Transcriptome analysis of biomineralisation-related genes within the pearl sac: host and donor oyster contribution.

Cultured pearl production is a complex biological process involving the implantation of a mantle graft from a donor pearl oyster along with a bead nucleus into the gonad of a second recipient host oyster. Therefore, pearl production potentially involves the genetic co-operation of two oyster genomes. Whilst many genes in the mantle tissue have been identified and linked to shell biomineralisation in pearl oysters, few studies have determined which of these biomineralisation genes are expressed in the pearl sac and potentially linked to pearl biomineralisation processes. It is also uncertain whether the host or donor oyster is primarily responsible for the expression of biomineralisation genes governing pearl formation, with only two shell matrix proteins previously identified as being expressed by the donor oyster in the pearl sac. To further our understanding of pearl formation, the pearl sac transcriptome of Pinctada maxima and Pinctada margaritifera was each sequenced to an equivalent 5× genome coverage with putative molluscan biomineralisation-related genes identified. Furthermore, the host and donor contribution of these expressed genes within the pearl sac were quantified using a novel approach whereby two pearl oyster species harbouring unique genomes, P. maxima or P. margaritifera, were used to produce xenografted pearl sacs. A total of 19 putative mollusc biomineralisation genes were identified and found to be expressed in the pearl sacs of P. maxima and P. margaritifera. From this list of expressed genes, species-diagnostic single nucleotide polymorphisms (SNP) were identified within seven of these genes; Linkine, N66, Perline, N44, MSI60, Calreticulin and PfCHS1. Based on the presence/absence of species diagnostic gene transcripts within xenografted pearl sacs, all seven genes were found to be expressed by the species used as the donor oyster. In one individual we also found that the host was expressing Linkine. These results convincingly show for the first time that the donor mantle tissue is primarily responsible for the expression of biomineralisation genes in the pearl sac.

In silico whole-genome EST analysis reveals 2322 novel microsatellites for the silver-lipped pearl oyster, Pinctada maxima.

Molecular stock improvement techniques such as marker assisted selection have great potential in accelerating selective breeding programmes for animal production industries. However, the discovery and application of trait/marker associations usually requires a large number of genome-wide polymorphic loci. Here, we present 2322 unique microsatellites for the silver-lipped pearl oyster, Pinctada maxima, a species of aquaculture importance throughout the Indo-Australian Archipelago for production of the highly valued South Sea pearl. More than 1.2 million Roche 454 expressed sequence tag (EST) reads were screened for microsatellite repeat motifs. A total of 12,604 sequences contained either a di, tri, tetra, penta or hexa microsatellite repeat motif (n ≥ 6), with 6435 of these sequences having sufficient flanking regions for primer development. All identified microsatellites with designed primers were condensed into 2322 unique clusters (i.e., unique loci) of which 360 were shown to be polymorphic based on multiple sequence reads with different repeat motifs. Genotyping of five microsatellite loci demonstrated that in silico evaluation of polymorphism levels was a very useful method for identification of polymorphic loci, with the variation uncovered being a lower bound. Gene Ontology annotations of sequences containing microsatellites suggest that most are derived from a diverse array of unique genes. This EST derived microsatellite database will be a valuable resource for future studies in genetic map construction, diversity analysis, quantitative trait loci analysis, association mapping and marker assisted selection, not only for P. maxima, but also closely related species within the genus Pinctada.

Molecular detection of hybridization between sympatric kangaroo species in south-eastern Australia.

Introgressive hybridization has traditionally been regarded as rare in many vertebrate groups, including mammals. Despite a propensity to hybridize in captivity, introgression has rarely been reported between wild sympatric macropodid marsupials. Here we investigate sympatric populations of western (Macropus fuliginosus) and eastern (Macropus giganteus) grey kangaroos through 12 autosomal microsatellite loci and 626 bp of the hypervariable mitochondrial DNA (mtDNA) control region. M. fuliginosus and M. giganteus within the region of sympatry corresponded, both genetically and morphologically, to their respective species elsewhere in their distributions. Of the 223 grey kangaroos examined, 7.6% displayed evidence of introgression, although no F1 hybrids were detected. In contrast to captive studies, there was no evidence for unidirectional hybridization in sympatric grey kangaroos. However, a higher portion of M. giganteus backcrosses existed within the sample compared with M. fuliginosus. Hybridization in grey kangaroos is reflective of occasional breakdowns in species boundaries, occurring throughout the region and potentially associated with variable conditions and dramatic reductions in densities. Such rare hybridization events allow populations to incorporate novel diversity while still retaining species integrity.

Predicting genetic merit for mastitis and fertility in dairy cattle using genome wide selection and high density SNP screens.

Two novel methods for genome wide selection (GWS) were examined for predicting the genetic merit of animals using SNP information alone. A panel of 1,546 dairy bulls with reliable EBVs was genotyped for 15,380 SNPs that spanned the whole bovine genome. Two complexity reduction methods were used, partial least squares (PLS) and regression using a genetic algorithm (GAR), to find optimal solutions of EBVs against SNP information. Extensive internal cross-validation was used tofind the best predictive models followed by external validation (without direct use of the pedigree or SNP location). Both PLS and GAR provided both accurate fit to the training data set for somatic cell count (SCC) (max r = 0.83) and fertility (max r = 0.88) and showed an accuracy of prediction of r = 0.47 for SCC, and r = 0.72 for fertility. This is the first empirical demonstration that genome wide selection can account for a very high proportion of additive genetic variation in fitness traits whilst exploiting only a small percentage of available SNP information, without use of pedigree or QTL mapping. PLS was computationally more efficient than GAR.

