Kyall R. Zenger

SSCP is not so difficult: the application and utility of single‐stranded conformation polymorphism in evolutionary biology and molecular ecology

All genetic markers are estimators of DNA nucleotide sequence variation. Rather than obtaining DNA sequence data, it is cheaper and faster to use techniques that estimate sequence variation, although this usually results in the loss of some information. SSCP (single‐stranded conformation polymorphism) offers a sensitive but inexpensive, rapid, and convenient method for determining which DNA samples in a set differ in sequence, so that only an informative subset need be sequenced. In short, most DNA sequence variation can be detected with relatively little sequencing. SSCP has been widely applied in medical diagnosis, yet few studies have been published in population genetics. The utility and convenience of SSCP is far from fully appreciated by molecular population biologists. We hope to help redress this by illustrating the application of a single simple SSCP protocol to mitochondrial genes, nuclear introns, microsatellites, and anonymous nuclear sequences, in a range of vertebrates and invertebrates.

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 rabbits 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 rabbits 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.

A primary assembly of a bovine haplotype block map based on a 15,036-single-nucleotide polymorphism panel genotyped in holstein-friesian cattle

Analysis of data on 1000 Holstein–Friesian bulls genotyped for 15,036 single-nucleotide polymorphisms (SNPs) has enabled genomewide identification of haplotype blocks and tag SNPs. A final subset of 9195 SNPs in Hardy–Weinberg equilibrium and mapped on autosomes on the bovine sequence assembly (release Btau 3.1) was used in this study. The average intermarker spacing was 251.8 kb. The average minor allele frequency (MAF) was 0.29 (0.05–0.5). Following recent precedents in human HapMap studies, a haplotype block was defined where 95% of combinations of SNPs within a region are in very high linkage disequilibrium. A total of 727 haplotype blocks consisting of ≥3 SNPs were identified. The average block length was 69.7 ± 7.7 kb, which is ∼5–10 times larger than in humans. These blocks comprised a total of 2964 SNPs and covered 50,638 kb of the sequence map, which constitutes 2.18% of the length of all autosomes. A set of tag SNPs, which will be useful for further fine-mapping studies, has been identified. Overall, the results suggest that as many as 75,000–100,000 tag SNPs would be needed to track all important haplotype blocks in the bovine genome. This would require ∼250,000 SNPs in the discovery phase.

Genetic diversity in remnant mainland and "pristine"’ island populations of three endemic Australian macropodids (Marsupialia): Macropus eugenii, Lagorchestes hirsutus and Petrogale lateralis

Since European settlement, mainland Australia has experienced a wave of mammal extinctions and population declines. However, some species have persisted on off-shore islands, which are now viewed as important wildlife refuges. In this study, we assessed the level of genetic diversity, at 7–11 microsatellite loci, in island and remnant mainland populations of three endemic species of macropodid marsupial; the tammar wallaby Macropus eugenii(n = 92); rufous hare-wallaby Lagorchestes hirsutus(n = 40) and black-footed rock-wallaby Petrogale lateralis(n = 164). There was a consistent pattern of significantly higher levels of microsatellite diversity in the remnant mainland population (A= 4.9–13.9; He= 0.61–0.86) of each species compared to conspecific “pristine” island populations (A= 1.2–3.7; He= 0.05–0.44). These marked differences are even apparent where island populations currently have a substantially larger census size. In addition, island populations were substantially inbred (Fe= 0.49–0.91). Although island populations have been insulated from the relatively recent threatening processes operating on the mainland, they have nevertheless been significantly impacted by increased inbreeding and the substantial erosion of genetic diversity. Despite the difficulties of ensuring the survival of remnant mainland populations, they appear to retain substantially more genetic diversity than their island counterparts and therefore are more likely to contribute to the long-term persistence of their species. These data also demonstrate that small remnant mainland populations (n < 10–20) are often capable of rapid recovery and are not necessarily genetically depauperate.

