Dataset Information

Breaking RAD: an evaluation of the utility of restriction site-associated DNA sequencing for genome scans of adaptation.

ABSTRACT: Understanding how and why populations evolve is of fundamental importance to molecular ecology. Restriction site-associated DNA sequencing (RADseq), a popular reduced representation method, has ushered in a new era of genome-scale research for assessing population structure, hybridization, demographic history, phylogeography and migration. RADseq has also been widely used to conduct genome scans to detect loci involved in adaptive divergence among natural populations. Here, we examine the capacity of those RADseq-based genome scan studies to detect loci involved in local adaptation. To understand what proportion of the genome is missed by RADseq studies, we developed a simple model using different numbers of RAD-tags, genome sizes and extents of linkage disequilibrium (length of haplotype blocks). Under the best-case modelling scenario, we found that RADseq using six- or eight-base pair cutting restriction enzymes would fail to sample many regions of the genome, especially for species with short linkage disequilibrium. We then surveyed recent studies that have used RADseq for genome scans and found that the median density of markers across these studies was 4.08 RAD-tag markers per megabase (one marker per 245 kb). The length of linkage disequilibrium for many species is one to three orders of magnitude less than density of the typical recent RADseq study. Thus, we conclude that genome scans based on RADseq data alone, while useful for studies of neutral genetic variation and genetic population structure, will likely miss many loci under selection in studies of local adaptation.

SUBMITTER: Lowry DB

PROVIDER: S-EPMC5446919 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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Breaking RAD: an evaluation of the utility of restriction site-associated DNA sequencing for genome scans of adaptation.

Lowry David B DB Hoban Sean S Kelley Joanna L JL Lotterhos Katie E KE Reed Laura K LK Antolin Michael F MF Storfer Andrew A

Molecular ecology resources 20161216 2

Understanding how and why populations evolve is of fundamental importance to molecular ecology. Restriction site-associated DNA sequencing (RADseq), a popular reduced representation method, has ushered in a new era of genome-scale research for assessing population structure, hybridization, demographic history, phylogeography and migration. RADseq has also been widely used to conduct genome scans to detect loci involved in adaptive divergence among natural populations. Here, we examine the capaci ...[more]

PMID: 27860289

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Project description:Background and Aims:Bee orchids (Ophrys) have become the most popular model system for studying reproduction via insect-mediated pseudo-copulation and for exploring the consequent, putatively adaptive, evolutionary radiations. However, despite intensive past research, both the phylogenetic structure and species diversity within the genus remain highly contentious. Here, we integrate next-generation sequencing and morphological cladistic techniques to clarify the phylogeny of the genus. Methods:At least two accessions of each of the ten species groups previously circumscribed from large-scale cloned nuclear ribosomal internal transcibed spacer (nrITS) sequencing were subjected to restriction site-associated sequencing (RAD-seq). The resulting matrix of 4159 single nucleotide polymorphisms (SNPs) for 34 accessions was used to construct an unrooted network and a rooted maximum likelihood phylogeny. A parallel morphological cladistic matrix of 43 characters generated both polymorphic and non-polymorphic sets of parsimony trees before being mapped across the RAD-seq topology. Key Results:RAD-seq data strongly support the monophyly of nine out of ten groups previously circumscribed using nrITS and resolve three major clades; in contrast, supposed microspecies are barely distinguishable. Strong incongruence separated the RAD-seq trees from both the morphological trees and traditional classifications; mapping of the morphological characters across the RAD-seq topology rendered them far more homoplastic. Conclusions:The comparatively high level of morphological homoplasy reflects extensive convergence, whereas the derived placement of the fusca group is attributed to paedomorphic simplification. The phenotype of the most recent common ancestor of the extant lineages is inferred, but it post-dates the majority of the character-state changes that typify the genus. RAD-seq may represent the high-water mark of the contribution of molecular phylogenetics to understanding evolution within Ophrys; further progress will require large-scale population-level studies that integrate phenotypic and genotypic data in a cogent conceptual framework.

Project description:Restriction site-associated DNA sequencing (RAD-Seq) is a genome complexity reduction technique that facilitates large-scale marker discovery and genotyping by sequencing. Recent applications of RAD-Seq have included linkage and QTL mapping with a particular focus on non-model species. In the current study, we have applied RAD-Seq to two Atlantic salmon families from a commercial breeding program. The offspring from these families were classified into resistant or susceptible based on survival/mortality in an Infectious Pancreatic Necrosis (IPN) challenge experiment, and putative homozygous resistant or susceptible genotype at a major IPN-resistance QTL. From each family, the genomic DNA of the two heterozygous parents and seven offspring of each IPN phenotype and genotype was digested with the SbfI enzyme and sequenced in multiplexed pools.Sequence was obtained from approximately 70,000 RAD loci in both families and a filtered set of 6,712 segregating SNPs were identified. Analyses of genome-wide RAD marker segregation patterns in the two families suggested SNP discovery on all 29 Atlantic salmon chromosome pairs, and highlighted the dearth of male recombination. The use of pedigreed samples allowed us to distinguish segregating SNPs from putative paralogous sequence variants resulting from the relatively recent genome duplication of salmonid species. Of the segregating SNPs, 50 were linked to the QTL. A subset of these QTL-linked SNPs were converted to a high-throughput assay and genotyped across large commercial populations of IPNV-challenged salmon fry. Several SNPs showed highly significant linkage and association with resistance to IPN, and population linkage-disequilibrium-based SNP tests for resistance were identified.We used RAD-Seq to successfully identify and characterise high-density genetic markers in pedigreed aquaculture Atlantic salmon. These results underline the effectiveness of RAD-Seq as a tool for rapid and efficient generation of QTL-targeted and genome-wide marker data in a large complex genome, and its possible utility in farmed animal selection programs.

