Characterizing restriction enzyme‐associated loci in historic ragweed (Ambrosia artemisiifolia) voucher specimens using custom‐designed RNA probes

Population genetic studies of nonmodel organisms frequently employ reduced representation library (RRL) methodologies, many of which rely on protocols in which genomic DNA is digested by one or more restriction enzymes. However, because high molecular weight DNA is recommended for these protocols, samples with degraded DNA are generally unsuitable for RRL methods. Given that ancient and historic specimens can provide key temporal perspectives to evolutionary questions, we explored how custom‐designed RNA probes could enrich for RRL loci (Restriction Enzyme‐Associated Loci baits, or REALbaits). Starting with genotyping‐by‐sequencing (GBS) data generated on modern common ragweed (Ambrosia artemisiifolia L.) specimens, we designed 20 000 RNA probes to target well‐characterized genomic loci in herbarium voucher specimens dating from 1835 to 1913. Compared to shotgun sequencing, we observed enrichment of the targeted loci at 19‐ to 151‐fold. Using our GBS capture pipeline on a data set of 38 herbarium samples, we discovered 22 813 SNPs, providing sufficient genomic resolution to distinguish geographic populations. For these samples, we found that dilution of REALbaits to 10% of their original concentration still yielded sufficient data for downstream analyses and that a sequencing depth of ~7m reads was sufficient to characterize most loci without wasting sequencing capacity. In addition, we observed that targeted loci had highly variable rates of success, which we primarily attribute to similarity between loci, a trait that ultimately interferes with unambiguous read mapping. Our findings can help researchers design capture experiments for RRL loci, thereby providing an efficient means to integrate samples with degraded DNA into existing RRL data sets.     

Long‐read sequence capture of the haemoglobin gene clusters across codfish species

Combining high‐throughput sequencing with targeted sequence capture has become an attractive tool to study specific genomic regions of interest. Most studies have so far focused on the exome using short‐read technology. These approaches are not designed to capture intergenic regions needed to reconstruct genomic organization, including regulatory regions and gene synteny. Here, we demonstrate the power of combining targeted sequence capture with long‐read sequencing technology for comparative genomic analyses of the haemoglobin (Hb) gene clusters across eight species separated by up to 70 million years. Guided by the reference genome assembly of the Atlantic cod (Gadus morhua) together with genome information from draft assemblies of selected codfishes, we designed probes covering the two Hb gene clusters. Use of custom‐made barcodes combined with PacBio RSII sequencing led to highly continuous assemblies of the LA (~100 kb) and MN (~200 kb) clusters, which include syntenic regions of coding and intergenic sequences. Our results revealed an overall conserved genomic organization of the Hb genes within this lineage, yet with several, lineage‐specific gene duplications. Moreover, for some of the species examined, we identified amino acid substitutions at two sites in the Hbb1 gene as well as length polymorphisms in its regulatory region, which has previously been linked to temperature adaptation in Atlantic cod populations. This study highlights the use of targeted long‐read capture as a versatile approach for comparative genomic studies by generation of a cross‐species genomic resource elucidating the evolutionary history of the Hb gene family across the highly divergent group of codfishes.     

Inferring the mode of origin of polyploid species from next‐generation sequence data

Many eukaryote organisms are polyploid. However, despite their importance, evolutionary inference of polyploid origins and modes of inheritance has been limited by a need for analyses of allele segregation at multiple loci using crosses. The increasing availability of sequence data for nonmodel species now allows the application of established approaches for the analysis of genomic data in polyploids. Here, we ask whether approximate Bayesian computation (ABC), applied to realistic traditional and next‐generation sequence data, allows correct inference of the evolutionary and demographic history of polyploids. Using simulations, we evaluate the robustness of evolutionary inference by ABC for tetraploid species as a function of the number of individuals and loci sampled, and the presence or absence of an outgroup. We find that ABC adequately retrieves the recent evolutionary history of polyploid species on the basis of both old and new sequencing technologies. The application of ABC to sequence data from diploid and polyploid species of the plant genus Capsella confirms its utility. Our analysis strongly supports an allopolyploid origin of C. bursa‐pastoris about 80 000 years ago. This conclusion runs contrary to previous findings based on the same data set but using an alternative approach and is in agreement with recent findings based on whole‐genome sequencing. Our results indicate that ABC is a promising and powerful method for revealing the evolution of polyploid species, without the need to attribute alleles to a homeologous chromosome pair. The approach can readily be extended to more complex scenarios involving higher ploidy levels.     

