Estimation of linkage disequilibrium and interspecific gene flow in Ficedula flycatchers by a newly developed 50k single‐nucleotide polymorphism array

With the access to draft genome sequence assemblies and whole‐genome resequencing data from population samples, molecular ecology studies will be able to take truly genome‐wide approaches. This now applies to an avian model system in ecological and evolutionary research: Old World flycatchers of the genus Ficedula, for which we recently obtained a 1.1 Gb collared flycatcher genome assembly and identified 13 million single‐nucleotide polymorphism (SNP)s in population resequencing of this species and its sister species, pied flycatcher. Here, we developed a custom 50K Illumina iSelect flycatcher SNP array with markers covering 30 autosomes and the Z chromosome. Using a number of selection criteria for inclusion in the array, both genotyping success rate and polymorphism information content (mean marker heterozygosity = 0.41) were high. We used the array to assess linkage disequilibrium (LD) and hybridization in flycatchers. Linkage disequilibrium declined quickly to the background level at an average distance of 17 kb, but the extent of LD varied markedly within the genome and was more than 10‐fold higher in ‘genomic islands’ of differentiation than in the rest of the genome. Genetic ancestry analysis identified 33 F₁ hybrids but no later‐generation hybrids from sympatric populations of collared flycatchers and pied flycatchers, contradicting earlier reports of backcrosses identified from much fewer number of markers. With an estimated divergence time as recently as <1 Ma, this suggests strong selection against F₁ hybrids and unusually rapid evolution of reproductive incompatibility in an avian system.     

Genomic distribution and estimation of nucleotide diversity in natural populations: perspectives from the collared flycatcher (Ficedula albicollis) genome

Properly estimating genetic diversity in populations of nonmodel species requires a basic understanding of how diversity is distributed across the genome and among individuals. To this end, we analysed whole‐genome resequencing data from 20 collared flycatchers (genome size ≈1.1 Gb; 10.13 million single nucleotide polymorphisms detected). Genomewide nucleotide diversity was almost identical among individuals (mean = 0.00394, range = 0.00384–0.00401), but diversity levels varied extensively across the genome (95% confidence interval for 200‐kb windows = 0.0013–0.0053). Diversity was related to selective constraint such that in comparison with intergenic DNA, diversity at fourfold degenerate sites was reduced to 85%, 3′ UTRs to 82%, 5′ UTRs to 70% and nondegenerate sites to 12%. There was a strong positive correlation between diversity and chromosome size, probably driven by a higher density of targets for selection on smaller chromosomes increasing the diversity‐reducing effect of linked selection. Simulations exploring the ability of sequence data from a small number of genetic markers to capture the observed diversity clearly demonstrated that diversity estimation from finite sampling of such data is bound to be associated with large confidence intervals. Nevertheless, we show that precision in diversity estimation in large outbred population benefits from increasing the number of loci rather than the number of individuals. Simulations mimicking RAD sequencing showed that this approach gives accurate estimates of genomewide diversity. Based on the patterns of observed diversity and the performed simulations, we provide broad recommendations for how genetic diversity should be estimated in natural populations.     

The easy road to genome‐wide medium density SNP screening in a non‐model species: development and application of a 10 K SNP‐chip for the house sparrow (Passer domesticus)

With the advent of next generation sequencing, new avenues have opened to study genomics in wild populations of non‐model species. Here, we describe a successful approach to a genome‐wide medium density Single Nucleotide Polymorphism (SNP) panel in a non‐model species, the house sparrow (Passer domesticus), through the development of a 10 K Illumina iSelect HD BeadChip. Genomic DNA and cDNA derived from six individuals were sequenced on a 454 GS FLX system and generated a total of 1.2 million sequences, in which SNPs were detected. As no reference genome exists for the house sparrow, we used the zebra finch (Taeniopygia guttata) reference genome to determine the most likely position of each SNP. The 10 000 SNPs on the SNP‐chip were selected to be distributed evenly across 31 chromosomes, giving on average one SNP per 100 000 bp. The SNP‐chip was screened across 1968 individual house sparrows from four island populations. Of the original 10 000 SNPs, 7413 were found to be variable, and 99% of these SNPs were successfully called in at least 93% of all individuals. We used the SNP‐chip to demonstrate the ability of such genome‐wide marker data to detect population sub‐division, and compared these results to similar analyses using microsatellites. The SNP‐chip will be used to map Quantitative Trait Loci (QTL) for fitness‐related phenotypic traits in natural populations.     

