL3. Pachycladon’s parental origins are not known exactly yet, despite that previous studies describe that one of the parental genomes is closely related to A. thaliana. Hence, as the first step, we set out to isolate the duplicated copies (homeologs) of the four genes of interest from each of the four study species. Those sequences were then analyzed to identify the progenitor origins of each homeolog for each gene to ‘assemble’ the progenitor pathways. The gene trees resulting from phylogenetic analyses were consistent in the placements of homeologs for each gene, positioning one of the copies (Type 1) in the Camelineae lineage (lineage I) and other copy (Type 2) outside of (and typically sister to) Lineage I. Categorization of the homeologs of each gene into progenitor Types facilitated the analysis of sequence divergence and expression studies to provide more insights into the molecular evolution of the pathway. The Type 2 copies of all genes were found to have greater variation than the Type 1 copies. Despite several mutations with likely functional effects being identified, Ka/Ks analyses suggested that both copies of most genes in most species have been under the influence of purifying selection. qPCR analyses of the relative expression of gene homeologs suggested similar gene expression patterns in four Pachycladon species and across two sample types, whole seedlings, and mature leaves. Based on the results, we assumed multiple molecular mechanisms overlap controlling the expression of the homeologs such as relative gene dosage balance, subgenome dominance, and pleiotropy. The most general finding was a trend showing preferential expression of Type 1 homeologs observed across genes and species (although there were exceptions to this pattern). Based on other studies, the Type 1 homeologs are likely to be of paternal origin, indicating a paternal bias in homeolog expression. In contrast to the other genes examined, the highly pleiotropic gene, TTG1, was found to have very similar levels of homeolog expression across all species. These results provide a detailed analysis of the molecular evolution of a genetic pathway following duplication via allopolyploidy.
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    Evolution of cytonuclear coordination in Tragopogon (Asteraceae) allopolyploids : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Plant Biology at Massey University, Manawatu, New Zealand
    (Massey University, 2023) Hussain, Sidra
    Cytonuclear coordination is an important aspect in plant evolution which involves the synchronized interactions between nuclear and organellar genomes. Allopolyploidy, resulting from interspecific hybridization and genome duplication, could result in cytonuclear incompatibilities. Therefore, to coordinate cytonuclear interactions, allopolyploids may undergo alterations in duplicated nuclear gene expression via incorporating maternally biased expression patterns. To investigate cytonuclear responses to allopolyploidy, in this study, expression patterns of duplicated nuclear genes and their organelle counterparts, implicated in cytonuclear enzyme complexes, as well as dual-targeted genes were investigated in the reciprocally formed young T. miscellus allopolyploids (90-100 years) and diploid parent species; T. dubius and T. pratensis. In addition, the effect of polyploidy on morphological traits of T. miscellus allopolyploids were examined and assessed relative to parent species. The expression data showed that T. miscellus allopolyploids are regulating expression at the homeolog level, primarily through T. pratensis bias, while maintaining the total gene expression levels as to parental levels. The morphological evaluation of allopolyploids and diploids demonstrated that both reciprocal forms of T. miscellus have significantly longer, but fewer leaves compared to the diploid parent species. These findings reflect that young T. miscellus allopolyploids are certainly undergoing homeolog expression regulation to accommodate cytonuclear interactions as well as displaying morphological responses to allopolyploidy. This study provides insights into polyploid genome evolution and contributes to further understanding of the cytonuclear coordination in allopolyploids.
