Eva-Maria Willing

Estimates of Genetic Differentiation Measured by FST Do Not Necessarily Require Large Sample Sizes When Using Many SNP Markers

Population genetic studies provide insights into the evolutionary processes that influence the distribution of sequence variants within and among wild populations. FST is among the most widely used measures for genetic differentiation and plays a central role in ecological and evolutionary genetic studies. It is commonly thought that large sample sizes are required in order to precisely infer FST and that small sample sizes lead to overestimation of genetic differentiation. Until recently, studies in ecological model organisms incorporated a limited number of genetic markers, but since the emergence of next generation sequencing, the panel size of genetic markers available even in non-reference organisms has rapidly increased. In this study we examine whether a large number of genetic markers can substitute for small sample sizes when estimating FST. We tested the behavior of three different estimators that infer FST and that are commonly used in population genetic studies. By simulating populations, we assessed the effects of sample size and the number of markers on the various estimates of genetic differentiation. Furthermore, we tested the effect of ascertainment bias on these estimates. We show that the population sample size can be significantly reduced (as small as n = 4–6) when using an appropriate estimator and a large number of bi-allelic genetic markers (k>1,000). Therefore, conservation genetic studies can now obtain almost the same statistical power as studies performed on model organisms using markers developed with next-generation sequencing.

Genome‐wide single nucleotide polymorphisms reveal population history and adaptive divergence in wild guppies

Adaptation of guppies (Poecilia reticulata) to contrasting upland and lowland habitats has been extensively studied with respect to behaviour, morphology and life history traits. Yet population history has not been studied at the whole‐genome level. Although single nucleotide polymorphisms (SNPs) are the most abundant form of variation in many genomes and consequently very informative for a genome‐wide picture of standing natural variation in populations, genome‐wide SNP data are rarely available for wild vertebrates. Here we use genetically mapped SNP markers to comprehensively survey genetic variation within and among naturally occurring guppy populations from a wide geographic range in Trinidad and Venezuela. Results from three different clustering methods, Neighbor‐net, principal component analysis (PCA) and Bayesian analysis show that the population substructure agrees with geographic separation and largely with previously hypothesized patterns of historical colonization. Within major drainages (Caroni, Oropouche and Northern), populations are genetically similar, but those in different geographic regions are highly divergent from one another, with some indications of ancient shared polymorphisms. Clear genomic signatures of a previous introduction experiment were seen, and we detected additional potential admixture events. Headwater populations were significantly less heterozygous than downstream populations. Pairwise FST values revealed marked differences in allele frequencies among populations from different regions, and also among populations within the same region. FST outlier methods indicated some regions of the genome as being under directional selection. Overall, this study demonstrates the power of a genome‐wide SNP data set to inform for studies on natural variation, adaptation and evolution of wild populations

Genetic linkage map of the guppy, Poecilia reticulata, and quantitative trait loci analysis of male size and colour variation

We report construction of a genetic linkage map of the guppy genome using 790 single nucleotide polymorphism markers, integrated from six mapping crosses. The markers define 23 linkage groups (LGs), corresponding to the known haploid number of guppy chromosomes. The map, which spans a genetic length of 899 cM, includes 276 markers linked to expressed genes (expressed sequence tag), which have been used to derive broad syntenic relationships of guppy LGs with medaka chromosomes. This combined linkage map should facilitate the advancement of genetic studies for a wide variety of complex adaptive phenotypes relevant to natural and sexual selection in this species. We have used the linkage data to predict quantitative trait loci for a set of variable male traits including size and colour pattern. Contributing loci map to the sex LG for many of these traits.

Paired-end RAD-seq for de novo assembly and marker design without available reference

MOTIVATION Next-generation sequencing technologies have facilitated the study of organisms on a genome-wide scale. A recent method called restriction site associated DNA sequencing (RAD-seq) allows to sample sequence information at reduced complexity across a target genome using the Illumina platform. Single-end RAD-seq has proven to provide a large number of informative genetic markers in reference as well as non-reference organisms. RESULTS Here, we present a method for de novo assembly of paired-end RAD-seq data in order to produce extended contigs flanking a restriction site. We were able to reconstruct one-tenth of the guppy genome represented by 200-500 bp contigs associated to EcoRI recognition sites. In addition, these contigs were used as reference allowing the detection of thousands of new polymorphic markers that are informative for mapping and population genetic studies in the guppy. AVAILABILITY A perl and C++ implementation of the method demonstrated in this article is available under http://guppy.weigelworld.org/weigeldatabases/radMarkers/ as package RApiD. CONTACT christine.dreyer@tuebingen.mpg.de SUPPLEMENTARY INFORMATION Supplementary data are available at Bioinformatics online.