Small‐ and large‐scale heterogeneity in genetic variation across the collard flycatcher genome: implications for estimating genetic diversity in nonmodel organisms

Population genetic studies in nonmodel organisms are often hampered by a lack of reference genomes that are essential for whole‐genome resequencing. In the light of this, genotyping methods have been developed to effectively eliminate the need for a reference genome, such as genotyping by sequencing or restriction site‐associated DNA sequencing (RAD‐seq). However, what remains relatively poorly studied is how accurately these methods capture both average and variation in genetic diversity across an organism's genome. In this issue of Molecular Ecology Resources, Dutoit et al. (2016) use whole‐genome resequencing data from the collard flycatcher to assess what factors drive heterogeneity in nucleotide diversity across the genome. Using these data, they then simulate how well different sequencing designs, including RAD sequencing, could capture most of the variation in genetic diversity. They conclude that for evolutionary and conservation‐related studies focused on the estimating genomic diversity, researchers should emphasize the number of loci analysed over the number of individuals sequenced.     

Helping decision making for reliable and cost‐effective 2b‐RAD sequencing and genotyping analyses in non‐model species

High‐throughput sequencing has revolutionized population and conservation genetics. RAD sequencing methods, such as 2b‐RAD, can be used on species lacking a reference genome. However, transferring protocols across taxa can potentially lead to poor results. We tested two different IIB enzymes (AlfI and CspCI) on two species with different genome sizes (the loggerhead turtle Caretta caretta and the sharpsnout seabream Diplodus puntazzo) to build a set of guidelines to improve 2b‐RAD protocols on non‐model organisms while optimising costs. Good results were obtained even with degraded samples, showing the value of 2b‐RAD in studies with poor DNA quality. However, library quality was found to be a critical parameter on the number of reads and loci obtained for genotyping. Resampling analyses with different number of reads per individual showed a trade‐off between number of loci and number of reads per sample. The resulting accumulation curves can be used as a tool to calculate the number of sequences per individual needed to reach a mean depth ≥20 reads to acquire good genotyping results. Finally, we demonstrated that selective‐base ligation does not affect genomic differentiation between individuals, indicating that this technique can be used in species with large genome sizes to adjust the number of loci to the study scope, to reduce sequencing costs and to maintain suitable sequencing depth for a reliable genotyping without compromising the results. Here, we provide a set of guidelines to improve 2b‐RAD protocols on non‐model organisms with different genome sizes, helping decision‐making for a reliable and cost‐effective genotyping.     

RAD‐sequencing for estimating genomic relatedness matrix‐based heritability in the wild: A case study in roe deer

Estimating the evolutionary potential of quantitative traits and reliably predicting responses to selection in wild populations are important challenges in evolutionary biology. The genomic revolution has opened up opportunities for measuring relatedness among individuals with precision, enabling pedigree‐free estimation of trait heritabilities in wild populations. However, until now, most quantitative genetic studies based on a genomic relatedness matrix (GRM) have focused on long‐term monitored populations for which traditional pedigrees were also available, and have often had access to knowledge of genome sequence and variability. Here, we investigated the potential of RAD‐sequencing for estimating heritability in a free‐ranging roe deer (Capreolous capreolus) population for which no prior genomic resources were available. We propose a step‐by‐step analytical framework to optimize the quality and quantity of the genomic data and explore the impact of the single nucleotide polymorphism (SNP) calling and filtering processes on the GRM structure and GRM‐based heritability estimates. As expected, our results show that sequence coverage strongly affects the number of recovered loci, the genotyping error rate and the amount of missing data. Ultimately, this had little effect on heritability estimates and their standard errors, provided that the GRM was built from a minimum number of loci (above 7,000). Genomic relatedness matrix‐based heritability estimates thus appear robust to a moderate level of genotyping errors in the SNP data set. We also showed that quality filters, such as the removal of low‐frequency variants, affect the relatedness structure of the GRM, generating lower h² estimates. Our work illustrates the huge potential of RAD‐sequencing for estimating GRM‐based heritability in virtually any natural population.     

