The establishment of a Living Genome Resource Bank utilising genetic analysis and assisted breeding technology: a case study on the koala (Phascolarctos cinereus)

Abstract

As species become progressively more threatened and nearer extinction, innovative conservation strategies must be designed to reduce the effects of deterministic and stochastic factors that contribute to their demise. One such conservation strategy is the use of ex situ populations within a captive or closely managed environment, which can be used to create a reservoir of genetic diversity that serves as a method of genetic exchange and maintenance. Although ex situ conservation can involve a frozen, or ‘suspended’, reservoir of gametes or embryos, this is not yet technically feasible for most wildlife species; therefore, an ex situ bank of living animals can be established as a living reservoir of genetic diversity. This concept has been termed a ‘Living Genome Resource Bank’ (LGRB) and aims to create an easily accessible repository of genetic material that is intended to bridge the gap between geographically isolated in situ and ex situ populations of a species. When possible, assisted breeding technologies can be used to further support genetic exchange between isolated colonies of animals. The research outlined in this Thesis explored the concept and underlying information required to establish a functional LGRB for the koala (Phascolarctos cinereus). A comprehensive genetic analysis of two in situ populations (Gold Coast, n= 94 and Moreton Bay, n= 49) and one ex situ koala colony (captive, n= 37) in South-East Queensland, Australia, was performed using two different molecular markers (single nucleotide polymorphisms and microsatellites) to determine the genetic variation and divergence of the koala populations analysed. DdRADseq identified 6,433 SNP loci in the koalas and analysis showed divergence between each population, with further subdivision and divergence of the Gold Coast population. There was within-population Bayesian clustering in each of the three populations. Population differentiation (FST analysis) showed greater divergence among the two wild populations (FST = 0.045) than between the wild and captive populations (FST = 0.022). Outlier analysis identified 42 SNP loci that were likely under selection. Of these, the surrounding sequence of 19 loci matched sequences in the koala genome and could be further identified at a protein-level. Five different biological pathways were associated with these outlier loci, including; environmental information processes, organismal systems, disease, metabolism and cellular processes. These alleles are potentially adaptive in koalas and warrant further investigation. Heterozygosity fitness correlations were calculated to indicate any decrease in population and individual fitness associated with inbreeding and inbreeding depression. Based on SNP data, the average observed heterozygosity for the captive, GC and MB populations were 0.31, 0.28 and 0.25, respectively. Microsatellite data on the Captive and GC populations calculated an average observed heterozygosity of 0.64 and 0.70, respectively. Based on SNP data, FIS of the captive, GC and MB populations were calculated as 0.20, 0.26 and 0.24, respectively. Microsatellite analysis of the captive and GC populations determined an FIS of 0.018 and 0.173, respectively. Heterozygosity was correlated with fitness, as indicated by body condition, body mass, joey success and sperm function in the captive population. There was also a significant, positive correlation between heterozygosity and skeletal development and resistance to infection with Chlamydia pecorum in the GC koala population.Assisted breeding technologies are an important factor to a LGRB, as they can be used to facilitate the genetic exchange, recovery and connectivity between isolated populations. Empirical research into the recovery and exchange of male koala gametes outlined evidence that koala spermatozoa, left undisturbed and stored at 5 °C, can maintain adequate levels of viability more than 35 days post-ejaculation; this included 40.5% (±7.3) motile, 35.8% (±7.1) live, and a 1.9 (±0.4) rate of motility and 24.7% (±2.2) with high mitochondrial membrane potential. This survival rate is four times longer than any other known mammalian spermatozoa. Further research into the possible mechanism for this longevity identified that koala spermatozoa do not primarily rely on mitochondrial function, anaerobic glycolysis, or the pentose phosphate pathway for energy production. This significant discovery was further supported by evidence that spermatozoa recovered from the epididymis of deceased or diseased koalas can be free of bacterial infection and survive at 5 °C (with no seminal plasma) 10 days post-collection and still maintain viability of progressive motility of up to 30%, rate of 2 (out if 5) for motility, and 51% live + high MMP, at a concentration of 76 x106 per mL.The significant empirical data provided in this thesis can serve as a base-line reference for future genetic and reproductive analyses of the koala, to aid in the management and conservation of the species and provide underlying information required to establish a LGRB for species conservation.