QTL mapping and GWAS reveal candidate genes controlling capsaicinoid content in Capsicum

Capsaicinoids are unique compounds produced only in peppers (Capsicum spp.). Several studies using classical quantitative trait loci (QTLs) mapping and genomewide association studies (GWAS) have identified QTLs controlling capsaicinoid content in peppers; however, neither the QTLs common to each population nor the candidate genes underlying them have been identified due to the limitations of each approach used. Here, we performed QTL mapping and GWAS for capsaicinoid content in peppers using two recombinant inbred line (RIL) populations and one GWAS population. Whole‐genome resequencing and genotyping by sequencing (GBS) were used to construct high‐density single nucleotide polymorphism (SNP) maps. Five QTL regions on chromosomes 1, 2, 3, 4 and 10 were commonly identified in both RIL populations over multiple locations and years. Furthermore, a total of 109 610 SNPs derived from two GBS libraries were used to analyse the GWAS population consisting of 208 C. annuum‐clade accessions. A total of 69 QTL regions were identified from the GWAS, 10 of which were co‐located with the QTLs identified from the two biparental populations. Within these regions, we were able to identify five candidate genes known to be involved in capsaicinoid biosynthesis. Our results demonstrate that QTL mapping and GBS‐GWAS represent a powerful combined approach for the identification of loci controlling complex traits.     

Abundant recent activity of retrovirus‐like retrotransposons within and among flycatcher species implies a rich source of structural variation in songbird genomes

Transposable elements (TEs) are genomic parasites capable of inserting virtually anywhere in the host genome, with manifold consequences for gene expression, DNA methylation and genomic stability. Notably, they can contribute to phenotypic variation and hence be associated with, for example, local adaptation and speciation. However, some organisms such as birds have been widely noted for the low densities of TEs in their genomes and this has been attributed to a potential dearth in transposition during their evolution. Here, we show that avian evolution witnessed diverse and abundant transposition on very recent timescales. First, we made an in‐depth repeat annotation of the collared flycatcher genome, including identification of 23 new, retrovirus‐like LTR retrotransposon families. Then, using whole‐genome resequencing data from 200 Ficedula flycatchers, we detected 11,888 polymorphic TE insertions (TE presence/absence variations, TEVs) that segregated within and among species. The density of TEVs was one every 1.5–2.5 Mb per individual, with heterozygosities of 0.12–0.16. The majority of TEVs belonged to some 10 different LTR families, most of which are specific to the flycatcher lineage. TEVs were validated by tracing the segregation of hundreds of TEVs across a three‐generation pedigree of collared flycatchers and also by their utility as markers recapitulating the phylogenetic relationships among flycatcher species. Our results suggest frequent germline invasions of songbird genomes by novel retroviruses as a rich source of structural variation, which may have had underappreciated phenotypic consequences for the diversification of this species‐rich group of birds.     

POPULATION DIVERGENCE IN SEXUAL ORNAMENTS: THE WHITE FOREHEAD PATCH OF NORWEGIAN PIED FLYCATCHERS IS SMALL AND UNSEXY

Models of sexual selection suggest that populations may easily diverge in male secondary sexual characters. Studies of a Spanish population of the pied flycatcher, Ficedula hypoleuca, and a Swedish population of the closely related collared flycatcher, F. albicollis, have indicated that the white forehead patch of males is a sexually selected trait. We studied the white forehead patch of male pied flycatchers (n = 487) in a Norwegian population over seven years. Males with large forehead patches were in general more brightly colored, but patch height was not correlated to body mass, body size, or parasite loads. Conditions during the nestling period did not seem to influence patch height as an adult. Patch height increased slightly from the first to the second year as adults, but then remained relatively constant at higher ages. Patch height was not related to survival. Year-to-year changes showed that males who increased in patch height also increased in body mass, suggesting that expression of the forehead patch may be partly condition dependent. However, changes in body mass explained only a small proportion of the variance in patch height between males. Thus, patch height would not be a good indicator of male quality. Furthermore, patch size was also not related to male ability to feed nestlings, indicating that females would not obtain direct benefits by choosing males with large patches. However, patch height could be a Fisher trait, but this requires heritability and there was no significant father-son resemblance in patch height. Comparisons of the males visited by each female during the mate sampling period indicated that chosen males did not have larger forehead patches than rejected males. Experimental manipulation of patch height did not affect male mating success. These results indicate that females do not use patch size as a mate choice cue. Finally, patch height did not predict the outcome of male contests for nestboxes, suggesting that the forehead patch is not an intrasexually selected cue of status. Norwegian pied flycatchers have smaller forehead patches than both Spanish pied flycatchers and Swedish collared flycatchers. We suggest that this pattern may be explained by the lack of sexual selection on the forehead patch in the Norwegian population as compared to the other populations, where the patch is apparently sexually selected. We discuss possible reasons for these population divergences, such as female choice on an alternative secondary sexual character (general plumage color) and speciation among Ficedula flycatchers.     

