Inheritance of molecular markers and sex in the Australian freshwater crayfish, Cherax destructor Clark

Inheritance of three kinds of molecular genetic markers (mtDNA, random-amplified polymorphic DNAs (RAPDs) and allozymes) and sex were investigated in crossbreeding experiments between three populations of the Australian freshwater crayfish Cherax destructor. Crossbreeding did not disrupt the ively maternally inherited, and allozyme and RAPD markers were transmitted following expected Mendelian principles for co-dominant and dominant traits respectively. Unlike these three markers, sex ratios were found to be distorted by crossbreeding in some families. Two crossbred families produced only females. The implications of these findings for freshwater crayfish population genetics, taxonomy and aquaculture are discussed.

Myoepithelial molecular markers in human breast carcinoma PMC42-LA cells are induced by extracellular matrix and stromal cells

The microenvironment plays a key role in the cellular differentiation of the two main cell lineages of the human breast, luminal epithelial, and myoepithelial. It is not clear, however, how the components of the microenvironment control the development of these cell lineages. To investigate how lineage development is regulated by 3-D culture and microenvironment components, we used the PMC42-LA human breast carcinoma cell line, which possesses stem cell characteristics. When cultured on a two-dimensional glass substrate, PMC42-LA cells formed a monolayer and expressed predominantly luminal epithelial markers, including cytokeratins 8, 18, and 19; E-cadherin; and sialomucin. The key myoepithelial-specific proteins alpha-smooth muscle actin and cytokeratin 14 were not expressed. When cultured within Engelbreth-Holm- Swarm sarcoma-derived basement membrane matrix (EHS matrix), PMC42-LA cells formed organoids in which the expression of luminal markers was reduced and the expression of other myoepithelial-specific markers (cytokeratin 17 and P-cadherin) was promoted. The presence of primary human mammary gland fibroblasts within the EHS matrix induced expression of the key myoepithelial-specific markers, alpha-smooth muscle actin and cytokeratin 14. Immortalized human skin fibroblasts were less effective in inducing expression of these key myoepithelial-specific markers. Confocal dual-labeling showed that individual cells expressed luminal or myoepithelial proteins, but not both. Conditioned medium from the mammary fibroblasts was equally effective in inducing myoepithelial marker expression. The results indicate that the myoepithelial lineage is promoted by the extracellular matrix, in conjunction with products secreted by breast-specific fibroblasts. Our results demonstrate a key role for the breast microenvironment in the regulation of breast lineage development.

Three molecular markers show no evidence of population genetic structure in the gouldian finch (Erythrura gouldiae)

Assessment of genetic diversity and connectivity between regions can inform conservation managers about risk of inbreeding, potential for adaptation and where population boundaries lie. The Gouldian finch (Erythrura gouldiae) is a threatened species in northern Australia, occupying the savannah woodlands of the biogeographically complex monsoon tropics. We present the most comprehensive population genetic analysis of diversity and structure the Gouldian finch using 16 microsatellite markers, mitochondrial control region and 3,389 SNPs from genotyping-by-sequencing. Mitochondrial diversity is compared across three related, co-distributed finches with different conservation threat-statuses. There was no evidence of genetic differentiation across the western part of the range in any of the molecular markers, and haplotype diversity but not richness was lower than a common co-distributed species. Individuals within the panmictic population in the west may be highly dispersive within this wide area, and we urge caution when interpreting anecdotal observations of changes to the distribution and/or flock sizes of Gouldian finch populations as evidence of overall changes to the population size of this species.

Molecular genetic markers for abalone aquaculture

Describes the development and mapping of molecular markers in the blacklip and greenlip abalones. By means of a genome scan using a novel selective DNA pooling strategy, markers associated with growth were discovered that could potentially be applied to increase genetic gain in abalone aquaculture, whilst minimising inbreeding.

Lack of genetic divergence found with microsatellite DNA markers in the tarakihi Nemadactylus macropterus

Three classes of molecular markers are commonly employed during population genetic studies of marine taxa: allozymes, mitochondrial DNA (mtDNA), and microsatellite DNA. These markers differ in their levels of polymorphism, and the ease and cost of their application. Nemadactylus macropterus is a commercially important marine fish from New Zealand and southern Australia that has been the subject of genetic (allozyme, mtDNA) and non-genetic (otolith microchemistry, larval advection) studies of stock structure. We collected microsatellite DNA data from this species to compare the utility of these molecular markers with those genetic methods previously applied to N. macropterus. Microsatellites did not indicate significant divergence among Australian samples, or between Australian and New Zealand samples. The latter is incongruent with the allozyme and mtDNA studies, and it is suggested that allelic homoplasy has hindered the resolution of population structure when using microsatellites.