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    Phylogenomics and evolution of polyploid Azorella (Apiaceae) in New Zealand : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Plant Biology at Massey University, Manawatū, New Zealand
    (Massey University, 2023) Ning, Weixuan
    Polyploid plants have more than the usual two sets of chromosomes in every cell. Analysing the macroevolutionary patterns of polyploid plants can provide further insight into the mechanisms of polyploidization or whole genome duplication (WGD) in driving species diversification. The polyploid-rich lineage, Azorella, in New Zealand (NZ) has two sections, Schizeilema and Stilbocarpa, with a total of 17 described polyploid taxa (species, subspecies, or varieties) in three known ploidy levels (4x, 6x and 10x). The divergent leaf morphologies and distinct distribution range of polyploid taxa in NZ Azorella makes this lineage an ideal system to investigate the macroevolutionary outcomes of WGD in a polyploid-rich lineage. This thesis aimed to 1) resolve the origins and species relationships of NZ Azorella using phylogenetic inference, and 2) compare the polyploidy-associated genomic, morphological, and ecological traits to understand the post-WGD diversification of Azorella polyploids. In this thesis (Chapter 1), I first reviewed the current phylogenomic approaches for resolving species relationships in groups that have complex evolutionary histories, including polyploidization and reticulation. To resolve the NZ Azorella phylogenetic relationships (Chapter 2), I applied Hyb-Seq of the Angiosperms353 bait set via Illumina sequencing to amplify 353 target-enriched single copy nuclear genes. Additionally, nrDNA and whole chloroplast DNA were recovered via genome-skimming reads to represent high copy genes/regions that are traditionally used in phylogenetics. Hyb-Seq of Angiosperms353 loci was combined with a PacBio sequencing run to improve homeologous gene extraction (Chapter 3). Finally, NZ Azorella post-polyploidization diversification patterns (Chapter 3) were assessed using the variation in genome sizes (via flow cytometry), stomatal guard cell length (using scanning electron microscopy), and ecological niches (using the R package ENMTools). Overall, from biogeographical analyses, I found two independent dispersal events of species in New Zealand Azorella sections Stilbocarpa and Schizeilema, respectively. Using the concordance factors among gene trees and single nucleotide polymorphisms from Hyb-Seq data, as well as the topological incongruence between single copy and high copy gene trees, the results indicated hybrid origins of several hexaploid (6x) species, reticulate relationships among tetraploids, and an allopolyploid origin of the 10x species A. colensoi. Furthermore, different post-polyploidization diversification patterns were compared among Azorella taxa in different ploidy levels, which showed that phylogenetic relationships (i.e., genome content), reticulate evolutionary histories, genomic modification processes (i.e., expansion or contraction), niche shifts, and the age of the polyploid species are all important factors to predict the macroevolutionary patterns of polyploid species.
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    Identification and characterisation of an enzyme from Monoglobus pectinilyticus associated with degradation of pectin from kiwiberry : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biological Sciences at Massey University, Palmerston North, New Zealand
    (Massey University, 2022) Lewis, Aymee
    Pectin is a complex polysaccharide and a very important source of dietary soluble fibre, present in a variety of fruit and vegetables. Pectin possesses a wide range of contributions to a healthy diet and a healthy human gut, such as lowering cholesterol, protecting against intestinal inflammation and maintaining digestive health. Yet pectin is unable to be degraded by human gastrointestinal enzymes, arriving in the large intestine mostly intact. Limited research has showed the domination of Gram-negative bacteria which possess a range of secreted extracellular cell-bound and cell-free pectin degrading enzymes. These enzymes attack the pectin backbone and the accessory side chains resulting in the isolation of mono/oligosaccharides for subsequent uptake. The recent novel discovery of Monoglobus pectinilyticus, a Gram-positive bacterium, expanded the narrow knowledge regarding specific pectinolytic degraders. Genomic studies showed this bacterium contains an arsenal of enzymes specifically for pectin degradation. However, several putative pectin-degrading enzymes produced by M. pectinilyticus were unable to be identified against up-to-date databases, thus suggesting that there may be potentially novel classes of CAZyme(s) to be discovered. The proposed study will attempt to identify such enzyme(s) using New Zealand cultivar kiwiberries as the source of pectin. The initial aim of this study was to investigate the enzyme(s) involved in β-1,4-galactan by growing M. pectinilyticus with a mixture of pectin substrates (0.5% citrus pectin, 0.5% kiwiberry pectin and 0.5% potato galactan), in hope to identify such enzyme(s). Unfortunately, no β-galactosidase/β-galactanase was found. However a potentially novel binding enzyme, protein 0050 was isolated, cloned and expressed, which may be involved in a pectin degrading version of a cellulosome complex. Further research into isolating a variety of secreted proteins activated in presence of galactan during M. pectinilyticus cultivation is needed to understand the complexity of pectin degradation.