Power to detect risk alleles using genome-wide tag SNP panels

Advances in high-throughput genotyping and the International HapMap Project have enabled association studies at the whole-genome level. We have constructed whole-genome genotyping panels of over 550,000 (HumanHap550) and 650,000 (HumanHap650Y) SNP loci by choosing tag SNPs from all populations genotyped by the International HapMap Project. These panels also contain additional SNP content in regions that have historically been overrepresented in diseases, such as nonsynonymous sites, the MHC region, copy number variant regions and mitochondrial DNA. We estimate that the tag SNP loci in these panels cover the majority of all common variation in the genome as measured by coverage of both all common HapMap SNPs and an independent set of SNPs derived from complete resequencing of genes obtained from SeattleSNPs. We also estimate that, given a sample size of 1,000 cases and 1,000 controls, these panels have the power to detect single disease loci of moderate risk (λ ~ 1.8–2.0). Relative risks as low as λ ~ 1.1–1.3 can be detected using 10,000 cases and 10,000 controls depending on the sample population and disease model. If multiple loci are involved, the power increases significantly to detect at least one locus such that relative risks 20%–35% lower can be detected with 80% power if between two and four independent loci are involved. Although our SNP selection was based on HapMap data, which is a subset of all common SNPs, these panels effectively capture the majority of all common variation and provide high power to detect risk alleles that are not represented in the HapMap data.     

Malaria infections reinforce competitive asymmetry between two Ficedula flycatchers in a recent contact zone

Parasites may influence the outcome of interspecific competition between closely related host species through lower parasite virulence in the host with which they share the longer evolutionary history. We tested this idea by comparing the prevalence of avian malaria (Haemosporidia) lineages and their association with survival in pied and collared flycatchers (Ficedula hypoleuca and F. albicollis) breeding in a recent contact zone on the Swedish island of Öland. A nested PCR protocol amplifying haemosporidian fragments of mtDNA was used to screen the presence of malaria lineages in 1048 blood samples collected during 6 years. Competitively inferior pied flycatchers had a higher prevalence of blood parasites, including the lineages that were shared between the two flycatcher species. Multistate mark–recapture models revealed a lower survival of infected versus uninfected female pied flycatchers, while no such effects were detected in male pied flycatchers or in collared flycatchers of either sex. Our results show that a comparatively new host, the collared flycatcher, appears to be less susceptible to a local northern European malarial lineage where the collared flycatchers have recently expanded their distribution. Pied flycatchers experience strong reproductive interference from collared flycatchers, and the additional impact of species‐specific blood parasite effects adds to this competitive exclusion. These results support the idea that parasites can strongly influence the outcome of interspecific competition between closely related host species, but that the invading species need not necessarily be more susceptible to local parasites.     

Single-nucleotide polymorphism characterization in species with limited available sequence information: high nucleotide diversity revealed in the avian genome

As a case study for single-nucleotide polymorphism (SNP) identification in species for which little or no sequence information is available, we investigated several approaches to identifying SNPs in two passerine bird species: pied and collared flycatchers (Ficedula hypoleuca and F. albicollis). All approaches were successful in identifying sequence polymorphism and over 50 candidate SNPs per species were identified from approximately 9.1 kb of sequence. In addition, 17 sites were identified in which the frequency of alternative bases differed by > 50% between species (termed interspecific SNPs). Interestingly, polymorphism of microsatellite/intron loci in the source species appeared to be a positive predictor of nucleotide diversity in homologous flycatcher sequences. The overall nucleotide diversity of flycatchers was 2.3-2.7 x 10(-3), which is approximately 3-6 times higher than observed in recent studies of human SNPs. Higher nucleotide diversity in the avian genome could be due to the relatively older age of flycatcher populations, compared with humans, and/or a higher long-term effective population size.     

