Indigenous Australian genomes show deep structure and rich novel variation.

The Indigenous peoples of Australia have a rich linguistic and cultural history. How this relates to genetic diversity remains largely unknown because of their limited engagement with genomic studies. Here we analyse the genomes of 159 individuals from four remote Indigenous communities, including people who speak a language (Tiwi) not from the most widespread family (Pama-Nyungan). This large collection of Indigenous Australian genomes was made possible by careful community engagement and consultation. We observe exceptionally strong population structure across Australia, driven by divergence times between communities of 26,000-35,000 years ago and long-term low but stable effective population sizes. This demographic history, including early divergence from Papua New Guinean (47,000 years ago) and Eurasian groups1, has generated the highest proportion of previously undescribed genetic variation seen outside Africa and the most extended homozygosity compared with global samples. A substantial proportion of this variation is not observed in global reference panels or clinical datasets, and variation with predicted functional consequence is more likely to be homozygous than in other populations, with consequent implications for medical genomics2. Our results show that Indigenous Australians are not a single homogeneous genetic group and their genetic relationship with the peoples of New Guinea is not uniform. These patterns imply that the full breadth of Indigenous Australian genetic diversity remains uncharacterized, potentially limiting genomic medicine and equitable healthcare for Indigenous Australians.

Equitable Expanded Carrier Screening Needs Indigenous Clinical and Population Genomic Data.

Expanded carrier screening (ECS) for recessive monogenic diseases requires prior knowledge of genomic variation, including DNA variants that cause disease. The composition of pathogenic variants differs greatly among human populations, but historically, research about monogenic diseases has focused mainly on people with European ancestry. By comparison, less is known about pathogenic DNA variants in people from other parts of the world. Consequently, inclusion of currently underrepresented Indigenous and other minority population groups in genomic research is essential to enable equitable outcomes in ECS and other areas of genomic medicine. Here, we discuss this issue in relation to the implementation of ECS in Australia, which is currently being evaluated as part of the national Government's Genomics Health Futures Mission. We argue that significant effort is required to build an evidence base and genomic reference data so that ECS can bring significant clinical benefit for many Aboriginal and/or Torres Strait Islander Australians. These efforts are essential steps to achieving the Australian Government's objectives and its commitment "to leveraging the benefits of genomics in the health system for all Australians." They require culturally safe, community-led research and community involvement embedded within national health and medical genomics programs to ensure that new knowledge is integrated into medicine and health services in ways that address the specific and articulated cultural and health needs of Indigenous people. Until this occurs, people who do not have European ancestry are at risk of being, in relative terms, further disadvantaged.

Eco-Metabolomics and Metabolic Modeling: Making the Leap From Model Systems in the Lab to Native Populations in the Field.

Experimental high-throughput analysis of molecular networks is a central approach to characterize the adaptation of plant metabolism to the environment. However, recent studies have demonstrated that it is hardly possible to predict in situ metabolic phenotypes from experiments under controlled conditions, such as growth chambers or greenhouses. This is particularly due to the high molecular variance of in situ samples induced by environmental fluctuations. An approach of functional metabolome interpretation of field samples would be desirable in order to be able to identify and trace back the impact of environmental changes on plant metabolism. To test the applicability of metabolomics studies for a characterization of plant populations in the field, we have identified and analyzed in situ samples of nearby grown natural populations of Arabidopsis thaliana in Austria. A. thaliana is the primary molecular biological model system in plant biology with one of the best functionally annotated genomes representing a reference system for all other plant genome projects. The genomes of these novel natural populations were sequenced and phylogenetically compared to a comprehensive genome database of A. thaliana ecotypes. Experimental results on primary and secondary metabolite profiling and genotypic variation were functionally integrated by a data mining strategy, which combines statistical output of metabolomics data with genome-derived biochemical pathway reconstruction and metabolic modeling. Correlations of biochemical model predictions and population-specific genetic variation indicated varying strategies of metabolic regulation on a population level which enabled the direct comparison, differentiation, and prediction of metabolic adaptation of the same species to different habitats. These differences were most pronounced at organic and amino acid metabolism as well as at the interface of primary and secondary metabolism and allowed for the direct classification of population-specific metabolic phenotypes within geographically contiguous sampling sites.

On the post-glacial spread of human commensal Arabidopsis thaliana.

