We present a new approach to genotyping based on multiplexed shotgun sequencing that can identify recombination breakpoints in a large number of individuals simultaneously at a resolution sufficient for most mapping purposes, such as quantitative trait locus (QTL) mapping and mapping of induced mutations. We first describe a simple library construction protocol that uses just 10 ng of genomic DNA per individual and makes the approach accessible to any laboratory with standard molecular biology equipment. Sequencing this library results in a large number of sequence reads widely distributed across the genomes of multiplexed bar-coded individuals. We develop a Hidden Markov Model to estimate ancestry at all genomic locations in all individuals using these data. We demonstrate the utility of the approach by mapping a dominant marker allele in D. simulans to within 105 kb of its true position using 96 F1-backcross individuals genotyped in a single lane on an Illumina Genome Analyzer. We further demonstrate the utility of our method by genetically mapping more than 400 previously unassembled D. simulans contigs to linkage groups and by evaluating the quality of targeted introgression lines. At this level of multiplexing and divergence between strains, our method allows estimation of recombination breakpoints to a median of 38-kb intervals. Our analysis suggests that higher levels of multiplexing and/or use of strains with lower levels of divergence are practicable.

Summary Morphology evolves often through changes in developmental genes, but the causal mutations, and their effects, remain largely unknown. The evolution of naked cuticle—rather than trichomes—on larvae of Drosophila sechellia resulted from changes in five transcriptional enhancers of shavenbaby, a gene encoding a transcription factor that governs trichome morphogenesis. Here we show that the function of one of these enhancers evolved through multiple single nucleotide substitutions that altered both the timing and level of shavenbaby expression. The consequences of these nucleotide substitutions on larval morphology were quantified with a novel functional assay. We found that each substitution had a relatively small phenotypic effect, and that many nucleotide changes account for this large morphological difference. In addition, we observed that the substitutions displayed non-additive effects to generate a large phenotypic change. These data provide unprecedented resolution of the phenotypic effects of substitutions and show how individual nucleotide changes in a transcriptional enhancer have caused morphological evolution.

A key regulatory gene in metamorphosing (holometabolous) insect life histories is the transcription factor broad (br), which specifies pupal development. To determine the role of br in a directdeveloping (hemimetabolous) insect that lacks a pupal stage, we cloned br from the milkweed bug, Oncopeltus fasciatus (Of'br). We find that, unlike metamorphosing insects, in which br expression is restricted to the larval-pupal transition, Of'br mRNA is expressed during embryonic development and is maintained at each nymphal molt but then disappears at the molt to the adult. Induction of a supernumerary nymphal stage with a juvenile hormone (JH) mimic prevented the disappearance of br mRNA. In contrast, induction of a precocious adult molt by application of precocene II to third-stage nymphs caused a loss of br mRNA at the precocious adult molt. Thus, JH is necessary to maintain br expression during the nymphal stages. Injection of Of'br dsRNA into either early third-or fourthstage nymphs caused a repetition of stage-specific pigmentation patterns and prevented the normal anisometric growth of the wing pads without affecting isometric growth or molting. Therefore, br is necessary for the mutable (heteromorphic) changes that occur during hemimetabolous development. Our results suggest that metamorphosis in insects arose as expression of br, which conveys competence for change, became restricted to one postembryonic instar. After this shift in br expression, the progressive changes that occur within the nymphal series in basal insects became compressed to the one short period of morphogenesis seen in the larva-to-pupa transition of holometabolous insects.evolution of metamorphosis ͉ heteromorphosis ͉ Oncopeltus ͉ juvenile hormone ͉ allometry L ife history strategies are highly plastic within animal phyla; some groups develop directly, whereas related taxa pass through a metamorphosis. Regulation of stage-specific differences may be under either environmental or hormonal control, but relatively little is known of the molecular switches involved or how changes in the timing of these switches can lead to evolutionary change (1). In insects, metamorphosis arose once from a direct-developing ancestor Ϸ300 million years ago (2). A key regulatory gene in metamorphosing (holometabolous) insect life histories is the transcription factor broad (br) (3-7). In both moths and flies, epidermal expression of br is restricted to the larval-pupal transition (3, 5-7), and its expression at this time is required for activation of pupal-specific gene expression, as well as suppression of larval-and adult-specific gene expression (3, 7). Accordingly, Drosophila null mutants never enter the pupal stage; instead, they remain in a prolonged larval state (8). In addition, gynander larvae mosaic for br null and br ϩ tissue produced mosaic larval and pupal tissue, respectively, at the larval-pupal transition (9). Loss of br also prevents the larvalpupal transition in the silkmoth; tissues that were transformed with a vector driving br RNA interferenc...

Background The Hemiptera (aphids, cicadas, and true bugs) are a key insect order, with high diversity for feeding ecology and excellent experimental tractability for molecular genetics. Building upon recent sequencing of hemipteran pests such as phloem-feeding aphids and blood-feeding bed bugs, we present the genome sequence and comparative analyses centered on the milkweed bug Oncopeltus fasciatus , a seed feeder of the family Lygaeidae. Results The 926-Mb Oncopeltus genome is well represented by the current assembly and official gene set. We use our genomic and RNA-seq data not only to characterize the protein-coding gene repertoire and perform isoform-specific RNAi, but also to elucidate patterns of molecular evolution and physiology. We find ongoing, lineage-specific expansion and diversification of repressive C2H2 zinc finger proteins. The discovery of intron gain and turnover specific to the Hemiptera also prompted the evaluation of lineage and genome size as predictors of gene structure evolution. Furthermore, we identify enzymatic gains and losses that correlate with feeding biology, particularly for reductions associated with derived, fluid nutrition feeding. Conclusions With the milkweed bug, we now have a critical mass of sequenced species for a hemimetabolous insect order and close outgroup to the Holometabola, substantially improving the diversity of insect genomics. We thereby define commonalities among the Hemiptera and delve into how hemipteran genomes reflect distinct feeding ecologies. Given Oncopeltus ’s strength as an experimental model, these new sequence resources bolster the foundation for molecular research and highlight technical considerations for the analysis of medium-sized invertebrate genomes. Electronic supplementary material The online version of this article (10.1186/s13059-019-1660-0) contains supplementary material, which is available to authorized users.