Advances in sequencing technology provide special opportunities for genotyping individuals with speed and thrift, but the lack of software to automate the calling of tens of thousands of genotypes over hundreds of individuals has hindered progress. Stacks is a software system that uses short-read sequence data to identify and genotype loci in a set of individuals either de novo or by comparison to a reference genome. From reduced representation Illumina sequence data, such as RAD-tags, Stacks can recover thousands of single nucleotide polymorphism (SNP) markers useful for the genetic analysis of crosses or populations. Stacks can generate markers for ultra-dense genetic linkage maps, facilitate the examination of population phylogeography, and help in reference genome assembly. We report here the algorithms implemented in Stacks and demonstrate their efficacy by constructing loci from simulated RAD-tags taken from the stickleback reference genome and by recapitulating and improving a genetic map of the zebrafish, Danio rerio.

https://webpages.uidaho.edu/hohenlohe/Catchenetal2011.pdf

Stacks: Building and Genotyping Loci De Novo From Short-Read Sequences

Marine stickleback fish have colonized and adapted to thousands of streams and lakes formed since the last ice age, providing an exceptional opportunity to characterize genomic mechanisms underlying repeated ecological adaptation in nature. Here we develop a high-quality reference genome assembly for threespine sticklebacks. By sequencing the genomes of twenty additional individuals from a global set of marine and freshwater populations, we identify a genome-wide set of loci that are consistently associated with marine-freshwater divergence. Our results indicate that reuse of globally shared standing genetic variation, including chromosomal inversions, has an important role in repeated evolution of distinct marine and freshwater sticklebacks, and in the maintenance of divergent ecotypes during early stages of reproductive isolation. Both coding and regulatory changes occur in the set of loci underlying marine-freshwater evolution, but regulatory changes appear to predominate in this well known example of repeated adaptive evolution in nature.

/pdf/the-genomic-basis-of-adaptive-evolution-in-threespine-27yq6s7i0b.pdf

The genomic basis of adaptive evolution in threespine sticklebacks

Lancelets ('amphioxus') are the modern survivors of an ancient chordate lineage, with a fossil record dating back to the Cambrian period. Here we describe the structure and gene content of the highly polymorphic approximately 520-megabase genome of the Florida lancelet Branchiostoma floridae, and analyse it in the context of chordate evolution. Whole-genome comparisons illuminate the murky relationships among the three chordate groups (tunicates, lancelets and vertebrates), and allow not only reconstruction of the gene complement of the last common chordate ancestor but also partial reconstruction of its genomic organization, as well as a description of two genome-wide duplications and subsequent reorganizations in the vertebrate lineage. These genome-scale events shaped the vertebrate genome and provided additional genetic variation for exploitation during vertebrate evolution.

/pdf/the-amphioxus-genome-and-the-evolution-of-the-chordate-2hi9ggv8nx.pdf

The amphioxus genome and the evolution of the chordate karyotype

Next-generation sequencing technology provides novel opportunities for gathering genome-scale sequence data in natural populations, laying the empirical foundation for the evolving field of population genomics. Here we conducted a genome scan of nucleotide diversity and differentiation in natural populations of threespine stickleback (Gasterosteus aculeatus). We used Illumina-sequenced RAD tags to identify and type over 45,000 single nucleotide polymorphisms (SNPs) in each of 100 individuals from two oceanic and three freshwater populations. Overall estimates of genetic diversity and differentiation among populations confirm the biogeographic hypothesis that large panmictic oceanic populations have repeatedly given rise to phenotypically divergent freshwater populations. Genomic regions exhibiting signatures of both balancing and divergent selection were remarkably consistent across multiple, independently derived populations, indicating that replicate parallel phenotypic evolution in stickleback may be occurring through extensive, parallel genetic evolution at a genome-wide scale. Some of these genomic regions co-localize with previously identified QTL for stickleback phenotypic variation identified using laboratory mapping crosses. In addition, we have identified several novel regions showing parallel differentiation across independent populations. Annotation of these regions revealed numerous genes that are candidates for stickleback phenotypic evolution and will form the basis of future genetic analyses in this and other organisms. This study represents the first high-density SNP–based genome scan of genetic diversity and differentiation for populations of threespine stickleback in the wild. These data illustrate the complementary nature of laboratory crosses and population genomic scans by confirming the adaptive significance of previously identified genomic regions, elucidating the particular evolutionary and demographic history of such regions in natural populations, and identifying new genomic regions and candidate genes of evolutionary significance.