Recent work has shown that Arabidopsis thaliana contains genetic groups originating from different ice age refugia, with one particular group comprising over 95% of the current worldwide population. In Europe, relicts of other groups can be found in local populations along the Mediterranean Sea. Here we provide evidence that these 'relicts' occupied post-glacial Eurasia first and were later replaced by the invading 'non-relicts', which expanded through the east-west axis of Eurasia, leaving traces of admixture in the north and south of the species range. The non-relict expansion was likely associated with human activity and led to a demographic replacement similar to what occurred in humans. Introgressed genomic regions from relicts are associated with flowering time and enriched for genes associated with environmental conditions, such as root cap development or metal ion trans-membrane transport, which suggest that admixture with locally adapted relicts helped the non-relicts colonize new habitats.

The Spontaneous Mutation Rate in the Fission Yeast Schizosaccharomyces pombe.

The rate at which new mutations arise in the genome is a key factor in the evolution and adaptation of species. Here we describe the rate and spectrum of spontaneous mutations for the fission yeast Schizosaccharomyces pombe, a key model organism with many similarities to higher eukaryotes. We undertook an ∼1700-generation mutation accumulation (MA) experiment with a haploid S. pombe, generating 422 single-base substitutions and 119 insertion-deletion mutations (indels) across the 96 replicates. This equates to a base-substitution mutation rate of 2.00 × 10(-10) mutations per site per generation, similar to that reported for the distantly related budding yeast Saccharomyces cerevisiae. However, these two yeast species differ dramatically in their spectrum of base substitutions, the types of indels (S. pombe is more prone to insertions), and the pattern of selection required to counteract a strong AT-biased mutation rate. Overall, our results indicate that GC-biased gene conversion does not play a major role in shaping the nucleotide composition of the S. pombe genome and suggest that the mechanisms of DNA maintenance may have diverged significantly between fission and budding yeasts. Unexpectedly, CpG sites appear to be excessively liable to mutation in both species despite the likely absence of DNA methylation.

Genetic architecture of natural variation of telomere length in Arabidopsis thaliana.

Telomeres represent the repetitive sequences that cap chromosome ends and are essential for their protection. Telomere length is known to be highly heritable and is derived from a homeostatic balance between telomeric lengthening and shortening activities. Specific loci that form the genetic framework underlying telomere length homeostasis, however, are not well understood. To investigate the extent of natural variation of telomere length in Arabidopsis thaliana, we examined 229 worldwide accessions by terminal restriction fragment analysis. The results showed a wide range of telomere lengths that are specific to individual accessions. To identify loci that are responsible for this variation, we adopted a quantitative trait loci (QTL) mapping approach with multiple recombinant inbred line (RIL) populations. A doubled haploid RIL population was first produced using centromere-mediated genome elimination between accessions with long (Pro-0) and intermediate (Col-0) telomere lengths. Composite interval mapping analysis of this population along with two established RIL populations (Ler-2/Cvi-0 and Est-1/Col-0) revealed a number of shared and unique QTL. QTL detected in the Ler-2/Cvi-0 population were examined using near isogenic lines that confirmed causative regions on chromosomes 1 and 2. In conclusion, this work describes the extent of natural variation of telomere length in A. thaliana, identifies a network of QTL that influence telomere length homeostasis, examines telomere length dynamics in plants with hybrid backgrounds, and shows the effects of two identified regions on telomere length regulation.

Staufen2 regulates neuronal target RNAs.

RNA-binding proteins play crucial roles in directing RNA translation to neuronal synapses. Staufen2 (Stau2) has been implicated in both dendritic RNA localization and synaptic plasticity in mammalian neurons. Here, we report the identification of functionally relevant Stau2 target mRNAs in neurons. The majority of Stau2-copurifying mRNAs expressed in the hippocampus are present in neuronal processes, further implicating Stau2 in dendritic mRNA regulation. Stau2 targets are enriched for secondary structures similar to those identified in the 3' UTRs of Drosophila Staufen targets. Next, we show that Stau2 regulates steady-state levels of many neuronal RNAs and that its targets are predominantly downregulated in Stau2-deficient neurons. Detailed analysis confirms that Stau2 stabilizes the expression of one synaptic signaling component, the regulator of G protein signaling 4 (Rgs4) mRNA, via its 3' UTR. This study defines the global impact of Stau2 on mRNAs in neurons, revealing a role in stabilization of the levels of synaptic targets.