PSMC analysis of effective population sizes in molecular ecology and its application to black‐and‐white Ficedula flycatchers

Climatic fluctuations during the Quaternary period governed the demography of species and contributed to population differentiation and ultimately speciation. Studies of these past processes have previously been hindered by a lack of means and genetic data to model changes in effective population size (N_e) through time. However, based on diploid genome sequences of high quality, the recently developed pairwise sequentially Markovian coalescent (PSMC) can estimate trajectories of changes in N_e over considerable time periods. We applied this approach to resequencing data from nearly 200 genomes of four species and several populations of the Ficedula species complex of black‐and‐white flycatchers. N_e curves of Atlas, collared, pied and semicollared flycatcher converged 1–2 million years ago (Ma) at an N_e of ≈ 200 000, likely reflecting the time when all four species last shared a common ancestor. Subsequent separate N_e trajectories are consistent with lineage splitting and speciation. All species showed evidence of population growth up until 100–200 thousand years ago (kya), followed by decline and then start of a new phase of population expansion. However, timing and amplitude of changes in N_e differed among species, and for pied flycatcher, the temporal dynamics of N_e differed between Spanish birds and central/northern European populations. This cautions against extrapolation of demographic inference between lineages and calls for adequate sampling to provide representative pictures of the coalescence process in different species or populations. We also empirically evaluate criteria for proper inference of demographic histories using PSMC and arrive at recommendations of using sequencing data with a mean genome coverage of ≥18X, a per‐site filter of ≥10 reads and no more than 25% of missing data.     

New resources for genetic studies in Populus nigra: genome‐wide SNP discovery and development of a 12k Infinium array

Whole genome resequencing of 51 Populus nigra (L.) individuals from across Western Europe was performed using Illumina platforms. A total number of 1 878 727 SNPs distributed along the P. nigra reference sequence were identified. The SNP calling accuracy was validated with Sanger sequencing. SNPs were selected within 14 previously identified QTL regions, 2916 expressional candidate genes related to rust resistance, wood properties, water‐use efficiency and bud phenology and 1732 genes randomly spread across the genome. Over 10 000 SNPs were selected for the construction of a 12k Infinium Bead‐Chip array dedicated to association mapping. The SNP genotyping assay was performed with 888 P. nigra individuals. The genotyping success rate was 91%. Our high success rate was due to the discovery panel design and the stringent parameters applied for SNP calling and selection. In the same set of P. nigra genotypes, linkage disequilibrium throughout the genome decayed on average within 5–7 kb to half of its maximum value. As an application test, ADMIXTURE analysis was performed with a selection of 600 SNPs spread throughout the genome and 706 individuals collected along 12 river basins. The admixture pattern was consistent with genetic diversity revealed by neutral markers and the geographical distribution of the populations. These newly developed SNP resources and genotyping array provide a valuable tool for population genetic studies and identification of QTLs through natural‐population based genetic association studies in P. nigra.     

Genomewide single nucleotide polymorphism discovery in Atlantic salmon (Salmo salar): validation in wild and farmed American and European populations

A considerable number of single nucleotide polymorphisms (SNPs) are required to elucidate genotype–phenotype associations and determine the molecular basis of important traits. In this work, we carried out de novo SNP discovery accounting for both genome duplication and genetic variation from American and European salmon populations. A total of 9 736 473 nonredundant SNPs were identified across a set of 20 fish by whole‐genome sequencing. After applying six bioinformatic filtering steps, 200 K SNPs were selected to develop an Affymetrix Axiom^® myDesign Custom Array. This array was used to genotype 480 fish representing wild and farmed salmon from Europe, North America and Chile. A total of 159 099 (79.6%) SNPs were validated as high quality based on clustering properties. A total of 151 509 validated SNPs showed a unique position in the genome. When comparing these SNPs against 238 572 markers currently available in two other Atlantic salmon arrays, only 4.6% of the SNP overlapped with the panel developed in this study. This novel high‐density SNP panel will be very useful for the dissection of economically and ecologically relevant traits, enhancing breeding programmes through genomic selection as well as supporting genetic studies in both wild and farmed populations of Atlantic salmon using high‐resolution genomewide information.     