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    Detection of loci associated with water-soluble carbohydrate accumulation and environmental adaptation in white clover (Trifolium repens L.) : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Plant Biology at Massey University, Palmerston North, New Zealand
    (Massey University, 2021) Pearson, Sofie
    White clover (Trifolium repens L.) is an economically important forage legume in New Zealand/Aotearoa (NZ). It provides quality forage and a source of bioavailable nitrogen fixed through symbiosis with soil Rhizobium bacteria. This thesis investigated the genetic basis of two traits of significant agronomic interest in white clover. These were foliar water-soluble carbohydrate (WSC) accumulation and soil moisture deficit (SMD) tolerance. Previously generated divergent WSC lines of white clover were characterised for foliar WSC and leaf size. Significant (p < 0.05) divergence in foliar WSC content was observed between five breeding pools. Little correlation was observed between WSC and leaf size, indicating that breeding for increased WSC content could be achieved in large and small leaf size classes of white clover in as few as 2 – 3 generations. Genotyping by sequencing (GBS) data were obtained for 1,113 white clover individuals (approximately 47 individuals from each of 24 populations). Population structure was assessed using discriminant analysis of principal components (DAPC) and individuals were assigned to 11 genetic clusters. Divergent selection created a structure that differentiated high and low WSC populations. Outlier detection methodologies using PCAdapt, BayeScan and KGD-FST applied to the GBS data identified 33 SNPs in diverse gene families that discriminated high and low WSC populations. One SNP associated with the starch biosynthesis gene, glgC was identified in a genome-wide association study (GWAS) of 605 white clover individuals. Transcriptome and proteome analyses also provided evidence to suggest that high WSC levels in different breeding pools were achieved through sorting of allelic variants of carbohydrate metabolism pathway genes. Transcriptome and proteome analyses suggested 14 gene models from seven carbohydrate gene families (glgC, WAXY, glgA, glgB, BAM, AMY and ISA3) had responded to artificial selection. Patterns of SNP variation in the AMY, glgC and WAXY gene families separated low and high WSC individuals. Allelic variants in these gene families represent potential targets for assisted breeding of high WSC levels. Overall, multiple lines of evidence corroborate the importance of glgC for increasing foliar WSC accumulation in white clover. Soil moisture deficit (SMD) tolerance was investigated in naturalised populations of white clover collected from 17 sites representing contrasting SMD across the South Island/Te Waipounamu of NZ. Weak genetic differentiation of populations was detected in analyses of GBS data, with three genetic clusters identified by ADMIXTURE. Outlier detection and environmental association analyses identified 64 SNPs significantly (p < 0.05) associated with environmental variation. Mapping of these SNPs to the white clover reference genome, together with gene ontology analyses, suggested some SNPs were associated with genes involved in carbohydrate metabolism and root morphology. A common set of allelic variants in a subset of the populations from high SMD environments may also identify targets for selective breeding, but this variation needs further investigation.