The advantages and limitations of trait analysis with GWAS: a review.

Over the last 10 years, high-density SNP arrays and DNA re-sequencing have illuminated the majority of the genotypic space for a number of organisms, including humans, maize, rice and Arabidopsis. For any researcher willing to define and score a phenotype across many individuals, Genome Wide Association Studies (GWAS) present a powerful tool to reconnect this trait back to its underlying genetics. In this review we discuss the biological and statistical considerations that underpin a successful analysis or otherwise. The relevance of biological factors including effect size, sample size, genetic heterogeneity, genomic confounding, linkage disequilibrium and spurious association, and statistical tools to account for these are presented. GWAS can offer a valuable first insight into trait architecture or candidate loci for subsequent validation.

Massive genomic variation and strong selection in Arabidopsis thaliana lines from Sweden.

Despite advances in sequencing, the goal of obtaining a comprehensive view of genetic variation in populations is still far from reached. We sequenced 180 lines of A. thaliana from Sweden to obtain as complete a picture as possible of variation in a single region. Whereas simple polymorphisms in the unique portion of the genome are readily identified, other polymorphisms are not. The massive variation in genome size identified by flow cytometry seems largely to be due to 45S rDNA copy number variation, with lines from northern Sweden having particularly large numbers of copies. Strong selection is evident in the form of long-range linkage disequilibrium (LD), as well as in LD between nearby compensatory mutations. Many footprints of selective sweeps were found in lines from northern Sweden, and a massive global sweep was shown to have involved a 700-kb transposition.

The genomic signature of splicing-coupled selection differs between long and short introns.

Understanding the function of noncoding regions in the genome, such as introns, is of central importance to evolutionary biology. One approach is to assay for the targets of natural selection. On one hand, the sequence of introns, especially short introns, appears to evolve in an almost neutral manner. Whereas on the other hand, a large proportion of intronic sequence is under selective constraint. This discrepancy is largely dependent on intron length and differences in the methods used to infer selection. We have used a method based on DNA strand asymmetery that does not require comparison with any putatively neutrally evolving sequence, nor sequence conservation between species, to detect selection within introns. The strongest signal we identify is associated with short introns. This signal comes from a family of motifs that could act as cryptic 5' splice sites during mRNA processing, suggesting a mechanistic justification underlying this signal of selection. Together with an analysis of intron length and splice site strength, we observe that the genomic signature of splicing-coupled selection differs between long and short introns.

DNA double-strand break repair and the evolution of intron density.

The density of introns is both an important feature of genome architecture and a highly variable trait across eukaryotes. This heterogeneity has posed an evolutionary puzzle for the last 30 years. Recent evidence is consistent with novel introns being the outcome of the error-prone repair of DNA double-stranded breaks (DSBs) via non-homologous end joining (NHEJ). Here we suggest that deletion of pre-existing introns could occur via the same pathway. We propose a novel framework in which species-specific differences in the activity of NHEJ and homologous recombination (HR) during the repair of DSBs underlie changes in intron density.

Syntaxin 5 is required for copper homeostasis in Drosophila and mammals.

Copper is essential for aerobic life, but many aspects of its cellular uptake and distribution remain to be fully elucidated. A genome-wide screen for copper homeostasis genes in Drosophila melanogaster identified the SNARE gene Syntaxin 5 (Syx5) as playing an important role in copper regulation; flies heterozygous for a null mutation in Syx5 display increased tolerance to high dietary copper. The phenotype is shown here to be due to a decrease in copper accumulation, a mechanism also observed in both Drosophila and human cell lines. Studies in adult Drosophila tissue suggest that very low levels of Syx5 result in neuronal defects and lethality, and increased levels also generate neuronal defects. In contrast, mild suppression generates a phenotype typical of copper-deficiency in viable, fertile flies and is exacerbated by co-suppression of the copper uptake gene Ctr1A. Reduced copper uptake appears to be due to reduced levels at the plasma membrane of the copper uptake transporter, Ctr1. Thus Syx5 plays an essential role in copper homeostasis and is a candidate gene for copper-related disease in humans.

Nonsense-mediated decay enables intron gain in Drosophila.