Whole‐exome targeted sequencing of the uncharacterized pine genome

The large genome size of many species hinders the development and application of genomic tools to study them. For instance, loblolly pine (Pinus taeda L.), an ecologically and economically important conifer, has a large and yet uncharacterized genome of 21.7 Gbp. To characterize the pine genome, we performed exome capture and sequencing of 14 729 genes derived from an assembly of expressed sequence tags. Efficiency of sequence capture was evaluated and shown to be similar across samples with increasing levels of complexity, including haploid cDNA, haploid genomic DNA and diploid genomic DNA. However, this efficiency was severely reduced for probes that overlapped multiple exons, presumably because intron sequences hindered probe:exon hybridizations. Such regions could not be entirely avoided during probe design, because of the lack of a reference sequence. To improve the throughput and reduce the cost of sequence capture, a method to multiplex the analysis of up to eight samples was developed. Sequence data showed that multiplexed capture was reproducible among 24 haploid samples, and can be applied for high‐throughput analysis of targeted genes in large populations. Captured sequences were de novo assembled, resulting in 11 396 expanded and annotated gene models, significantly improving the knowledge about the pine gene space. Interspecific capture was also evaluated with over 98% of all probes designed from P. taeda that were efficient in sequence capture, were also suitable for analysis of the related species Pinus elliottii Engelm.     

Barley whole exome capture: a tool for genomic research in the genus Hordeum and beyond

Advanced resources for genome‐assisted research in barley (Hordeum vulgare) including a whole‐genome shotgun assembly and an integrated physical map have recently become available. These have made possible studies that aim to assess genetic diversity or to isolate single genes by whole‐genome resequencing and in silico variant detection. However such an approach remains expensive given the 5 Gb size of the barley genome. Targeted sequencing of the mRNA‐coding exome reduces barley genomic complexity more than 50‐fold, thus dramatically reducing this heavy sequencing and analysis load. We have developed and employed an in‐solution hybridization‐based sequence capture platform to selectively enrich for a 61.6 megabase coding sequence target that includes predicted genes from the genome assembly of the cultivar Morex as well as publicly available full‐length cDNAs and de novo assembled RNA‐Seq consensus sequence contigs. The platform provides a highly specific capture with substantial and reproducible enrichment of targeted exons, both for cultivated barley and related species. We show that this exome capture platform provides a clear path towards a broader and deeper understanding of the natural variation residing in the mRNA‐coding part of the barley genome and will thus constitute a valuable resource for applications such as mapping‐by‐sequencing and genetic diversity analyzes.     

Targeted multiplex next‐generation sequencing: advances in techniques of mitochondrial and nuclear DNA sequencing for population genomics

Next‐generation sequencing (NGS) is emerging as an efficient and cost‐effective tool in population genomic analyses of nonmodel organisms, allowing simultaneous resequencing of many regions of multi‐genomic DNA from multiplexed samples. Here, we detail our synthesis of protocols for targeted resequencing of mitochondrial and nuclear loci by generating indexed genomic libraries for multiplexing up to 100 individuals in a single sequencing pool, and then enriching the pooled library using custom DNA capture arrays. Our use of DNA sequence from one species to capture and enrich the sequencing libraries of another species (i.e. cross‐species DNA capture) indicates that efficient enrichment occurs when sequences are up to about 12% divergent, allowing us to take advantage of genomic information in one species to sequence orthologous regions in related species. In addition to a complete mitochondrial genome on each array, we have included between 43 and 118 nuclear loci for low‐coverage sequencing of between 18 kb and 87 kb of DNA sequence per individual for single nucleotide polymorphisms discovery from 50 to 100 individuals in a single sequencing lane. Using this method, we have generated a total of over 500 whole mitochondrial genomes from seven cetacean species and green sea turtles. The greater variation detected in mitogenomes relative to short mtDNA sequences is helping to resolve genetic structure ranging from geographic to species‐level differences. These NGS and analysis techniques have allowed for simultaneous population genomic studies of mtDNA and nDNA with greater genomic coverage and phylogeographic resolution than has previously been possible in marine mammals and turtles.     

