Characterising MHC diversity in the platypus (Ornithorhynchus anatinus): An approach to species conservation

The Major Histocompatibility Complex is a multigene family that plays a vital role in the innate and adaptive immune response of jawed vertebrates, as an interface between the immune system and infectious agents. Investigation of population diversity at the MHC genes is gaining attention to infer immunological fitness and adaptability of vulnerable populations to a changing environment. Microsatellite markers located in close proximity to MHC loci presents a simple, inexpensive and high throughput method of inferring genetic diversity at the MHC due to tight linkage with specific MHC alleles. The platypus (Ornithorhynchus anatinus) faces an uncertain future from the impact of a variety of potential threats, including climate change, human encroachment causing habitat degradation and the infectious disease, mucormycosis. With platypus populations spread across the eastern and south-eastern Australian mainland and the islands of Tasmania, King Island and Kangaroo Island, the selective maintenance of MHC alleles is thought to vary between populations and characterisation of MHC diversity will prove valuable to current conservation efforts to infer the inherent evolutionary potential and immunological fitness of each surveyed population

Spatiotemporal variations in retrovirus-host interactions among Darwin's finches

Endogenous retroviruses (ERVs) are inherited remnants of retroviruses that colonized host germline over millions of years, providing a sampling of retroviral diversity across time. Here, we utilize the strength of Darwin's finches, a system synonymous with evolutionary studies, for investigating ERV history, revealing recent retrovirus-host interactions in natural populations. By mapping ERV variation across all species of Darwin's finches and comparing with outgroup species, we highlight geographical and historical patterns of retrovirus-host occurrence, utilizing the system for evaluating the extent and timing of retroviral activity in hosts undergoing adaptive radiation and colonization of new environments. We find shared ERVs among all samples indicating retrovirus-host associations pre-dating host speciation, as well as considerable ERV variation across populations of the entire Darwin's finches' radiation. Unexpected ERV variation in finch species on different islands suggests historical changes in gene flow and selection. Non-random distribution of ERVs along and between chromosomes, and across finch species, suggests association between ERV accumulation and the rapid speciation of Darwin's finches.Endogenous retroviruses (ERV) are inherited remains of retroviruses that have colonized host genomes during evolution. Here the authors observe considerable species-specific ERV variation among Darwin's finches, reflecting historic retrovirus-host interactions

Optimizing Staining Protocols for Laser Microdissection of Specific Cell Types from the Testis Including Carcinoma In Situ

Microarray and RT-PCR based methods are important tools for analysis of gene expression; however, in tissues containing many different cells types, such as the testis, characterization of gene expression in specific cell types can be severely hampered by noise from other cells. The laser microdissection technology allows for enrichment of specific cell types. However, when the cells are not morphologically distinguishable, it is necessary to use a specific staining method for the target cells. In this study we have tested different fixatives, storage conditions for frozen sections and staining protocols, and present two staining protocols for frozen sections, one for fast and specific staining of fetal germ cells, testicular carcinoma in situ cells, and other cells with embryonic stem cell-like properties that express the alkaline phosphatase, and one for specific staining of lipid droplet-containing cells, which is useful for isolation of the androgen-producing Leydig cells. Both protocols retain a morphology that is compatible with laser microdissection and yield RNA of a quality suitable for PCR and microarray analysis

Major histocompatibility complex diversity in anurans

The Major Histocompatibility Complex (MHC) is a large gene complex vital to the vertebrate immune response. The antigen-presenting molecules of the MHC class I and class II are involved in the immune surveillance of intracellular and extracellular pathogens, respectively. The regions of the MHC involved in peptide binding and presentation are often highly polymorphic and have high allelic variation within populations. High MHC diversity is theorised to provide immunogenetic competence to a population, and these genes have become a popular adaptive genetic marker in population studies, often with a conservation context. This thesis presents the characterisation of MHC diversity in two anuran species undergoing very different population histories: the cane toad (Rhinella marina), undergoing range expansions over the course of the Australian invasion; and the New Zealand endemic Hochstetter’s frog (Leiopelma hochstetteri), where populations are highly fragmented. In both cases, I investigate MHC diversity alongside neutral genetic diversity to infer the relative influences of neutral genetic forces, predominately drift, and selection in shaping allelic variation. This involved characterisation of the class I and class II in the cane toad, prior to completing a diversity study utilising genetic markers from both classes. I also characterised a class II beta gene in the Hochstetter’s frog to characterise diversity across 5 populations. The characterisation of the cane toad class I revealed a single classical locus, and an expansion of non-classical loci. This is similar to the class I organisation found in the model anuran, Xenopus laevis and across its subfamily Xenopodinae. The cane toad and X. laevis diverged around 230MYA. The characterisation of the cane toad class II revealed four class II alpha loci and three class II beta loci. It also revealed the expression of MHC class II splice isoforms at several alpha and beta genes. This is the first observation of alternative splicing in the MHC of any anuran species. The diversity of the classical class I (UA) and class II beta gene (DAB) was studied in the source of the Australian introduction (Hawaii), in a long-colonised site (Cairns) and a site on the invasion front (Timber Creek). I found that little diversity was lost as a result of the introduction and that Cairns was highly genetically representative of Hawaii. I found genetic drift acting at the invasion front, decaying genetic diversity at microsatellite markers and the UA locus in the dispersing cane toads. DAB diversity was maintained, however. Selection has retained all three DAB alleles across the toad invasion, likely mediated by pathogenic bacteria and parasites infecting the population. I found very high MHC class II DAB polymorphism in the Hochstetter’s frog across 5 sampled populations. Populations showed extreme differentiation; only two DAB alleles were shared by more than one population. Populations generally had high DAB diversity, except Otawa. The Otawa population had only two DAB alleles present. Combined with low microsatellite diversity, Otawa has likely undergone a recent decline, which has impacted genetic diversity. Low immunogenetic diversity in Otawa may predispose this population to a greater risk of extinction from emergent disease

