Single nucleotide polymorphism (SNP) discovery and genotyping are essential to genetic mapping. There remains a need for a simple, inexpensive platform that allows high-density SNP discovery and genotyping in large populations. Here we describe the sequencing of restriction-site associated DNA (RAD) tags, which identified more than 13,000 SNPs, and mapped three traits in two model organisms, using less than half the capacity of one Illumina sequencing run. We demonstrated that different marker densities can be attained by choice of restriction enzyme. Furthermore, we developed a barcoding system for sample multiplexing and fine mapped the genetic basis of lateral plate armor loss in threespine stickleback by identifying recombinant breakpoints in F2 individuals. Barcoding also facilitated mapping of a second trait, a reduction of pelvic structure, by in silico re-sorting of individuals. To further demonstrate the ease of the RAD sequencing approach we identified polymorphic markers and mapped an induced mutation in Neurospora crassa. Sequencing of RAD markers is an integrated platform for SNP discovery and genotyping. This approach should be widely applicable to genetic mapping in a variety of organisms.

Most adaptation is thought to occur through the fixation of numerous alleles at many different loci. Consequently, the independent evolution of similar phenotypes is predicted to occur through different genetic mechanisms. The genetic basis of adaptation is still largely unknown, however, and it is unclear whether adaptation to new environments utilizes ubiquitous small-effect polygenic variation or large-effect alleles at a small number of loci. To address this question, we examined the genetic basis of bony armor loss in three freshwater populations of Alaskan threespine stickleback, Gasterosteus aculeatus, that evolved from fully armored anadromous populations in the last 14,000 years. Crosses between complete-armor and low-armor populations revealed that a single Mendelian factor governed the formation of all but the most anterior lateral plates, and another independently segregating factor largely determined pelvic armor. Genetic mapping localized the Mendelian genes to different chromosomal regions, and crosses among these same three widely separated populations showed that both bony plates and pelvic armor failed to fully complement, implicating the same Mendelian armor reduction genes. Thus, rapid and repeated armor loss in Alaskan stickleback populations appears to be occurring through the fixation of largeeffect variants in the same genes.A central tenet of evolutionary theory is that adaptation in the wild, like artificial selection, occurs gradually through the sequential fixation of small-effect variants (1). Consequently, the independent evolution of similar phenotypes is expected to use unique combinations of genes and alleles (2). New populations, however, are often established in novel environments at the edge of an organism's range, and selective pressures faced in these new habitats are often an important causative factor for adaptive radiations (3). Importantly, novel environments may also have immediate disruptive effects on developmental processes that can expose novel genetic variants, some of which may have large effects on evolving phenotypes (4, 5). The importance of genes of major effect is currently the focus of renewed research (6, 7). The role of major effect genes during adaptation, however, is still unclear, as is the frequency with which recurrent phenotypic evolution occurs through changes in the same (8-11) or different (8,12,13) genes. In addition, the genetics of adaptation has most often been studied in the laboratory (14), with much less work in natural populations (13). To address these problems, we have taken advantage of a unique natural system, the rapid postglacial diversification of threespine stickleback, Gasterosteus aculeatus (15). Thousands of coastal freshwater populations of stickleback have derived independently from anadromous (sea-run) ancestors. Phenotypically similar throughout their range, anadromous stickleback are protected with bony armor including lateral plates and a robust set of dorsal and pelvic spines (Fig. 1D). In contrast, derived lacustrine pop...

Population genomic studies are beginning to provide a more comprehensive view of dynamic genomescale processes in evolution. Patterns of genomic architecture, such as genomic islands of increased divergence, may be important for adaptive population differentiation and speciation. We used nextgeneration sequencing data to examine the patterns of local and long-distance linkage disequilibrium (LD) across oceanic and freshwater populations of threespine stickleback, a useful model for studies of evolution and speciation. We looked for associations between LD and signatures of divergent selection, and assessed the role of recombination rate variation in generating LD patterns. As predicted under the traditional biogeographic model of unidirectional gene flow from ancestral oceanic to derived freshwater stickleback populations, we found extensive local and long-distance LD in fresh water. Surprisingly, oceanic populations showed similar patterns of elevated LD, notably between large genomic regions previously implicated in adaptation to fresh water. These results support an alternative biogeographic model for the stickleback radiation, one of a metapopulation with appreciable bidirectional gene flow combined with strong divergent selection between oceanic and freshwater populations. As predicted by theory, these processes can maintain LD within and among genomic islands of divergence. These findings suggest that the genomic architecture in oceanic stickleback populations may provide a mechanism for the rapid re-assembly and evolution of multi-locus genotypes in newly colonized freshwater habitats, and may help explain genetic mapping of parallel phenotypic variation to similar loci across independent freshwater populations.

Understanding how genetic variation is partitioned across genomes within and among populations is a fundamental problem in ecological and evolutionary genetics. To address this problem we are studied the threespine stickleback fish, which has repeatedly undergone parallel phenotypic and genetic differentiation when oceanic fish have invaded freshwater habitats. While significant evolutionary genetic research has been performed using stickleback from geographic regions that have been de-glaciated in the last 20,000 years, less research has focused on freshwater populations that predate the last glacial maximum. We performed RAD-seq based population genomic analyses on stickleback from across Oregon, which was not glaciated during the last maximum. We sampled stickleback from coastal, Willamette Basin, and central Oregon sites, analyzed their genetic diversity using RAD-seq, performed STRUCTURE analyses, reconstructed their phylogeographic history, and tested the hypothesis of recent stickleback introduction into central Oregon, where incidence of this species was only recently documented. Our results showed a clear phylogeographic break between coastal and inland populations, with oceanic populations exhibiting the lowest levels of divergence from one another. Willamette Basin and central Oregon populations formed a clade of closely related populations, a finding consistent with a recent introduction of stickleback into central Oregon. Finally, genome wide analysis of genetic diversity (π) and correlations of alleles within individuals in subpopulations (F IS ) supported a role for introgressive hybridization in coastal populations and a recent expansion in central Oregon. Our results exhibit the power of next generation sequencing genomic approaches such as RAD-seq to identify both historical population structure and recent colonization history.