Intron number varies considerably among genomes, but despite their fundamental importance, the mutational mechanisms and evolutionary processes underlying the expansion of intron number remain unknown. Here we show that Drosophila, in contrast to most eukaryotic lineages, is still undergoing a dramatic rate of intron gain. These novel introns carry significantly weaker splice sites that may impede their identification by the spliceosome. Novel introns are more likely to encode a premature termination codon (PTC), indicating that nonsense-mediated decay (NMD) functions as a backup for weak splicing of new introns. Our data suggest that new introns originate when genomic insertions with weak splice sites are hidden from selection by NMD. This mechanism reduces the sequence requirement imposed on novel introns and implies that the capacity of the spliceosome to recognize weak splice sites was a prerequisite for intron gain during eukaryotic evolution.

Malvolio is a copper transporter in Drosophila melanogaster.

Divalent metal ion transporter 1 (DMT1; also known as SLC11A2) can transport several metals including Fe and Cu in mammalian systems. We set out to determine whether Malvolio (Mvl), the Drosophila melanogaster orthologue of DMT1, can also transport Cu. Overexpression of Mvl caused Cu accumulation in Drosophila S2 cultured cells and conversely dsRNAi knockdown of endogenous Mvl reduced cellular Cu levels. Cell viability under Cu limiting conditions was reduced following dsRNAi knockdown. A homozygous viable Mvl loss-of-function mutant (Mvl(97f)) was sensitive to excess Cu and female Mvl(97f) flies were also sensitive to Cu limitation. An MtnA-EYFP reporter was used as a proxy measure of Cu distribution within Mvl(97f/+) larvae. Under basal conditions Cu levels were reduced in the anterior midgut and proventriculus relative to control larvae. These results demonstrate Mvl is a functional Cu transporter and that despite partial functional redundancy with the Ctr1 proteins, Cu uptake through this pathway is necessary for optimal viability at the cellular and organismal levels.

Essential roles in development and pigmentation for the Drosophila copper transporter DmATP7.

Defects in the mammalian Menkes and Wilson copper transporting P-type ATPases cause severe copper homeostasis disease phenotypes in humans. Here, we find that DmATP7, the sole Drosophila orthologue of the Menkes and Wilson genes, is vital for uptake of copper in vivo. Analysis of a DmATP7 loss-of-function allele shows that DmATP7 is essential in embryogenesis, early larval development, and adult pigmentation and is probably required for copper uptake from the diet. These phenotypes are analogous to those caused by mutation in the mouse and human Menkes genes, suggesting that like Menkes, DmATP7 plays at least two roles at the cellular level: delivering copper to cuproenzymes required for pigmentation and neuronal function and removing excess cellular copper via facilitated efflux. DmATP7 displays a dynamic and unexpected expression pattern in the developing embryo, implying novel functions for this copper pump and the lethality observed in DmATP7 mutant flies is the earliest seen for any copper homeostasis gene.

Yan regulates Lozenge during Drosophila eye development.

The Drosophila eye offers an excellent opportunity to understand how general developmental processes are subtly altered to result in specific cell fates. Numerous transcription factors have been characterized in the developing eye; most of these are active in overlapping subsets of cells. Mechanisms used to regulate transcription factors act at many levels, and include competition for cognate binding sites, post translational modification, transcriptional regulation and cofactor availability. In undifferentiated cells of the larval eye imaginal disc, the transcriptional repressor Yan outcompetes the transcriptional activator Pointed for ETS binding sites on the prosperoenhancer. During differentiation, the Ras signaling cascade alters the Yan/Pointed dynamic through protein phosphorylation, effecting a developmental switch. In this way, Yan and Pointed are essential for prospero regulation. Hyperstable Yan (ACT) cannot be phosphorylated and blocks prospero expression. Lozenge is expressed in undifferentiated cells, and is required for prospero regulation. We sequenced the eye-specific enhancer of lozenge in three Drosophila species spanning 17 million years of evolution and found complete conservation of three ETS consensus binding sites. We show that lozengeexpression increases as cells differentiate, and that Yan (ACT) blocks this upregulation at the level of transcription. We find that expression of Lozenge via an alternate enhancer alters the temporal expression of Prospero, and is sufficient to rescue Prospero expression in the presence of Yan (ACT). These results suggest that Lozenge is involved in the Yan/Pointed dynamic in a Ras-dependent manner. We propose that upregulated Lozenge acts as a cofactor to alter Pointed affinity, by a mechanism that is recapitulated in mammalian development.