Genome-wide genetic diversity of Holstein Friesian cattle reveals new insights into Australian and global population variability, including impact of selection.

Past breeding strategies for dairy cattle have been very effective in producing rapid genetic gain to achieve industry targets and raise profitability. Such gains have been largely facilitated by intense selection of sires combined with the use of artificial insemination. However, this practice can potentially limit the level of genetic diversity through inbreeding and selection plateaus. The rate of inbreeding in Australia is increasing, primarily as a result of semen importation from a small number of prominent bulls from the USA. The effect of this genetic influx in the Australian dairy cattle population is poorly understood both in terms of diversity and local adaptation/divergence. This study uses 845 genome-wide SNP genetic markers and 431 bulls to characterize the level of genetic diversity and genetic divergence within the Australian and international Holstein Friesian dairy population. No significant differences in genetic diversity (as measured by heterozygosity [H(o)] and allelic richness [A]) were observed over the 25-year time period (1975-1999) for bulls used in Australia. The importation of foreign semen into Australia has increased the effective population size until it was in effect a sub-sample of the global population. Our data indicate that most individuals are equally closely related to one another, regardless of country of origin and year of birth. In effect, the global population can be considered as one single population unit. These results indicate that inbreeding, genetic drift and selection has had little effect at reducing genetic diversity and differentiating the Australian Holstein Friesian population at a genome-wide level.

Technical note: Whole-genome amplification of DNA extracted from cattle semen samples.

The bovine genome sequence project and the discovery of many thousands of bovine single nucleotide polymorphisms has opened the door for large-scale genotyping studies to identify genes that contribute to economically important traits with relevance to the beef and dairy industries. Large amounts of DNA will be required for these research projects. This study reports the use of the whole-genome amplification (WGA) method to create an unlimited supply of DNA for use in genotyping studies and long-term storage for future gene discovery projects. Two commercial WGA kits (GenomiPhi, Amersham Biosciences, Sydney, Australia, and REPLI-g, Qiagen, Doncaster, Australia) were used to amplify DNA from straws of bull semen, resulting in an average of 7.2 and 67 microg of DNA per reaction, respectively. The comparison of 3.5 kb of sequences from the amplified and unamplified DNA indicated no detectable DNA differences. Similarly, gene marker analysis conducted on genomic DNA and DNA after WGA indicated no difference in marker amplification or clarity and accuracy of scoring for approximately 10,000 single nucleotide polymorphism markers when compared with WGA samples genotyped in duplicate. These results illustrate that WGA is a suitable method for the amplification and recovery of DNA from bull semen samples for routine genomic investigations.

Intraspecific variation, sex-biased dispersal and phylogeography of the eastern grey kangaroo (Macropus giganteus).

Genetic information has played an important role in the development of management units by focusing attention on the evolutionary properties and genetics of populations. Wildlife authorities cannot hope to manage species effectively without knowledge of geographical boundaries and demic structure. The present investigation provides an analysis of mitochondrial DNA and microsatellite data, which is used to infer both historical and contemporary patterns of population structuring and dispersal in the eastern grey kangaroo (Macropus giganteus) in Australia. The average level of genetic variation across sample locations was one of the highest observed for marsupials (h=0.95, HE=0.82). Contrary to ecological studies, both genic and genotypic analyses reveal weak genetic structure of populations, where high levels of dispersal may be inferred up to 230 km. The movement of individuals was predominantly male-biased (average Nem=22.61, average Nfm=2.73). However, neither sex showed significant isolation by distance. On a continental scale, there was strong genetic differentiation and phylogeographic distinction between southern (TAS, VIC and NSW) and northern (QLD) populations, indicating a current and/or historical restriction of gene flow. In addition, it is evident that northern populations are historically more recent, and were derived from a small number of southern founders. Phylogenetic comparisons between M. g. giganteus and M. g. tasmaniensis indicated that the current taxonomic status of these subspecies should be revised as there was a lack of genetic differentiation between the populations sampled.

A rapid population expansion retains genetic diversity within European rabbits in Australia.

The well documented historical translocations of the European rabbit (Oryctolagus cuniculus) offer an excellent framework to test the genetic effects of reductions in effective population size. It has been proposed that rabbits went through an initial bottleneck at the time of their establishment in Australia, as well as multiple founder events during the rabbit's colonization process. To test these hypotheses, genetic variation at seven microsatellite loci was measured in 252 wild rabbits from five populations across Australia. These populations were compared to each other and to data from Europe. No evidence of a genetic bottleneck was observed with the movement of 13 rabbits from Europe to Australia when compared to French data. Within Australia the distribution of genetic diversity did not reflect the suggested pattern of sequential founder effects. In fact, the current pattern of genetic variation in Australia is most likely a result of multiple factors including mutation, genetic drift and geographical differentiation. The absence of reduced genetic diversity is almost certainly a result of the rabbit's rapid population expansion at the time of establishment in Australia. These results highlight the importance of population growth following a demographic bottleneck, which largely determines the severity of genetic loss.