Manual on application of molecular tools in aquaculture and inland fisheries management. Part I : conceptual basis of population genetic approaches

The aim of this manual is to provide a comprehensive practical tool for the generation and analysis of genetic data for subsequent application in aquatic resources management in relation to genetic stock identification in inland fisheries and aquaculture. The material only covers general background on genetics in relation to aquaculture and fisheries resource management, the techniques and relevant methods of data analysis that are commonly used to address questions relating to genetic resource characterisation and population genetic analyses. No attempt is made to include applications of genetic improvement techniques e.g. selective breeding or producing genetically modified organisms (GMOs). The manual includes two ‘stand-alone’ parts, of which this is the first volume: Part 1 – Conceptual basis of population genetic approaches: will provide a basic foundation on genetics in general, and concepts of population genetics. Issues on the choices of molecular markers and project design are also discussed. Part 2 – Laboratory protocols, data management and analysis: will provide step-by-step protocols of the most commonly used molecular genetic techniques utilised in population genetics and systematic studies. In addition, a brief discussion and explanation of how these data are managed and analysed is also included. This manual is expected to enable NACA member country personnel to be trained to undertake molecular genetic studies in their own institutions, and as such is aimed at middle and higher level technical grades. The manual can also provide useful teaching material for specialised advanced level university courses in the region and postgraduate students. The manual has gone through two development/improvement stages. The initial material was tested at a regional workshop and at the second stage feedback from participants was used to improve the contents.

Manual on application of molecular tools in aquaculture and inland fisheries management. Part I : laboratory protocols, data management and analysis

The aim of this manual is to provide a comprehensive practical tool for the generation and analysis of genetic data for subsequent application in aquatic resources management in relation to genetic stock identification in inland fisheries and aquaculture. The material only covers general background on genetics in relation to aquaculture and fisheries resource management, the techniques and relevant methods of data analysis that are commonly used to address questions relating to genetic resource characterisation and population genetic analyses. No attempt is made to include applications of genetic improvement techniques e.g. selective breeding or producing genetically modified organisms (GMOs). The manual includes two ‘stand-alone’ parts, of which this is the second volume: Part 1 – Conceptual basis of population genetic approaches: will provide a basic foundation on genetics in general, and concepts of population genetics. Issues on the choices of molecular markers and project design are also discussed. Part 2 – Laboratory protocols, data management and analysis: will provide step-by-step protocols of the most commonly used molecular genetic techniques utilised in population genetics and systematic studies. In addition, a brief discussion and explanation of how these data are managed and analysed is also included. This manual is expected to enable NACA member country personnel to be trained to undertake molecular genetic studies in their own institutions, and as such is aimed at middle and higher level technical grades. The manual can also provide useful teaching material for specialised advanced level university courses in the region and postgraduate students. The manual has gone through two development/improvement stages. The initial material was tested at a regional workshop and at the second stage feedback from participants was used to improve the contents.

Cancer cachexia: a molecular nutrition approach to cancer-related muscle loss

The project identified changes in molecular markers of antioxidant status and muscle atrophy during cancer-associated weight-loss, and confirmed omega-3 EPA as an effective treatment for muscle-loss in experimental cancer cachexia. The project also identified the PG-SGA nutrition tool as a powerful predictor of patient outcomes and survival in clinical cachexia.

Non-invasive sampling techniques : applications for mapping the generic variation of powerful owls

The Powerful Owl (Ninox strenua) is endemic to Australia, being resident in the three eastern mainland states and the Australian Capital Territory. It is classified nationally as of conservation significance and vulnerable in the state of Victoria. The elusive nature of this owl, along with its dispersed distribution, low population density and difficulty in identifying individual birds, limit the collection of ecological data. Molecular methods can be used to obtain crucial ecological information, essential for Powerful Owl conservation.

Non-invasive sampling is a relatively new method used for obtaining genetic material from free-ranging animals. This type of sampling however, is generally overlooked as a potential DNA source. Shed hair and feathers, faeces, urine, skins and eggshells are all potential sources of DNA. Non-invasive sampling regimes may be the only alternative for the genetic analysis of endangered and/or elusive species that are difficult to sample otherwise.

Powerful Owls moult annually. Shed feathers therefore, can be collected from under roost trees and used for genetic analysis. Feathers collected provide DNA that is unique to the individual and can provide additional ecological knowledge of the species.

In this study we collected shed Powerful Owl feathers during 2003 and 2004. In order to obtain samples from across the owl's large distribution, public awareness about the project via the way of flyers, mail-outs, media sources (radio, newspapers and magazines), email lists and public seminars was initiated. Overall, the collection strategy was very successful with over 500 Powerful Owl feather samples being collected.

Genetic information obtained from the analysis of DNA from feathers can enable a more rigorous assessment of population viability of the Powerful Owl. Specifically designed molecular markers will facilitate unequivocal identification of individual birds ("DNA fingerprinting"). Through the application of molecular techniques we can collect ecological information about the Powerful Owl such as, genetic divergence, population structure, dispersal patterns, migration and inbreeding. These questions can not be addressed via traditional data collection and will contribute significantly to the successful conservation of the Powerful Owl and potentially other raptor species.