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    Pachycladon species evolved traits to adapt to New Zealand habitats : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Plant Biology at Massey University, Manawatū, New Zealand
    (Massey University, 2021) Dong, Yanni
    P. cheesemanii is a close relative of A. thaliana and is an allotetraploid perennial herb that is widespread in the South Island of New Zealand. It grows at altitudes of up to 1,000 m where it is subject to relatively high levels of UV-B radiation. However, to date the origin of this species and the mechanisms underlying its tolerance to its harsh living environmental conditions such as moderate–high UV-B radiation, cold and drought is unclear. To gain the first insights into how Pachycladon copes with UV-B stress, I sequenced the P. cheesemanii genome and compared the UV-B tolerance of plants from Wye Creek (~300-m altitude) and Kingston (~500-m altitude) with that of A. thaliana from Col-0 (~100-m altitude) and Kondara (1,000–1,100-m altitude). A high-quality draft genome of P. cheesemanii was assembled with a high percentage of conserved single-copy plant orthologues. A synteny analysis involving genomes from other species of the Brassicaceae family suggested that the two subgenomes of P. cheesemanii may have the same origin as species from Brassicaceae Lineage I and EII. While UV-B radiation caused greater growth reduction in A. thaliana Col-0 and Kondara than in P. cheesemanii Wye Creek, growth was not reduced in P. cheesemanii Kingston. Homologues of the A. thaliana UV-B radiation response genes have multiple copies in P. cheesemanii, and an expression analysis of those genes indicated that the tolerance mechanism in P. cheesemanii Wye Creek and Kingston may differ from that in A. thaliana. Although the P. cheesemanii genome shows close similarity with that of A. thaliana, the uniqueness of the strongly UV-B-induced UVR8-independent pathway in P. cheesemanii may help this species to tolerate relatively high UV-B radiation. Next, to understand the different stress responses of A. thaliana and P. cheesemanii, I designed a project to build multiple-stress transcriptomes for A. thaliana and P. cheesemanii. Since plant responses to salt and drought are related and have overlapping mechanisms, and salt stress can easily be applied in the laboratory, high salinity rather than drought stress was used to stress A. thaliana and P. cheesemanii plants in this study. Transcriptomes of A. thaliana and P. cheesemanii plants in response to cold, salt and UV-B radiation stresses were created. A high-quality de novo transcriptome assembly of allopolyploid P. cheesemanii was obtained by using multiple assemblers with further downstream processing. Differential expression analysis revealed a strong bias, in terms of the number of DEGs, towards upregulation in both A. thaliana and P. cheesemanii in responding to salt stress, as well as in P. cheesemanii’s cold and UV-B treatment responses. Meanwhile, in each species, a number of DEGs was shared between stresses, although the majority were unique in responding to each stress in upregulation and downregulation, respectively. Further, GO enrichment analysis revealed that these responsive genes were involved in some biological processes shared by A. thaliana and P. cheesemanii. Immune system processes, response to stimuli, signalling, developmental processes, growth, negative regulation of biological processes, multi-organism processes, biological regulation, secondary metabolic processes, cell communication, and cellular aromatic compound metabolic processes were common in the responses of both A. thaliana and P. cheesemanii to all three stresses. In both A. thaliana and P. cheesemanii, a number of these biological processes were also stress specific. First of all, in A. thaliana, cold stress may easily affect photomorphogenesis in cold responses, while the majority of the P. cheesemanii unique cold responses occurred in root differentiation, floral whorl development and regulation of programmed cell death. Second, A. thaliana responses to salt stress affected starch metabolism and lipid modification, whereas disaccharide and polysaccharide metabolism, as well as microtubule structure, were affected by salt stress uniquely in P. cheesemanii. Finally, A. thaliana responses to UV-B radiation involved a combination of physical and biological defences, including cell wall modification defence, stomatal movement, vitamin B6 metabolic processes and oxygen metabolic processes. In contrast, seed germination biological regulation was affected in P. cheesemanii under UV-B radiation stress. Further, P. cheesemanii had a larger number of unique GO enrichments in cold responses than did A. thaliana. There was a wide range of crosstalk among the biological processes in responding to the three stresses in A. thaliana, while only one main cluster was identified in crosstalk for the three stress responses in P. cheesemanii. In this main cluster, the biosynthetic process for anthocyanins was in the centre position, and it was found that multiple stress-responsive biological processes probably involved anthocyanins in P. cheesemanii. Thus, although the P. cheesemanii genome shows close similarity with that of A. thaliana, it appears to have evolved novel strategies such as a highly UV-B-activated UVR8-independent pathway, allowing the plant to tolerate relatively high UV-B radiation. The stress process is highly conserved in plant species under various stresses, but species also develop a few unique characteristics that may help them adapt to their own ecological niche and survive particular environmental stresses.