Genome analysis of the platypus

The sequencing of the platypus genome represents a significant milestone in the study of mammalian evolution. The platypus is an egg-laying mammal, a member of the order Monotremata, the most divergent mammalian clade. It is the first monotreme to have its genome sequenced. Its unique evolutionary position, as an offshoot between the early divergence of birds and the later emergence of therian (eutherian and marsupials) mammals, provides an unrivalled opportunity to understand the evolution of all mammals. Much like the physical characteristics of the platypus, the platypus genome shows traces of an amalgamation of mammalian and reptilian traits. Key findings to emerge from the genome project include the complex evolution of mammalian sex chromosomes, the transition from egg-laying to live birth in mammals, and the evolution of immune genes and venom molecules in this unique species

Expansion of a retrovirus lineage in the koala genome

Retroviruses have left their legacy in host genomes over millions of years as endogenous retroviruses (ERVs), and their structure, diversity, and prevalence provide insights into the historical dynamics of retrovirus–host interactions. In bioinformatic analyses of koala (Phascolarctos cinereus) whole-genome sequences, we identify a recently expanded ERV lineage (phaCin-ß) that is related to the New World squirrel monkey retrovirus. This ERV expansion shares many parallels with the ongoing koala retrovirus (KoRV) invasion of the koala genome, including highly similar and mostly intact sequences, and polymorphic ERV loci in the sampled koala population. The recent phaCin-ß ERV colonization of the koala genome appears to predate the current KoRV invasion, but polymorphic ERVs and divergence comparisons between these two lineages predict a currently uncharacterized, possibly still extant, phaCin-ß retrovirus. The genomics approach to ERV-guided discovery of novel retroviruses in host species provides a strong incentive to search for phaCin-ß retroviruses in the Australasian fauna

Bidirectional Selection for Body Weight on Standing Genetic Variation in a Chicken Model

Experimental populations of model organisms provide valuable opportunities to unravel the genomic impact of selection in a controlled system. The Virginia body weight chicken lines represent a unique resource to investigate signatures of selection in a system where long-term, single-trait, bidirectional selection has been carried out for more than 60 generations. At 55 generations of divergent selection, earlier analyses of pooled genome resequencing data from these lines revealed that 14.2% of the genome showed extreme differentiation between the selected lines, contained within 395 genomic regions. Here, we report more detailed analyses of these data exploring the regions displaying within- and between-line genomic signatures of the bidirectional selection applied in these lines. Despite the strict selection regime for opposite extremes in body weight, this did not result in opposite genomic signatures between the lines. The lines often displayed a duality of the sweep signatures, where an extended region of homozygosity in one line, in contrast to mosaic pattern of heterozygosity in the other line. These haplotype mosaics consisted of short, distinct haploblocks of variable between-line divergence, likely the results of a complex demographic history involving bottlenecks, introgressions and moderate inbreeding. We demonstrate this using the example of complex haplotype mosaicism in the growth1 QTL. These mosaics represent the standing genetic variation available at the onset of selection in the founder population. Selection on standing genetic variation can thus result in different signatures depending on the intensity and direction of selection

Whole genome sequencing reveals high differentiation, low levels of genetic diversity and short runs of homozygosity among Swedish wels catfish

The use of genetic markers in the context of conservation is largely being outcompeted by whole-genome data. Comparative studies between the two are sparse, and the knowledge about potential effects of this methodology shift is limited. Here, we used whole-genome sequencing data to assess the genetic status of peripheral populations of the wels catfish (Silurus glanis), and discuss the results in light of a recent microsatellite study of the same populations. The Swedish populations of the wels catfish have suffered from severe declines during the last centuries and persists in only a few isolated water systems. Fragmented populations generally are at greater risk of extinction, for example due to loss of genetic diversity, and may thus require conservation actions. We sequenced individuals from the three remaining native populations (Båven, Emån, and Möckeln) and one reintroduced population of admixed origin (Helge å), and found that genetic diversity was highest in Emån but low overall, with strong differentiation among the populations. No signature of recent inbreeding was found, but a considerable number of short runs of homozygosity were present in all populations, likely linked to historically small population sizes and bottleneck events. Genetic substructure within any of the native populations was at best weak. Individuals from the admixed population Helge å shared most genetic ancestry with the Båven population (72%). Our results are largely in agreement with the microsatellite study, and stresses the need to protect these isolated populations at the northern edge of the distribution of the species

Phylogenetic trees for MHC class I alpha 1 domain and alpha 2 domain.

<p>Maximum likelihood phylogenetic trees using General Time Reversible model of nucleotide substitution with invariant sites (rooted on midpoint) comparing <i>Rhinella marina</i> (Rhma) to other anuran species (<i>Xenopus laevis</i>, Xela; <i>Silurana tropicalis</i>, Sitr; <i>Rana temporaria</i>, Rate; <i>Agalychnis callidryas</i>, Agca; <i>Espadarana prosoblepon</i>, Espr; <i>Smilisca phaeota</i>, Smph; <i>Lithobates catesbeianus</i>, Lica; <i>L. clamitans</i>, Licl; <i>L. yavapaiensis</i>, Liya) and axolotl (<i>Ambystoma mexicanium</i>, Amme). Sequence titles include Genbank accessions.</p