/pdf/population-genomics-of-parallel-adaptation-in-threespine-1byih44we4.pdf

Population Genomics of Parallel Adaptation in Threespine Stickleback using Sequenced RAD Tags

Major phenotypic changes evolve in parallel in nature by molecular mechanisms that are largely unknown. Here, we use positional cloning methods to identify the major chromosome locus controlling armor plate patterning in wild threespine sticklebacks. Mapping, sequencing, and transgenic studies show that the Ectodysplasin (EDA) signaling pathway plays a key role in evolutionary change in natural populations and that parallel evolution of stickleback low-plated phenotypes at most freshwater locations around the world has occurred by repeated selection of Eda alleles derived from an ancestral low-plated haplotype that first appeared more than two million years ago. Members of this clade of low-plated alleles are present at low frequencies in marine fish, which suggests that standing genetic variation can provide a molecular basis for rapid, parallel evolution of dramatic phenotypic change in nature.

/pdf/widespread-parallel-evolution-in-sticklebacks-by-repeated-3ud074f5er.pdf

Widespread Parallel Evolution in Sticklebacks by Repeated Fixation of Ectodysplasin Alleles

Genetic screens in zebrafish (Danio rerio) have identified mutations that define the roles of hundreds of essential vertebrate genes. Genetic maps can link mutant phenotype with gene sequence by providing candidate genes for mutations and polymorphic genetic markers useful in positional cloning projects. Here we report a zebrafish genetic map comprising 4073 polymorphic markers, with more than twice the number of coding sequences localized in previously reported zebrafish genetic maps. We use this map in comparative studies to identify numerous regions of synteny conserved among the genomes of zebrafish, Tetraodon, and human. In addition, we use our map to analyze gene duplication in the zebrafish and Tetraodon genomes. Current evidence suggests that a whole-genome duplication occurred in the teleost lineage after it split from the tetrapod lineage, and that only a subset of the duplicates have been retained in modern teleost genomes. It has been proposed that differential retention of duplicate genes may have facilitated the isolation of nascent species formed during the vast radiation of teleosts. We find that different duplicated genes have been retained in zebrafish and Tetraodon, although similar numbers of duplicates remain in both genomes. Finally, we use comparative mapping data to address the proposal that the common ancestor of vertebrates had a genome consisting of 12 chromosomes. In a three-way comparison between the genomes of zebrafish, Tetraodon, and human, our analysis delineates the gene content for 11 of these 12 proposed ancestral chromosomes.

/pdf/the-zebrafish-gene-map-defines-ancestral-vertebrate-4jdo4b0a37.pdf

The zebrafish gene map defines ancestral vertebrate chromosomes

The dramatic radiation of sticklebacks in different post-glacial environments provides a unique opportunity to study the molecular mechanisms that underlie rapid evolutionary change in vertebrates. We have developed a number of genomic and genetic tools to facilitate further study of a wide range of morphological, physiological and behavioral traits in sticklebacks. A large collection of microsatellite markers has previously been developed for use in genome-wide linkage mapping of interesting traits in crosses between different stickleback forms. cDNA libraries have been generated and EST sequencing projects have begun to isolate stickleback homologs of developmental control genes. Large insert BAC libraries have been built to compare chromosome regions of interest from both anadromous and freshwater stickleback populations. Large scale fingerprinting of one of these libraries has been used to assemble overlapping contigs of BAC clones for chromosome walking and positional cloning. Together with recent development of methods to make transgenic sticklebacks, these tools should make it possible to identify the molecular basis of many different evolutionary traits in stickleback, and to begin to answer longstanding questions about the numbers and types of mutations that control the appearance of new morphological, physiological, and behavioral traits during vertebrate evolution.

Brian Friedlander

Papers

The zebrafish gene map defines ancestral vertebrate chromosomes

New genomic tools for molecular studies of evolutionary change in threespine sticklebacks