Contrasting patterns of genome‐wide polymorphism in the native and invasive range of the marine mollusc Crepidula fornicata

Selection processes are believed to be an important evolutionary driver behind the successful establishment of nonindigenous species, for instance through adaptation for invasiveness (e.g. dispersal mechanisms and reproductive allocation). However, evidence supporting this assumption is still scarce. Genome scans have often identified loci with atypical patterns of genetic differentiation (i.e. outliers) indicative of selection processes. Using microsatellite‐ and AFLP‐based genome scans, we looked for evidence of selection following the introduction of the mollusc Crepidula fornicata. Native to the northwestern Atlantic, this gastropod has become an emblematic invader since its introduction during the 19th and 20th centuries in the northeastern Atlantic and northeastern Pacific. We examined 683 individuals from seven native and 15 introduced populations spanning the latitudinal introduction and native ranges of the species. Our results confirmed the previously documented high genetic diversity in native and introduced populations with little genetic structure between the two ranges, a pattern typical of marine invaders. Analysing 344 loci, no outliers were detected between the introduced and native populations or in the introduced range. The genomic sampling may have been insufficient to reveal selection especially if it acts on traits determined by a few genes. Eight outliers were, however, identified within the native range, underlining a genetic singularity congruent with a well‐known biogeographical break along the Florida. Our results call into question the relevance of AFLP genome scans in detecting adaptation on the timescale of biological invasions: genome scans often reveal long‐term adaptation involving numerous genes throughout the genome but seem less effective in detecting recent adaptation from pre‐existing variation on polygenic traits. This study advocates other methods to detect selection effects during biological invasions—for example on phenotypic traits, although genome scans may remain useful for elucidating introduction histories.     

SimRAD: an R package for simulation‐based prediction of the number of loci expected in RADseq and similar genotyping by sequencing approaches

Application of high‐throughput sequencing platforms in the field of ecology and evolutionary biology is developing quickly with the introduction of efficient methods to reduce genome complexity. Numerous approaches for genome complexity reduction have been developed using different combinations of restriction enzymes, library construction strategies and fragment size selection. As a result, the choice of which techniques to use may become cumbersome, because it is difficult to anticipate the number of loci resulting from each method. We developed SimRAD, an R package that performs in silico restriction enzyme digests and fragment size selection as implemented in most restriction site associated DNA polymorphism and genotyping by sequencing methods. In silico digestion is performed on a reference genome or on a randomly generated DNA sequence when no reference genome sequence is available. SimRAD accurately predicts the number of loci under alternative protocols when a reference genome sequence is available for the targeted species (or a close relative) but may be unreliable when no reference genome is available. SimRAD is also useful for fine‐tuning a given protocol to adjust the number of targeted loci. Here, we outline the functionality of SimRAD and provide an illustrative example of the use of the package (available on the CRAN at http://cran.r-project.org/web/packages/SimRAD ).     

Heterogeneity and concordance in locus‐specific differentiation and introgression between species of towhees

The maintenance or breakdown of reproductive isolation is an observable outcome of secondary contact between species. In cases where hybrids beyond the F1 are formed, the representation of each species' ancestry can vary dramatically among genomic regions. This genomic heterogeneity in ancestry and introgression can offer insight into evolutionary processes, particularly if introgression is compared in multiple hybrid zones. Similarly, considerable heterogeneity exists across the genome in the extent to which populations and species have diverged, reflecting the combined effects of different evolutionary processes on genetic variation. We studied hybridization across two hybrid zones of two phenotypically well‐differentiated bird species in Mexico (Pipilo maculatus and P. ocai), to investigate genomic heterogeneity in differentiation and introgression. Using genotyping‐by‐sequencing (GBS) and hierarchical Bayesian models, we genotyped 460 birds at over 41 000 single nucleotide polymorphism (SNP) loci. We identified loci exhibiting extreme introgression relative to the genome‐wide expectation using a Bayesian genomic cline model. We also estimated locus‐specific F_ST and identified loci with exceptionally high genetic divergence between the parental species. We found some concordance of locus‐specific introgression in the two independent hybrid zones (6–20% of extreme loci shared across zones), reflecting areas of the genome that experience similar gene flow when the species interact. Additionally, heterogeneity in introgression and divergence across the genome revealed another subset of loci under the influence of locally specific factors. These results are consistent with a history in which reproductive isolation has been influenced by a common set of loci in both hybrid zones, but where local environmental and stochastic factors also lead to genomic differentiation.     