On the origin and evolutionary consequences of gene body DNA methylation

Significance DNA methylation in plants is found at CG, CHG, and CHH sequence contexts. In plants, CG DNA methylation is enriched in the transcribed regions of many constitutively expressed genes (gene body methylation; gbM) and shows correlations with several chromatin modifications. Contrary to other types of DNA methylation, the evolution and function of gbM are largely unknown. Here we show two independent concomitant losses of the DNA methyltransferase CHROMOMETHYLASE 3 (CMT3) and gbM without the predicted disruption of transcription and of modifications to chromatin. This result suggests that CMT3 is required for the establishment of gbM in actively transcribed genes, and that gbM is dispensable for normal transcription as well as for the composition and modification of plant chromatin. In plants, CG DNA methylation is prevalent in the transcribed regions of many constitutively expressed genes (gene body methylation; gbM), but the origin and function of gbM remain unknown. Here we report the discovery that Eutrema salsugineum has lost gbM from its genome, to our knowledge the first instance for an angiosperm. Of all known DNA methyltransferases, only CHROMOMETHYLASE 3 (CMT3) is missing from E. salsugineum. Identification of an additional angiosperm, Conringia planisiliqua, which independently lost CMT3 and gbM, supports that CMT3 is required for the establishment of gbM. Detailed analyses of gene expression, the histone variant H2A.Z, and various histone modifications in E. salsugineum and in Arabidopsis thaliana epigenetic recombinant inbred lines found no evidence in support of any role for gbM in regulating transcription or affecting the composition and modification of chromatin over evolutionary timescales.

RACK1 scaffold proteins influence miRNA abundance in Arabidopsis

MicroRNAs (miRNAs) regulate plant development by post-transcriptional regulation of target genes. In Arabidopsis thaliana, DCL1 processes precursors (pri-miRNAs) to miRNA duplexes, which associate with AGO1. Additional proteins act in concert with DCL1 (e.g. HYL1 and SERRATE) or AGO1 to facilitate efficient and precise pri-miRNA processing and miRNA loading, respectively. In this study, we show that the accumulation of plant microRNAs depends on RECEPTOR FOR ACTIVATED C KINASE 1 (RACK1), a scaffold protein that is found in all higher eukaryotes. miRNA levels are reduced in rack1 mutants, and our data suggest that RACK1 affects the microRNA pathway via several distinct mechanisms involving direct interactions with known microRNA factors: RACK1 ensures the accumulation and processing of some pri-miRNAs, directly interacts with SERRATE and is part of an AGO1 complex. As a result, mutations in RACK1 lead to over-accumulation of miRNA target mRNAs, which are important for ABA responses and phyllotaxy, for example. In conclusion, our study identified complex functioning of RACK1 proteins in the Arabidopsis miRNA pathway; these proteins are important for miRNA production and therefore plant development.

Improving and correcting the contiguity of long-read genome assemblies of three plant species using optical mapping and chromosome conformation capture data

Long-read sequencing can overcome the weaknesses of short reads in the assembly of eukaryotic genomes; however, at present additional scaffolding is needed to achieve chromosome-level assemblies. We generated Pacific Biosciences (PacBio) long-read data of the genomes of three relatives of the model plant Arabidopsis thaliana and assembled all three genomes into only a few hundred contigs. To improve the contiguities of these assemblies, we generated BioNano Genomics optical mapping and Dovetail Genomics chromosome conformation capture data for genome scaffolding. Despite their technical differences, optical mapping and chromosome conformation capture performed similarly and doubled N50 values. After improving both integration methods, assembly contiguity reached chromosome-arm-levels. We rigorously assessed the quality of contigs and scaffolds using Illumina mate-pair libraries and genetic map information. This showed that PacBio assemblies have high sequence accuracy but can contain several misassemblies, which join unlinked regions of the genome. Most, but not all, of these misjoints were removed during the integration of the optical mapping and chromosome conformation capture data. Even though none of the centromeres were fully assembled, the scaffolds revealed large parts of some centromeric regions, even including some of the heterochromatic regions, which are not present in gold standard reference sequences.