Isolation and genetic diversity of endangered grey nurse shark (Carcharias taurus) populations

Anthropogenic impacts are believed to be the primary threats to the eastern Australian population of grey nurse sharks (Carcharias taurus), which is listed as critically endangered, and the most threatened population globally. Analyses of 235 polymorphic amplified fragment length polymorphisms (AFLP) loci and 700 base pairs of mitochondrial DNA control region provide the first account of genetic variation and geographical partitioning (east and west coasts of Australia, South Africa) in C. taurus. Assignment tests, analysis of relatedness and Fst values all indicate that the Australian populations are isolated from South Africa, with negligible migration between the east and west Australian coasts. There are significant differences in levels of genetic variation among regions. Australian C. taurus, particularly the eastern population, has significantly less AFLP variation than the other sampling localities. Further, the eastern Australian sharks possess only a single mitochondrial haplotype, also suggesting a small number of founding individuals. Therefore, historical, rather than anthropogenic processes most likely account for their depauperate genetic variation. These findings have implications for the viability of the eastern Australian population of grey nurse sharks.

Landscape discontinuities influence gene flow and genetic structure in a large, vagile Australian mammal, Macropus fuliginosus

Large vagile mammals typically exhibit little genetic structuring across their range, particularly when their habitat is essentially continuous. We investigated the population genetic structure of a large vagile Australian macropodid, Macropus fuliginosus, which is continuously distributed across most of southern Australia, using nine highly polymorphic nuclear microsatellite loci. Five distinct genetic units were identified across the range, four on the mainland and one on Kangaroo Island. In addition to the predicted historic Nullarbor Plain Barrier, two unexpected mainland barriers to gene flow were identified. Both were associated with landscape discontinuities (Swan River, Flinders Ranges), which appear within the dispersal capabilities of M. fuliginosus. Typical of large vagile mammals, M. fuliginosus displays high genetic diversity (with the exception of an insular population) and weak genetic structuring (within genetic units). However, the expansion of M. fuliginosus from southwestern Australia during the Pleistocene has resulted in significantly reduced genetic diversity in eastern populations. No significant sex‐biased dispersal was detected, although differences in habitat, densities and climatic conditions between the eastern and western regions of the range appear to influence dispersal with the effects of isolation by distance only evident in the west. These results suggest that the biogeography of southern Australia is more complex than previously thought and reveal that seemingly minor landscape features can significantly impact genetic structuring in large vagile mammals.

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 mitochondiral 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 First-Generation Metric Linkage Disequilibrium Map of Bovine Chromosome 6

We constructed a metric linkage disequilibrium (LD) map of bovine chromosome 6 (BTA6) on the basis of data from 220 SNPs genotyped on 433 Australian dairy bulls. This metric LD map has distances in LD units (LDUs) that are analogous to centimorgans in linkage maps. The LD map of BTA6 has a total length of 8.9 LDUs. Within the LD map, regions of high LD (represented as blocks) and regions of low LD (steps) are observed, when plotted against the integrated map in kilobases. At the most stringent block definition, namely a set of loci with zero LDU increase over the span of these markers, BTA6 comprises 40 blocks, accounting for 41% of the chromosome. At a slightly lower stringency of block definition (a set of loci covering a maximum of 0.2 LDUs on the LD map), up to 81% of BTA6 is spanned by 46 blocks and with 13 steps that are likely to reflect recombination hot spots. The mean swept radius (the distance over which LD is likely to be useful for mapping) is 13.3 Mb, confirming extensive LD in Holstein–Friesian dairy cattle, which makes such populations ideal for whole-genome association studies.

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.

Mapping quantitative trait loci (QTL) in sheep. I. A new male framework linkage map and QTL for growth rate and body weight.

A male sheep linkage map comprising 191 microsatellites was generated from a single family of 510 Awassi-Merino backcross progeny. Except for ovine chromosomes 1, 2, 10 and 17, all other chromosomes yielded a LOD score difference greater than 3.0 between the best and second-best map order. The map is on average 11% longer than the Sheep Linkage Map v4.7 male-specific map. This map was employed in quantitative trait loci (QTL) analyses on body-weight and growth-rate traits between birth and 98 weeks of age. A custom maximum likelihood program was developed to map QTL in half-sib families for non-inbred strains (QTL-MLE) and is freely available on request. The new analysis package offers the advantage of enabling QTL × fixed effect interactions to be included in the model. Fifty-four putative QTL were identified on nine chromosomes. Significant QTL with sex-specific effects (i.e. QTL × sex interaction) in the range of 0.4 to 0.7 SD were found on ovine chromosomes 1, 3, 6, 11, 21, 23, 24 and 26.