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Item Phylogenetic analysis and population structure of the New Zealand Huntaway and heading dog : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Animal Science at Massey University, Al Rae Centre, Hamilton, New Zealand(Massey University, 2025) Henry, Millicent FrancesThe New Zealand Huntaway and Heading Dog are vital to the success of New Zealand’s livestock farming industry. This study presents one of the first large-scale genomic analyses of these two unique working breeds. Utilising phylogenetic and population structure methodology, the primary objectives of this research were to investigate the genetic relationships of the New Zealand Huntaway and Heading Dog with international dog breeds, formalise the major historical breed contributions that have shaped their development, and assess their population structure and diversity. This study utilised a dataset of 1,735 individuals, representing 129 dog breeds and three wild canid species, and included 211 Huntaways and 246 Heading Dog genomic datasets generated as part of this project. Genomic analyses revealed that both breeds can be traced back to ancestral UK rural working dog populations, occupying positions within the UK rural subclade on the domestic canine phylogeny. Baseline inbreeding scores suggest that neither the New Zealand Huntaway or Heading Dog populations express significantly high inbreeding levels, and the scores obtained are similar to those seen in other formally recognised dog breeds. A list of genes under putative positive selection in both populations is presented and could be used for future genetic selection in these dog populations. This study highlights the Huntaway and Heading Dog as genetically distinct breeds adapted to the unique demands of New Zealand’s pastoral farming systems. These results not only contribute to the current understanding of breed development in geographically isolated environments such as New Zealand but also highlights the opportunity of integrating genomics into working dog management and breeding practices. The genetic resources generated through this study will serve as a valuable reference for future research in the “Right Dog for the Job” project, aimed at preserving the health, diversity and functionality of the New Zealand Huntaway and Heading Dog.Item Genomic characterisation of New Zealand working farm dogs : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Animal Science at Massey University, Manawatū, New Zealand(Massey University, 2025) Smith, FlorenceThe majority of working farm dogs in New Zealand belong to either the Huntaway or Heading Dog breeds. These breeds are unique to New Zealand and are vital to efficient livestock farming, yet no large-scale DNA sequencing studies have been conducted on them to date. The Right Dog for the Job project aims to improve the health and performance of the farm dog population by analysing the genetic and phenotypic information from a sample of 2400 Huntaways and Heading Dogs, including 250 whole genome sequences. As the first use of this dataset, this project aimed to characterise the genetic variation in the sample of whole genome sequences. To this end, sequence bioinformatics tools were used to align sequence reads, then call, filter, and annotate 20 million genetic variants. The dataset was intersected with 395 OMIA-reported functional loci to identify 27 previously described Mendelian variants segregating in the population. Five of these (in the SOD1, VWF, CUBN, CLN8, and SGSH genes) were highlighted as compelling candidates for diagnostic selection. Next, the dataset was surveyed for high impact variants segregating within 132 genes previously shown to harbour phenotype causing variants. The aim here was to use variant effect prediction to identify novel causal variants within functionally relevant genes. This analysis yielded nine causal hypotheses (in the CNGB1, ABCA4, CNP, SLC3A1, CCDC66, GLB1, CYP1A2, and STK36 genes) for future association testing. The second stage of the project leveraged a sample of 299 dogs that were genotyped with the AxiomTM Canine HD Array. Of these dogs, 188 had also been whole genome sequenced. A linkage disequilibrium analysis was conducted to identify predictive markers on the array for several of the 27 Mendelian variants, with a view to supporting future marker-assisted selection. Next, the whole genome sequences were used as a reference to impute missing genotypes in the sample of 111 dogs genotyped with only the Axiom SNP chip, expanding the genomic dataset. Finally, GWAS were performed to identify genetic associations with four body size traits of interest: height, length, chest circumference, and muzzle circumference. Height was significantly associated with a region near LCORL, a gene known to regulate body size in various species. Despite representing one of the first analyses of the ‘Right Dog’ research programme, this project has identified genes and variants that should be of immediate practical use to working dog breeders and owners. The project has also helped to generate an extensive genomic dataset that will underpin future