The ability to efficiently and accurately determine genotypes is a keystone technology in modern genetics, crucial to studies ranging from clinical diagnostics, to genotype-phenotype association, to reconstruction of ancestry and the detection of selection. To date, high capacity, low cost genotyping has been largely achieved via “SNP chip” microarray-based platforms which require substantial prior knowledge of both genome sequence and variability, and once designed are suitable only for those targeted variable nucleotide sites. This method introduces substantial ascertainment bias and inherently precludes detection of rare or population-specific variants, a major source of information for both population history and genotype-phenotype association. Recent developments in reduced-representation genome sequencing experiments on massively parallel sequencers (commonly referred to as RAD-tag or RADseq) have brought direct sequencing to the problem of population genotyping, but increased cost and procedural and analytical complexity have limited their widespread adoption. Here, we describe a complete laboratory protocol, including a custom combinatorial indexing method, and accompanying software tools to facilitate genotyping across large numbers (hundreds or more) of individuals for a range of markers (hundreds to hundreds of thousands). Our method requires no prior genomic knowledge and achieves per-site and per-individual costs below that of current SNP chip technology, while requiring similar hands-on time investment, comparable amounts of input DNA, and downstream analysis times on the order of hours. Finally, we provide empirical results from the application of this method to both genotyping in a laboratory cross and in wild populations. Because of its flexibility, this modified RADseq approach promises to be applicable to a diversity of biological questions in a wide range of organisms.

Natural populations of beach mice exhibit a characteristic color pattern, relative to their mainland conspecifics, driven by natural selection for crypsis. We identified a derived, charge-changing amino acid mutation in the melanocortin-1 receptor ( Mc1r ) in beach mice, which decreases receptor function. In genetic crosses, allelic variation at Mc1r explains 9.8% to 36.4% of the variation in seven pigmentation traits determining color pattern. The derived Mc1r allele is present in Florida's Gulf Coast beach mice but not in Atlantic coast mice with similar light coloration, suggesting that different molecular mechanisms are responsible for convergent phenotypic evolution. Here, we link a single mutation in the coding region of a pigmentation gene to adaptive quantitative variation in the wild.

The study of pigmentation has played an important role in the intersection of evolution, genetics, and developmental biology. Pigmentation's utility as a visible phenotypic marker has resulted in over 100 years of intense study of coat color mutations in laboratory mice, thereby creating an impressive list of candidate genes and an understanding of the developmental mechanisms responsible for the phenotypic effects. Variation in color and pigment patterning has also served as the focus of many classic studies of naturally occurring phenotypic variation in a wide variety of vertebrates, providing some of the most compelling cases for parallel and convergent evolution. Thus, the pigmentation model system holds much promise for understanding the nature of adaptation by linking genetic changes to variation in fitness-related traits. Here, I first discuss the historical role of pigmentation in genetics, development and evolutionary biology. I then discuss recent empirically based studies in vertebrates, which rely on these historical foundations to make connections between genotype and phenotype for ecologically important pigmentation traits. These studies provide insight into the evolutionary process by uncovering the genetic basis of adaptive traits and addressing such long-standing questions in evolutionary biology as (1) are adaptive changes predominantly caused by mutations in regulatory regions or coding regions? (2) is adaptation driven by the fixation of dominant mutations? and (3) to what extent are parallel phenotypic changes caused by similar genetic changes? It is clear that coloration has much to teach us about the molecular basis of organismal diversity, adaptation and the evolutionary process.

A central challenge in evolutionary biology is to identify genes underlying ecologically important traits and describe the fitness consequences of naturally occurring variation at these loci. To address this goal, several novel approaches have been developed, including ‘population genomics,’ where a large number of molecular markers are scored in individuals from different environments with the goal of identifying markers showing unusual patterns of variation, potentially due to selection at linked sites. Such approaches are appealing because of (1) the increasing ease of generating large numbers of genetic markers, (2) the ability to scan the genome without measuring phenotypes and (3) the simplicity of sampling individuals without knowledge of their breeding history. Although such approaches are inherently applicable to non-model systems, to date these studies have been limited in their ability to uncover functionally relevant genes. By contrast, quantitative genetics has a rich history, and more recently, quantitative trait locus (QTL) mapping has had some success in identifying genes underlying ecologically relevant variation even in novel systems. QTL mapping, however, requires (1) genetic markers that specifically differentiate parental forms, (2) a focus on a particular measurable phenotype and (3) controlled breeding and maintenance of large numbers of progeny. Here we present current advances and suggest future directions that take advantage of population genomics and quantitative genetic approaches – in both model and non-model systems. Specifically, we discuss advantages and limitations of each method and argue that a combination of the two provides a powerful approach to uncovering the molecular mechanisms responsible for adaptation.

Identifying the genes underlying adaptation is a major challenge in evolutionary biology. Here, we describe the molecular changes underlying adaptive coat color variation in a natural population of rock pocket mice,Chaetodipus intermedius. Rock pocket mice are generally light-colored and live on light-colored rocks. However, populations of dark (melanic) mice are found on dark lava, and this concealing coloration provides protection from avian and mammalian predators. We conducted association studies by using markers in candidate pigmentation genes and discovered four mutations in the melanocortin-1-receptor gene,Mc1r, that seem to be responsible for adaptive melanism in one population of lava-dwelling pocket mice. Interestingly, another melanic population of these mice on a different lava flow shows no association withMc1rmutations, indicating that adaptive dark color has evolved independently in this species through changes at different genes.