Single‐nucleotide polymorphism discovery and diversity in the model legume Medicago truncatula

Extensive genomic resources are available in the model legume Medicago truncatula. Here, we present the discovery and design of the first array of single‐nucleotide polymorphism (SNP) markers in M. truncatula through large‐scale Sanger resequencing of genomic fragments spanning the genome, in a diverse panel of 16 M. truncatula accessions. Both anonymous fragments and fragments targeting candidate genes for flowering phenology and symbiosis were surveyed for nucleotide variation in almost 230 kb of unique genomic regions. A set of 384 SNP markers was designed for an Illumina's GoldenGate assay, genotyped on a collection of 192 inbred lines (CC192) representing the geographical range of the species and used to survey the diversity of two natural populations. Finally, 86% of the tested SNPs were of high quality and exhibited polymorphism in the CC192 collection. Even at the population level, we detected polymorphism for more than 50% of the selected SNPs. Analysis of the allele frequency spectrum in the CC192 showed a reduced ascertainment bias, mostly limited to very rare alleles (frequency <0.01). The substantial polymorphism detected at the species and population levels, the high marker quality and the potential to survey large samples of individuals make this set of SNP markers a valuable tool to improve our understanding of the effect of demographic and selective factors that shape the natural genetic diversity within the selfing species Medicago truncatula.     

SNP panel development for genetic management of wild and domesticated white bass (Morone chrysops)

White bass (Morone chrysops), striped bass and their interspecific hybrid are important game fishes, whereas the hybrid striped bass is an important aquaculture species in the US. Numerous state, federal and private hatcheries, therefore, rear these species for stocking purposes as well as for food fish. Although striped bass populations (both wild and domesticated) have been extensively evaluated, relatively little effort has been directed toward the study and improvement of white bass. In this study, we developed SNP resources to examine the genetic relationships among a long‐term domesticated white bass line and five potential founder stocks for selective breeding collected from drainages in Arkansas, Texas and Alabama. Using genotyping‐by‐sequencing, we generated 13 872 genome‐wide SNP loci across the six populations. Stringent filtering of SNP‐calling parameters identified 426 informative SNP loci. Population genetic and structure analyses using these loci revealed only moderate genetic differentiation between populations (global F_st = 0.083) and indicated two major genetic clusters. A final 57‐SNP assay was successfully designed and validated using the MassARRAY system. The developed SNP panel assigned 96 additional genotyped individuals to their population of origin with 100% accuracy. The SNP resources developed in this study should facilitate ongoing efforts in selective breeding and conservation of white bass.     

Outlier SNP markers reveal fine‐scale genetic structuring across European hake populations (Merluccius merluccius)

Shallow population structure is generally reported for most marine fish and explained as a consequence of high dispersal, connectivity and large population size. Targeted gene analyses and more recently genome‐wide studies have challenged such view, suggesting that adaptive divergence might occur even when neutral markers provide genetic homogeneity across populations. Here, 381 SNPs located in transcribed regions were used to assess large‐ and fine‐scale population structure in the European hake (Merluccius merluccius), a widely distributed demersal species of high priority for the European fishery. Analysis of 850 individuals from 19 locations across the entire distribution range showed evidence for several outlier loci, with significantly higher resolving power. While 299 putatively neutral SNPs confirmed the genetic break between basins (F_CT = 0.016) and weak differentiation within basins, outlier loci revealed a dramatic divergence between Atlantic and Mediterranean populations (F_CT range 0.275–0.705) and fine‐scale significant population structure. Outlier loci separated North Sea and Northern Portugal populations from all other Atlantic samples and revealed a strong differentiation among Western, Central and Eastern Mediterranean geographical samples. Significant correlation of allele frequencies at outlier loci with seawater surface temperature and salinity supported the hypothesis that populations might be adapted to local conditions. Such evidence highlights the importance of integrating information from neutral and adaptive evolutionary patterns towards a better assessment of genetic diversity. Accordingly, the generated outlier SNP data could be used for tackling illegal practices in hake fishing and commercialization as well as to develop explicit spatial models for defining management units and stock boundaries.     