UVR2 ensures transgenerational genome stability under simulated natural UV-B in Arabidopsis thaliana

Ground levels of solar UV-B radiation induce DNA damage. Sessile phototrophic organisms such as vascular plants are recurrently exposed to sunlight and require UV-B photoreception, flavonols shielding, direct reversal of pyrimidine dimers and nucleotide excision repair for resistance against UV-B radiation. However, the frequency of UV-B-induced mutations is unknown in plants. Here we quantify the amount and types of mutations in the offspring of Arabidopsis thaliana wild-type and UV-B-hypersensitive mutants exposed to simulated natural UV-B over their entire life cycle. We show that reversal of pyrimidine dimers by UVR2 photolyase is the major mechanism required for sustaining plant genome stability across generations under UV-B. In addition to widespread somatic expression, germline-specific UVR2 activity occurs during late flower development, and is important for ensuring low mutation rates in male and female cell lineages. This allows plants to maintain genome integrity in the germline despite exposure to UV-B.

Arabidopsis thaliana RNase H2 Deficiency Counteracts the Needs for the WEE1 Checkpoint Kinase but Triggers Genome Instability

Absence of the RNAse H2 complex, mediating the excision of ribonucleotides from DNA, was found to overcome the growth inhibitory phenotype of plants lacking a functional replication checkpoint because of tolerating the substitution of deoxynucleotides with ribonucleotides. This substitution, however, results in replication errors, demonstrating the need of RNase H2 activity for genome stability. The WEE1 kinase is an essential cell cycle checkpoint regulator in Arabidopsis thaliana plants experiencing replication defects. Whereas under non-stress conditions WEE1-deficient plants develop normally, they fail to adapt to replication inhibitory conditions, resulting in the accumulation of DNA damage and loss of cell division competence. We identified mutant alleles of the genes encoding subunits of the ribonuclease H2 (RNase H2) complex, known for its role in removing ribonucleotides from DNA-RNA duplexes, as suppressor mutants of WEE1 knockout plants. RNase H2 deficiency triggered an increase in homologous recombination (HR), correlated with the accumulation of γ-H2AX foci. However, as HR negatively impacts the growth of WEE1-deficient plants under replication stress, it cannot account for the rescue of the replication defects of the WEE1 knockout plants. Rather, the observed increase in ribonucleotide incorporation in DNA indicates that the substitution of deoxynucleotide with ribonucleotide abolishes the need for WEE1 under replication stress. Strikingly, increased ribonucleotide incorporation in DNA correlated with the occurrence of small base pair deletions, identifying the RNase H2 complex as an important suppressor of genome instability.

Reasons for the Invasive Success of a Guppy (Poecilia reticulata) Population in Trinidad

The introduction of non-native species into new habitats poses a major threat to native populations. Of particular interest, though often overlooked, are introductions of populations that are not fully reproductively isolated from native individuals and can hybridize with them. To address this important topic we used different approaches in a multi-pronged study, combining the effects of mate choice, shoaling behaviour and genetics. Here we present evidence that behavioural traits such as shoaling and mate choice can promote population mixing if individuals do not distinguish between native and foreign conspecifics. We examined this in the context of two guppy (Poecilia reticulata) populations that have been subject to an introduction and subsequent population mixing event in Trinidad. The introduction of Guanapo River guppies into the Turure River more than 50 years ago led to a marked reduction of the original genotype. In our experiments, female guppies did not distinguish between shoaling partners when given the choice between native and foreign individuals. Introduced fish are therefore likely to benefit from the protection of a shoal and will improve their survival chances as a result. The additional finding that male guppies do not discriminate between females on the basis of origin will further increase the process of population mixing, especially if males encounter mixed shoals. In a mesocosm experiment, in which the native and foreign populations were allowed to mate freely, we found, as expected on the basis of these behavioural interactions, that the distribution of offspring genotypes could be predicted from the proportions of the two types of founding fish. This result suggests that stochastic and environmental processes have reinforced the biological ones to bring about the genetic dominance of the invading population in the Turure River. Re-sampling the Turure for genetic analysis using SNP markers confirmed the population mixing process and showed that it is an on-going process in this river and has led to the nearly complete disappearance of the original genotype.