research as part of the broader programme, and ultimately contribute to the health, welfare, and performance of New Zealand’s iconic Huntaway and Heading Dog breeds.Item The dynamics of drug resistance evolution and diagnosis in Mycobacterium tuberculosis : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in Genetics/Genomics, Massey University, Manawatu, New Zealand(Massey University, 2024-07-30) Fong, Yang (Richard)Tuberculosis (TB) remains a critical global health challenge with over 10.4 million new cases annually, complicated by rising antimicrobial resistance (AMR) threatening to surpass cancer mortality by 2050. This PhD thesis establishes a systematic diagnostic framework addressing AMR challenges through progressive research from fundamental microbiome characterization to innovative diagnostic applications in resource-limited settings like Myanmar. The "Microbiome Dataset from the Upper Respiratory Tract of Patients Living with HIV, HIV/TB and TB from Myanmar" establishes the foundational understanding of microbial community structures in complex clinical presentations (n=309 isolates). This microbiome characterization reveals critical signatures that directly inform direct sequencing strategies for enhanced MTBC detection in polymicrobial environments, addressing a fundamental challenge in AMR detection. Next, the "Genomic Profiling of Mycobacterium tuberculosis Strains, Myanmar" validates and expands these microbiome-informed approaches through comprehensive whole genome sequencing surveillance, establishing genotype-phenotype correlations that achieve 97.8% concordance with phenotypic testing. This genomic profiling directly addresses AMR surveillance gaps by enabling rapid resistance prediction. Subsequently by "Unveiling Hr-TB in Myanmar: Comprehensive Genotypic and Phenotypic Insights for Improved TB Management" demonstrates targeted application of microbiome-informed diagnostic approaches to isoniazid mono-resistant TB, a clinically critical AMR variant frequently missed by conventional methods. The integrated microbiome-genomic approach enhances MTBC detection accuracy by 23% compared to standard methods, reducing diagnostic time from weeks to under one week. Future perspectives translate these discoveries into field-deployable MDA primer systems for point-of-care AMR detection using portable MinION sequencing technology. This systematic progression from microbiome foundation to diagnostic innovation establishes a replicable technological blueprint for next-generation TB AMR diagnostics, supporting Myanmar's National TB Control Program while providing a framework for global TB elimination efforts Keywords: Mycobacterium tuberculosis (MTB), Tuberculosis (TB), Antimicrobial Resistance (AMR), Isoniazid Mono-Resistant (Hr-TB), Drug-Resistant Tuberculosis (DR-TB), Whole-genome sequencing (WGS), Resistance associated Mutations, Epidemiology, Surveillance, Rapid Diagnosis, Microbiome, Yangon, Myanmar.Item Genetic and genomic studies of production, composition, and processability characteristics of milk from dairy sheep : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Animal Science at Massey University, Manawatū, New Zealand(Massey University, 2024-10-20) Marshall, Ana CarolinaThe main objective of this study was to perform genetic and genomic studies of sheep milk production, composition, and processability, particularly concerning cheese-making aptitude (milk coagulation properties) of sheep milk. The milk traits were investigated in a flock of 169 lactating ewes producing on a pasture-based system in Masterton, New Zealand. The total yield during the 2021-2022 season was 86.1 kg/ewe in 130 days of milking after the suckling period. The effects of animal factors on the lactation curves, and on milk composition and processability (milk coagulation properties, individual laboratory cheese yield, and heat coagulation time) throughout the season were investigated and discussed. Stage of lactation significantly (p < 0.05) influenced processability. In late lactation, the rennet coagulation time was longer, the curd at 30 minutes after rennet addition was softer, and milk heat stability was lower. In addition, milk protein polymorphisms were shown to influence milk composition and protein composition. Particularly, heat stability was affected by β-lactoglobulin polymorphism. The milk processability traits were also significantly associated with protein composition. The heritability estimates for milk production, composition, and processability traits ranged from 0.12 to 0.48. The genetic correlations obtained indicate that genetic improvement of this flock for higher yields of fat, and protein, and for lower somatic cell score, should indirectly improve milk coagulation traits in this flock. However, other traits such as milk pH, percentage traits (protein, casein, and lactose percentages), ratio of casein to protein, calcium, and ratio of casein to calcium were more strongly correlated with processability. The genome-wide association study performed on 149 dairy sheep genotyped with 50K SNPs Bead Chips, revealed a total of 87 SNPs and 55 candidate genes across Ovis aries autosomes 