Project description:BackgroundLinkage maps are an integral resource for dissection of complex genetic traits in plant and animal species. Canonical map construction follows a well-established workflow: an initial discovery phase where genetic markers are mined from a small pool of individuals, followed by genotyping of selected mapping populations using sets of marker panels. A newly developed sequence-based marker technology, Restriction site Associated DNA (RAD), enables synchronous single nucleotide polymorphism (SNP) marker discovery and genotyping using massively parallel sequencing. The objective of this research was to assess the utility of RAD markers for linkage map construction, employing barley as a model system. Using the published high density EST-based SNP map in the Oregon Wolfe Barley (OWB) mapping population as a reference, we created a RAD map using a limited set of prior markers to establish linakge group identity, integrated the RAD and prior data, and used both maps for detection of quantitative trait loci (QTL).ResultsUsing the RAD protocol in tandem with the Illumina sequence by synthesis platform, a total of 530 SNP markers were identified from initial scans of the OWB parental inbred lines--the "dominant" and "recessive" marker stocks--and scored in a 93 member doubled haploid (DH) mapping population. RAD sequence data from the structured population was converted into allele genotypes from which a genetic map was constructed. The assembled RAD-only map consists of 445 markers with an average interval length of 5 cM, while an integrated map includes 463 RAD loci and 2383 prior markers. Sequenced RAD markers are distributed across all seven chromosomes, with polymorphic loci emanating from both coding and noncoding regions in the Hordeum genome. Total map lengths are comparable and the order of common markers is identical in both maps. The same large-effect QTL for reproductive fitness traits were detected with both maps and the majority of these QTL were coincident with a dwarfing gene (ZEO) and the VRS1 gene, which determines the two-row and six-row germplasm groups of barley.ConclusionsWe demonstrate how sequenced RAD markers can be leveraged to produce high quality linkage maps for detection of single gene loci and QTLs. By combining SNP discovery and genotyping into parallel sequencing events, RAD markers should be a useful molecular breeding tool for a range of crop species. Expected improvements in cost and throughput of second and third-generation sequencing technologies will enable more powerful applications of the sequenced RAD marker system, including improvements in de novo genome assembly, development of ultra-high density genetic maps and association mapping.

Project description:BACKGROUND:Restriction site-Associated DNA sequencing (RAD-Seq) is widely applied to generate genome-wide sequence and genetic marker datasets. RAD-Seq has been extensively utilised, both at the population level and across species, for example in the construction of phylogenetic trees. However, the consistency of RAD-Seq data generated in different laboratories, and the potential use of cross-species orthologous RAD loci in the estimation of genetic relationships, have not been widely investigated. This study describes the use of SbfI RAD-Seq data for the estimation of evolutionary relationships amongst ten teleost fish species, using previously established phylogeny as a benchmark. RESULTS:The number of orthologous SbfI RAD loci identified decreased with increasing evolutionary distance between the species, with several thousand loci conserved across five salmonid species (divergence ~50 MY), and several hundred conserved across the more distantly related teleost species (divergence ~100-360 MY). The majority (>70%) of loci identified between the more distantly related species were genic in origin, suggesting that the bias of SbfI towards genic regions is useful for identifying distant orthologs. Interspecific single nucleotide variants at each orthologous RAD locus were identified. Evolutionary relationships estimated using concatenated sequences of interspecific variants were congruent with previously published phylogenies, even for distantly (divergence up to ~360 MY) related species. CONCLUSION:Overall, this study has demonstrated that orthologous SbfI RAD loci can be identified across closely and distantly related species. This has positive implications for the repeatability of SbfI RAD-Seq and its potential to address research questions beyond the scope of the original studies. Furthermore, the concordance in tree topologies and relationships estimated in this study with published teleost phylogenies suggests that similar meta-datasets could be utilised in the prediction of evolutionary relationships across populations and species with readily available RAD-Seq datasets, but for which relationships remain uncharacterised.

Dataset Information

Breaking RAD: an evaluation of the utility of restriction site-associated DNA sequencing for genome scans of adaptation.

Publications

Breaking RAD: an evaluation of the utility of restriction site-associated DNA sequencing for genome scans of adaptation.

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