Development and characterisation of 20 microsatellite loci isolated from the large bent-wing bat, Miniopterus schreibersii (Chiroptera: Miniopteridae) and their cross-taxa utility in the family Miniopteridae

The large bent-wing bat, Miniopterus schreibersii (Kuhl 1819), has a long history of taxonomic uncertainty and many populations are known to be in a state of decline. Microsatellite loci were developed for the taxonomic and population genetic assessment of the Australian complex of this species. Of the 33 primer sets designed for this research, seven (21%) were deemed suitably polymorphic for population-level analyses of the Australian taxa, with five (71%) of these loci revealing moderate to high levels of polymorphism (PIC = 0.56 to 0.91). The cross-taxa utility of the M. schreibersii microsatellite markers was assessed in the microbat (Chiroptera) family Miniopteridae. Sub-species and species covering the Miniopteridae’s global distribution (with the exception of the Middle East) were selected, numbering 25 taxa in total. Amplification was successful for 26 loci, of which 20 (77%) were polymorphic. High cross-taxa utility of markers was observed with amplification achieved for all taxa for between four (20%) and 20 (100%) loci, and polymorphism was considered moderate to high (PIC = 0.47–0.91) for 12 (60%) of these loci. The high cross-taxa utility of the microsatellites reported herein reveal versatile and cost-effective molecular markers, contributing an important genetic resource for the research and conservation of Miniopteridae species worldwide.

Unravelling migratory connectivity in marine turtles using multiple methods

1. Comprehensive knowledge of the fundamental spatial ecology of marine species is critical to allow the identification of key habitats and the likely sources of anthropogenic threats, thus informing effective conservation strategies. 2. Research on migratory marine vertebrates has lagged behind many similar terrestrial animal groups, but studies using electronic tagging systems and molecular techniques offer great insights. 3. Marine turtles have complex life history patterns, spanning wide spatio-temporal scales. As a result of this multidimensional complexity, and despite extensive effort, there are no populations for which a truly holistic understanding of the spatial aspects of the life history has been attained. There is a particular lack of information regarding the distribution and habitats utilized during the first few years of life. 4. We used satellite tracking technology to track individual turtles following nesting at the green turtle Chelonia mydas nesting colony at Poilão Island, Guinea Bissau; the largest breeding aggregation in the eastern Atlantic. 5. We further contextualize these data with pan-Atlantic molecular data and oceanographic current modelling to gain insights into likely dispersal patterns of hatchlings and small pelagic juveniles. 6. All adult turtles remained in the waters of West Africa, with strong connectivity demonstrated with Banc D’Arguin, Mauritania. 7. Despite shortcomings in current molecular markers, we demonstrate evidence for profound sub-structuring of marine turtle stocks across the Atlantic; with a high likelihood based on oceanographic modelling that most turtles from Guinea-Bissau are found in the eastern Atlantic. 8. Synthesis and applications. There is an increased need for a better understanding of spatial distribution of marine vertebrates demonstrating life histories with spatio-temporal complexity. We propose the synergistic use of the technologies and modelling used here as a working framework for the future rapid elucidation of the range and likely key habitats used by the different life stages from such species.

Sequential polyandry through divorce and re-pairing in a cooperatively breeding bird reduces helper-offspring relatedness

Polyandry is an important component of both sexual selection and kin structuring within cooperatively breeding species. A female may have multiple partners within a single reproductive attempt (simultaneous polyandry) or across multiple broods within and/or across years (sequential polyandry). Both types of polyandry confer a range of costs and benefits to individuals and alter the genetic structure of social groups over time. To date, many molecular studies of cooperative breeders have examined the evolution of cooperative breeding in relation to simultaneous polyandry. However, cooperatively breeding vertebrates are iteroparous, and thus sequential polyandry is also likely, but more rarely considered in this context. We examined sequential polyandry in a cooperatively breeding bird that has a low level of within-brood polyandry. Over a 5-year period (2006–2010), we monitored individual mating relationships using molecular markers in a population of individually marked apostlebirds (Struthidea cinerea). Divorce occurred between reproductive seasons in 17 % (8/48) of pairs and appeared to be female-driven. The level of sequential polyandry was also driven by the disappearance of males after breeding, and over 90 % of females, for whom we had suitable data, bred with multiple males over the period of study. This sequential polyandry significantly altered the relatedness of group members to the offspring in the nest. However, in about half of the cases, the second male was related (first- or second-order relative) to the first male of a sequentially polyandrous female and this alleviated the reduction in relatedness caused by polyandry. Our findings suggest that even in species with high within-brood parentage certainty, helper-offspring relatedness values can quickly erode through sequential polyandry.