2, 3, 6, 16, 18, 20, 25, and 26. The genetic architecture of milk coagulation traits was similar to that of the ratio of casein to calcium, pH, lactose, and the ratio of casein to protein. The genetic correlations and identification of potential genes associated with the control of these milk traits provide valuable insights for the selection of superior dairy sheep in New Zealand. The findings of this thesis need to be validated with a systematic large-scale recording scheme before developing a selection index for dairy sheep populations in New Zealand.Item Genome-wide association study for skin thickness and skin temperature traits in FocusPrimeᵀᴹ New Zealand sheep : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Animal Science at Massey University, Manawatū, New Zealand(Massey University, 2024) Hurring, Michelle ErinThis study investigated the genetic basis of skin traits in New Zealand FocusPrime sheep and their implications for lamb survival, with a particular focus on skin thickness and temperature, both of which play critical roles in the thermoregulation and overall survivability of lambs during the early stages of life. The primary objective was to identify genetic markers associated with these skin traits and to understand their heritability and correlation, thereby providing insights that could inform selective breeding programs aimed at improving lamb survival rates. The research employed a comprehensive genome-wide association analysis to analyse a large dataset of New Zealand FocusPrime sheep, meticulously measuring skin thickness and skin temperature, and correlating these phenotypic traits with genotypic data. The study found significant heritability estimates for both skin thickness and skin temperature, suggesting that these traits are genetically controlled and can be effectively targeted in breeding strategies. Specifically, the heritability of skin thickness was found to be high (values that exceed 0.40), indicating that a considerable proportion of the variation in this trait is due to genetic differences among individuals. Similarly, the heritability of skin temperature was also significant, underscoring the potential for genetic improvement through selective breeding. A key finding of this study was the negative correlation between skin thickness and skin temperature. Thicker skin, while beneficial in providing a barrier against environmental stressors, tends to be associated with lower skin temperatures, indicating a complex interplay in the thermoregulatory mechanisms of lambs. This finding highlights the need for a balanced approach in selective breeding, where both traits must be considered to optimize lamb survival. Breeding programs that focus solely on increasing skin thickness may inadvertently affect the thermoregulatory efficiency of lambs, thereby underscoring the importance of a holistic breeding strategy. The genome wide association study identified several significant genetic markers associated with skin thickness and skin temperature, providing valuable targets for future genetic selection. These markers offer a promising avenue for improving lamb survival through genetic means. The study also delves into the practical applications of these findings, suggesting that by incorporating these genetic markers into a breeding program, it is possible to enhance the resilience of lambs against harsh environmental conditions, thereby improving animal welfare and economic outcomes for sheep farmers. This research contributes to a broader understanding of the genetic factors influencing lamb survival, a topic of critical importance given the economic and welfare implications of lamb mortality. The findings have the potential to transform breeding practices in the New Zealand sheep industry, moving towards more scientifically informed strategies that enhance the overall sustainability and profitability of sheep farming. The implications of this research extend beyond New Zealand, offering insights that can be applied to sheep populations globally. Future research should continue to refine the genetic associations identified in this study and explore their practical implementation in diverse sheep populations. Further studies could also investigate the interaction of these genetic markers with environmental factors, providing a more comprehensive understanding of the determinants of lamb survival. By integrating genetic analysis with practical breeding strategies, there is significant potential to reduce lamb mortality rates and improve the resilience of sheep to environmental challenges. In conclusion, this study provides a robust framework for understanding the genetic basis of key skin traits in New Zealand sheep and their impact on lamb survival. The identification of heritable genetic markers associated with skin thickness and skin temperature paves the way for targeted breeding programs that can enhance lamb resilience and reduce mortality rates. This research not only advances scientific knowledge in the field of animal genetics but also offers practical solutions for improving the sustainability and economic viability of the sheep industry in New