Genomic variation in cline shape across a hybrid zone

Hybrid zones are unique biological interfaces that reveal both population level and species level evolutionary processes. A genome‐scale approach to assess gene flow across hybrid zones is vital, and now possible. In Mexican towhees (genus Pipilo), several morphological hybrid gradients exist. We completed a genome survey across one such gradient (9 populations, 140 birds) using mitochondrial DNA, 28 isozyme, and 377 AFLP markers. To assess variation in introgression among loci, cline parameters (i.e., width, center) for the 61 clinally varying loci were estimated and compiled into genomic distributions for tests against three empirical models spanning the range of observed cline shape. No single model accounts for observed variation in cline shape among loci. Numerous backcross individuals near the gradient center confirm a hybrid origin for these populations, contrary to a previous hypothesis based on social mimicry and character displacement. In addition, the observed variation does not bin into well‐defined categories of locus types (e.g., neutral vs. highly selected). Our multi‐locus analysis reveals cross‐genomic variation in selective constraints on gene flow and locus‐specific flexibility in the permeability of the interspecies membrane.     

Universal target‐enrichment baits for anthozoan (Cnidaria) phylogenomics: New approaches to long‐standing problems

Anthozoans (e.g., corals, anemones) are an ecologically important and diverse group of marine metazoans that occur from shallow to deep waters worldwide. However, our understanding of the evolutionary relationships among the ~7,500 species within this class is hindered by the lack of phylogenetically informative markers that can be reliably sequenced across a diversity of taxa. We designed and tested 16,306 RNA baits to capture 720 ultraconserved element loci and 1,071 exon loci. Library preparation and target enrichment were performed on 33 taxa from all orders within the class Anthozoa. Following Illumina sequencing and Trinity assembly, we recovered 1,774 of 1,791 targeted loci. The mean number of loci recovered from each species was 638 ± 222, with more loci recovered from octocorals (783 ± 138 loci) than hexacorals (475 ± 187 loci). Parsimony informative sites ranged from 26 to 49% for alignments at differing hierarchical taxonomic levels (e.g., Anthozoa, Octocorallia, Hexacorallia). The per cent of variable sites within each of three genera (Acropora, Alcyonium, and Sinularia) for which multiple species were sequenced ranged from 4.7% to 30%. Maximum‐likelihood analyses recovered highly resolved trees with topologies matching those supported by other studies, including the monophyly of the order Scleractinia. Our results demonstrate the utility of this target‐enrichment approach to resolve phylogenetic relationships from relatively old to recent divergences. Redesigning the baits with improved affinities to capture loci within each subclass will provide a valuable toolset to address systematic questions, further our understanding of the timing of diversifications and help resolve long‐standing controversial relationships in the class Anthozoa.     

Chromosome‐level genome assembly of Triplophysa tibetana,a fish adapted to the harsh high‐altitude environment of the Tibetan Plateau