The importance of replicating genomic analyses to verify phylogenetic signal for recently evolved lineages

Genomewide SNP data generated by nontargeted methods such as RAD and GBS are increasingly being used in phylogenetic and phylogeographic analyses. When these methods are used in the absence of a reference genome, however, little is known about the locations and evolution of the SNPs. In using such data to address phylogenetic questions, researchers risk drawing false conclusions, particularly if a representative number of SNPs is not obtained. Here, we empirically test the robustness of phylogenetic inference based on SNP data for closely related lineages. We conducted a genomewide analysis of 75 712 SNPs, generated via GBS, of southern bull‐kelp (Durvillaea). Durvillaea chathamensis co‐occurs with D. antarctica on Chatham Island, but the two species have previously been found to be so genetically similar that the status of the former has been questioned. Our results show that D. chathamensis, which differs from D. antarctica ecologically as well as morphologically, is indeed a reproductively isolated species. Furthermore, our replicated analyses show that D. chathamensis cannot be reliably distinguished phylogenetically from closely related D. antarctica using subsets (ranging in size from 400 to 10 000 sites) of the 40 912 parsimony‐informative SNPs in our data set and that bootstrap values alone can give misleading impressions of the strength of phylogenetic inferences. These results highlight the importance of independently replicating SNP analyses to verify that phylogenetic inferences based on nontargeted SNP data are robust. Our study also demonstrates that modern genomic approaches can be used to identify cases of recent or incipient speciation that traditional approaches (e.g. Sanger sequencing of a few loci) may be unable to detect or resolve.     

A resource of genome‐wide single‐nucleotide polymorphisms generated by RAD tag sequencing in the critically endangered European eel

Reduced representation genome sequencing such as restriction‐site‐associated DNA (RAD) sequencing is finding increased use to identify and genotype large numbers of single‐nucleotide polymorphisms (SNPs) in model and nonmodel species. We generated a unique resource of novel SNP markers for the European eel using the RAD sequencing approach that was simultaneously identified and scored in a genome‐wide scan of 30 individuals. Whereas genomic resources are increasingly becoming available for this species, including the recent release of a draft genome, no genome‐wide set of SNP markers was available until now. The generated SNPs were widely distributed across the eel genome, aligning to 4779 different contigs and 19 703 different scaffolds. Significant variation was identified, with an average nucleotide diversity of 0.00529 across individuals. Results varied widely across the genome, ranging from 0.00048 to 0.00737 per locus. Based on the average nucleotide diversity across all loci, long‐term effective population size was estimated to range between 132 000 and 1 320 000, which is much higher than previous estimates based on microsatellite loci. The generated SNP resource consisting of 82 425 loci and 376 918 associated SNPs provides a valuable tool for future population genetics and genomics studies and allows for targeting specific genes and particularly interesting regions of the eel genome.     

Population structure and local selection yield high genomic variation in Mimulus guttatus

Across western North America, Mimulus guttatus exists as many local populations adapted to site‐specific environmental challenges. Gene flow between locally adapted populations will affect genetic diversity both within demes and across the larger metapopulation. Here, we analyse 34 whole‐genome sequences from the intensively studied Iron Mountain population (IM) in conjunction with sequences from 22 Mimulus individuals sampled from across western North America. Three striking features of these data address hypotheses about migration and selection in a locally adapted population. First, we find very high levels of intrapopulation polymorphism (synonymous π = 0.033). Variation outside of genes is likely even higher but difficult to estimate because excessive divergence reduces the efficiency of read mapping. Second, IM exhibits a significantly positive genomewide average for Tajima's D. This indicates allele frequencies are typically more intermediate than expected from neutrality, opposite the pattern observed in many other species. Third, IM exhibits a distinctive haplotype structure with a genomewide excess of positive associations between rarer alleles at linked loci. This suggests an important effect of gene flow from other Mimulus populations, although a residual effect of population founding might also contribute. The combination of multiple analyses, including a novel tree‐based analytic method, illustrates how the balance of local selection, limited dispersal and metapopulation dynamics manifests across the genome. The overall genomic pattern of sequence diversity suggests successful gene flow of divergent immigrant genotypes into IM. However, many loci show patterns indicative of local adaptation, particularly at SNPs associated with chromosomal inversions.