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    Chloroplast genome evolution in New Zealand mycoheterotrophic Orchidaceae : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Plant Biology at Massey University, Manawatu, New Zealand
    (Massey University, 2019) Murray, Katherine Jane Hope
    The plastid genomes, or plastomes, of most photosynthetic land plants are highly similar. In contrast, those of non-photosynthetic, heterotrophic land plants are often reduced in both size and gene content. The apparent degradation of mycoheterotrophic plant plastomes has been attributed to a functionally-driven stepwise pattern of loss. However, the number of complete plastome sequences available for mycoheterotrophic plants is small and taxonomic coverage is biased. In this thesis, the plastomes of two mycoheterotrophic orchid species endemic to New Zealand, Corybas cryptanthus Hatch (Diurideae) and Danhatchia australis Garay & Christenson (Goodyerinae), as well as those of an albino and several photosynthetic representatives of Corybas are reported. Beyond increasing the number of mycoheterotrophic plastomes available for evaluating broad hypotheses about plastome evolution in non-photosynthetic plants, these data also provide insights into two little studied aspects of plastome evolution in mycoheterotrophs; intraspecific variation in the plastomes of mycoheterotrophs and the differences between mycoheterotrophs and their closest photosynthetic relatives. The plastomes of C. cryptanthus and D. australis differ in the extent to which they are degraded. Perhaps unexpectedly, the plastome of C. cryptanthus, which has close photosynthetic relatives and therefore is likely to have arisen more recently than the taxonomically isolated D. australis, is more reduced. Specifically, the plastomes of C. cryptanthus are approximately half the size and have half the gene content of the other Corybas sequenced whereas the plastome of D. australis is similar to those available for photosynthetic relatives. This contrast may reflect underlying differences between the two genera; the photosynthetic relatives of D. australis have plastomes containing NADH dehydrogenase (ndh) genes whereas those of photosynthetic Corybas have lost their ndh genes and their small single copy regions are highly reduced. These features may have predisposed the ancestor of C. cryptanthus to rapid genome degradation. Finally, observations on these results strongly suggest that plastome degradation follows, rather than precedes, the shift to mycoheterotrophy.
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    The genetic architecture of the divaricate growth form : a QTL mapping approach in Sophora (Fabaceae) : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Plant Biology at Massey University, Manawatu, New Zealand
    (Massey University, 2019) Pilkington, Kay
    Divarication is a plant growth form described, in its simplest form, as a tree or shrub with interlaced branches, wide branch angles and small, widely spaced, leaves giving the appearance of a densely tangled shrub. This growth form is a unique feature in the New Zealand flora that is present in ~ 10% of the woody plant species, a much higher frequency than that of other regional floras. While several hypotheses have been developed to explain why this growth form has evolved multiple times within New Zealand, to our knowledge, no work has addressed the genetic basis of the divaricating form. Sophora is one of several genera in New Zealand that possesses divaricate species. Among the factors making this an ideal system for a genetic investigation of divarication is an existing F₂ population formed from reciprocal crosses between the divaricating S. prostrata and the non-divaricating S. tetraptera. Using this segregating population and newly developed molecular markers, the first linkage maps for Sophora were generated, providing a new genetic resource in Sophora. These linkage maps allowed for quantitative trait locus (QTL) mapping for traits associated with the divaricate form in the segregating population. Multiple QTL were mapped to seven of the divaricate traits with many QTL co-locating for multiple traits, indicating that the divaricate growth form is genetically controlled by many loci, potentially including pleiotropic loci, that each contribute to the overall divaricate phenotype in Sophora. The strigolactone biosynthesis and perception pathway is a good candidate for involvement in control of the divaricate form based on mutant phenotypes in Pisum that display similarities to the divaricate growth form, such as increased branching, shorter plant height and smaller leaves. QTL, for multiple traits, were mapped to two candidate genes investigated, RMS1 and RMS4. An amino acid replacement was identified in RMS1, in S. prostrata, that is predicted to be deleterious suggesting it may be non-functional in S. prostrata. These results support RMS1 as a strong candidate gene for future work on divarication. This study is the first to investigate the genetic architecture of the divaricate growth form and contributes to further understanding of this unique feature in the New Zealand flora and of plant architecture generally.