Triplophysa is an endemic fish genus of the Tibetan Plateau in China. Triplophysa tibetana, which lives at a recorded altitude of ~4,000 m and plays an important role in the highland aquatic ecosystem, serves as an excellent model for investigating high‐altitude environmental adaptation. However, evolutionary and conservation studies of T. tibetana have been limited by scarce genomic resources for the genus Triplophysa. In the present study, we applied PacBio sequencing and the Hi‐C technique to assemble the T. tibetana genome. A 652‐Mb genome with 1,325 contigs with an N50 length of 3.1 Mb was obtained. The 1,137 contigs were further assembled into 25 chromosomes, representing 98.7% and 80.47% of all contigs at the base and sequence number level, respectively. Approximately 260 Mb of sequence, accounting for ~39.8% of the genome, was identified as repetitive elements. DNA transposons (16.3%), long interspersed nuclear elements (12.4%) and long terminal repeats (11.0%) were the most repetitive types. In total, 24,372 protein‐coding genes were predicted in the genome, and ~95% of the genes were functionally annotated via a search in public databases. Using whole genome sequence information, we found that T. tibetana diverged from its common ancestor with Danio rerio ~121.4 million years ago. The high‐quality genome assembled in this work not only provides a valuable genomic resource for future population and conservation studies of T. tibetana, but it also lays a solid foundation for further investigation into the mechanisms of environmental adaptation of endemic fishes in the Tibetan Plateau.     

Resistance gene enrichment sequencing (RenSeq) enables reannotation of the NB‐LRR gene family from sequenced plant genomes and rapid mapping of resistance loci in segregating populations

RenSeq is a NB‐LRR (nucleotide binding‐site leucine‐rich repeat) gene‐targeted, Resistance gene enrichment and sequencing method that enables discovery and annotation of pathogen resistance gene family members in plant genome sequences. We successfully applied RenSeq to the sequenced potato Solanum tuberosum clone DM, and increased the number of identified NB‐LRRs from 438 to 755. The majority of these identified R gene loci reside in poorly or previously unannotated regions of the genome. Sequence and positional details on the 12 chromosomes have been established for 704 NB‐LRRs and can be accessed through a genome browser that we provide. We compared these NB‐LRR genes and the corresponding oligonucleotide baits with the highest sequence similarity and demonstrated that ~80% sequence identity is sufficient for enrichment. Analysis of the sequenced tomato S. lycopersicum ‘Heinz 1706’ extended the NB‐LRR complement to 394 loci. We further describe a methodology that applies RenSeq to rapidly identify molecular markers that co‐segregate with a pathogen resistance trait of interest. In two independent segregating populations involving the wild Solanum species S. berthaultii (Rpi‐ber2) and S. ruiz‐ceballosii (Rpi‐rzc1), we were able to apply RenSeq successfully to identify markers that co‐segregate with resistance towards the late blight pathogen Phytophthora infestans. These SNP identification workflows were designed as easy‐to‐adapt Galaxy pipelines.     

Linkage disequilibrium network analysis (LDna) gives a global view of chromosomal inversions,local adaptation and geographic structure

Recent advances in sequencing allow population‐genomic data to be generated for virtually any species. However, approaches to analyse such data lag behind the ability to generate it, particularly in nonmodel species. Linkage disequilibrium (LD, the nonrandom association of alleles from different loci) is a highly sensitive indicator of many evolutionary phenomena including chromosomal inversions, local adaptation and geographical structure. Here, we present linkage disequilibrium network analysis (LDna), which accesses information on LD shared between multiple loci genomewide. In LD networks, vertices represent loci, and connections between vertices represent the LD between them. We analysed such networks in two test cases: a new restriction‐site‐associated DNA sequence (RAD‐seq) data set for Anopheles baimaii, a Southeast Asian malaria vector; and a well‐characterized single nucleotide polymorphism (SNP) data set from 21 three‐spined stickleback individuals. In each case, we readily identified five distinct LD network clusters (single‐outlier clusters, SOCs), each comprising many loci connected by high LD. In A. baimaii, further population‐genetic analyses supported the inference that each SOC corresponds to a large inversion, consistent with previous cytological studies. For sticklebacks, we inferred that each SOC was associated with a distinct evolutionary phenomenon: two chromosomal inversions, local adaptation, population‐demographic history and geographic structure. LDna is thus a useful exploratory tool, able to give a global overview of LD associated with diverse evolutionary phenomena and identify loci potentially involved. LDna does not require a linkage map or reference genome, so it is applicable to any population‐genomic data set, making it especially valuable for nonmodel species.