Zealand and beyond.Item 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, UsamaAllopolyploidy 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.Item Bananas grown in Aotearoa for feeding the infant gut microbiota : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biological Sciences at the College of Sciences, Massey University, Manawatū, New Zealand(Massey University, 2024) Frame, Simone FrancesThe human gut microbiota is established immediately at birth and thereafter is modulated by a range of environmental influences including the nutrients available within breast milk (and microbiota) or formula which support selective colonisation. Upon weaning to solid foods, the infant gut microbiota begins to become more akin to that of an adult in both microbial composition and metabolite production. Bananas are recommended by New Zealand health officials as an appropriate first food, however at present the only commercial variety available is Cavendish, imported largely from Ecuador and the Philippines. Although, in Northland and Gisborne regions other banana varieties are successfully cultivated, they have not been recognised by the commercial sector as valuable produce. Here we identify the nutritional composition of an array of banana cultivars grown in New Zealand, which are hybrids of acuminata (A) and balbisiana (B) species of banana and how these varieties impact SCFA production by the infant gut microbial population. We further identified the distinct shifts in microbial populations between three banana cultivar genome groups represented as AAA, AAB and ABB. Cavendish is AAA. Genomic mapping of unknown banana cultivars provided insight into the diversity of banana cultivars growing in New Zealand, in which the ecophysiology of bananas growing in this climate has presented different phenotypic characteristics to those of countries where banana horticulture is prevalent. These findings will contribute toward particularly, Māori communities building an emerging commercial banana industry and creating a nutritional database for New Zealand grown banana.Item ABAtE : active bacteriophages for AFB eradication : a thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Genetics at Massey University, Auckland, New Zealand(Massey University, 2023) Kok, DanielleThe European honey bee (Apis mellifera) is one of the most important livestock animals in New Zealand. Their value comes from a combination of pollination services and the production of honey for export, notably mānuka honey. American Foulbrood (AFB) is a disease of honey bee larvae and pupae and is caused by the bacterial pathogen, Paenibacillus larvae. AFB is the most serious disease that infects honey bees and is present in almost all countries where honey bees are found. AFB has been present in New Zealand since 1877 and spread to all parts of the country within 10 years. Unlike other countries, the use of antibiotics in hives infected with P. larvae is prohibited under New Zealand law and infected hives must be destroyed immediately. Bacteriophages (phages) are a well-studied alternative to antibiotics. Phages are simple viruses that kill specific bacteria and are highly abundant in the environment with an estimated 1031 globally. Phages have been shown to work effectively as a prophylactic to infection from certain diseases. With the growing antimicrobial resistance crisis, phages are becoming a well-studied and promising alternative to antibiotics. The aim of this research was to investigate the use of phages as a preventative measure against AFB. Previous work undertaken in other laboratories around the world has shown that phages can be isolated from healthy hives and nearby soil and that AFB pathogens are susceptible to destruction by these phages. In this work, we collected soil samples using citizen led science from hives throughout New Zealand. From soil samples provided we isolated 26 novel phages that are destructive to P. larvae. Selected phages were combined into a cocktail and tested against vegetative forms of P. larvae in in-lab testing. All phages were also sequenced and annotated and compared to other P. larvae phages that have been isolated around the world. This project: ABAtE (Active Bacteriophages for AFB Elimination), provides the groundwork study for an innovative approach to naturally protecting NZ beehives against AFB.Item Identification and characterization of effector proteins from pine needle pathogens : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in Genetics at Massey University, Manawatū, New Zealand(Massey University, 2022) Massoco Tarallo, MarianaCollectively, Dothistroma septosporum, Cyclaneusma minus and Phytophthora pluvialis cause serious foliar diseases on Pinus radiata in New Zealand and on many other pine species worldwide. Considering the ecological and economic importance of forest trees, understanding how these pathogens interact with their hosts on a molecular level is critical as it could lead to new and durable approaches to control the diseases they cause. Pathogens have the ability to deliver proteinaceous virulence factors, termed effectors, into the apoplast and cell cytoplasm of their host plants. Effectors typically promote host colonization through suppression of the plant immune system. However, in resistant host plants, one or more of these effectors can be recognized by corresponding immune receptors to activate the plant immune system. Often, one of the main outputs of this immune system is a localised cell death reaction, termed the hypersensitive response (HR), which renders the pathogen unable to cause disease (avirulent). The general goal of this thesis was to identify shared candidate effector (CE) proteins between the three foliar pine pathogens and to characterise their virulence (or avirulence) functions. This is important because disease resistance based on core effectors that are vital for a pathogen’s ability to cause disease is more likely to be durable. Using a combination of “omics” information and bioinformatic tools, two sets of orthologous CE proteins were identified between D. septosporum, C. minus and P. pluvialis, while several other sets were identified between the two fungal pathogens. Some of these CEs had the ability to trigger cell death responses in non-host Nicotiana plants, and some were shown to activate Nicotiana benthamiana genes involved in pathogen-associated molecular pattern-triggered immunity and HR. CEs were also screened in the host, Pn. radiata, using a method developed in this thesis, where it was determined that some of these CEs also trigger cell death. Two conserved cell death elicitor families, Ecp20 and Ecp32, were identified from D. septosporum and its close relative Fulvia fulvum, and the cell death triggered by some family members in N. benthamiana was shown to require membrane-localized receptor-like proteins. Tertiary structure predictions of CEs provided insights into the possible roles and host targets of these proteins during pine infection. Moreover, a shared β-trefoil fold was found between sequence-unrelated CE proteins from the three pine pathogens, along with evidence that they are also present in many other fungal species. A CRISPR/Cas9 gene editing methodology was applied to D. septosporum for the first time, which allowed for the functional characterization of three D. septosporum CE genes, two of which are also present in C. minus and P. pluvialis. Collectively, this thesis provides a significant advance in our understanding of pine-pathogen interactions at the molecular level and provides a blueprint for similar studies in other forest pathosystems.Item DNA barcoding Aotearoa New Zealand’s ray-finned fishes (Actinopterygii) : a reference database and use case : this thesis is completed in partial fulfilment of a Masters of Biological Science Degree, Massey University, New Zealand.(Massey University, 2022) MacLean Stones, MaddieDNA barcode reference databases have been created for the fish biodiversity of many nations, providing a resource to facilitate rapid species identification, biodiversity assessment, and ultimately greater awareness and understanding of freshwater and marine fish fauna. Aotearoa New Zealand (NZ) has a wide diversity of marine, estuarine, and freshwater habitats that comprise a diverse fish fauna, and a high proportion of endemic fish species. Even so, a DNA barcode reference database for the fishes of NZ has not yet been created. In this thesis, I curated a DNA barcode reference database for NZ fishes based on the Cytochrome Oxidase I (COI) gene region using previously published sequences from open-access repositories (i.e., Nucleotide Sequence Database Collaboration, and the Barcode of Life Data System) and novel sequences generated for species not previously sequenced (Chapter 2). To demonstrate the utility of this database, I then provide a use case to genetically identify larval fishes collected off the Northeast Coast of the North Island of NZ and compare these identities to those based on morphology (Chapter 3). To ensure representativeness and integrity of the sequence data within the NZ Fish Barcode Database, I preferentially generated sequences from fish specimens that had been identified by an expert taxonomist or held in museum collections. Furthermore, for widespread species that I did not have sequences for, I sought sequences from specimens collected as geographically close to NZ as possible. In Chapter 2, I was able to generate and retrieve sequences for 965 of the 1320 fish species recorded in NZ (73%); I provide a summary of our progress toward generating a DNA barcode reference database for NZ’s fish biodiversity and report interspecies genetic divergences (based on Kimura-2-pairwise distance) between species. In Chapter 3, the database use case, I found that larvae were often able to be identified to their taxonomic family based on morphological features, but in some cases their taxonomic affinities were unknown. DNA barcoding enabled us to identify the species identity of the larval fishes with reference to the NZ Fish Barcode Database. Overall, our generated DNA barcode reference database is a practical resource of value for future environmental DNA studies, biodiversity monitoring, and managing fisheries and commercial fisheries derived products.
