Genetic mapping of mutations in model systems has facilitated the identification of genes contributing to fundamental biological processes including human diseases. However, this approach has historically required the prior characterization of informative markers. Here we report a fast and cost-effective method for genetic mapping using next-generation sequencing that combines single nucleotide polymorphism discovery, mutation localization, and potential identification of causal sequence variants. In contrast to prior approaches, we have developed a hidden Markov model to narrowly define the mutation area by inferring recombination breakpoints of chromosomes in the mutant pool. In addition, we created an interactive online software resource to facilitate automated analysis of sequencing data and demonstrate its utility in the zebrafish and mouse models. Our novel methodology and online tools will make next-generation sequencing an easily applicable resource for mutation mapping in all model systems.[Supplemental material is available for this article.]There can be little argument that genetic mapping has made a substantial contribution to our understanding of biology. For many years these studies used phenotypically defined markers, such as those used by Morgan in Drosophila and Haldane in mice (Morgan 1911;Haldane et al. 1915). The modern era of genetic analysis was heralded by the recognition that variation in genomic DNA sequence itself could be used as a facile assay for mapping (Botstein et al. 1980). This was initially accomplished using analysis of restriction fragmentlength polymorphisms, which were later replaced by microsatellites and subsequently by single nucleotide polymorphisms (SNPs). Despite the remarkable technological advances, these approaches hold in common with those of Morgan and Haldane the utilization of prespecified markers. Next-generation sequencing (NGS) technology enables simultaneous discovery of very dense sets of informative markers and actual gene mapping in the same experiment. Here, we present a strategy and computational tools to map genes in model organisms using sequencing of pooled samples. The approach can be applied to any model organism with a characterized genome and also to both spontaneous and induced mutants. We demonstrate the utility of the strategy and efficiency of the computational approach by mapping spontaneous and ethylnitrosourea (ENU)-induced developmental mutants in zebrafish and mouse.Large-scale forward mutagenesis screens in zebrafish have been used with success to investigate fundamental developmental processes. While the recent completion of the zebrafish genome has greatly aided in the identification of genes, mapping analyses continue to rely on the use of traditional microsatellite markers. However, the utilization of SNPs for mapping of zebrafish mutants was proposed almost a decade ago (Stickney et al. 2002), large numbers of SNPs have been identified (Guryev et al. 2006;Bradley et al. 2007), and the application of NGS for SNP discovery and mutat...

SUMMARY Defects in the availability of heme substrates or the catalytic activity of the terminal enzyme in heme biosynthesis, ferrochelatase (Fech), impair heme synthesis, and thus cause human congenital anemias1,2. The inter-dependent functions of regulators of mitochondrial homeostasis and enzymes responsible for heme synthesis are largely unknown. To uncover this unmet need, we utilized zebrafish genetic screens and cloned mitochondrial ATPase inhibitory factor 1 (atpif1) from a zebrafish mutant with profound anemia, pinotage (pnt tq209). We now report a direct mechanism establishing that Atpif1 regulates the catalytic efficiency of vertebrate Fech to synthesize heme. The loss of Atpif1 impairs hemoglobin synthesis in zebrafish, mouse, and human hematopoietic models as a consequence of diminished Fech activity, and elevated mitochondrial pH. To understand the relationship among mitochondrial pH, redox potential, [2Fe-2S] clusters, and Fech activity, we used (1) genetic complementation studies of Fech constructs with or without [2Fe-2S] clusters in pnt, and (2) pharmacological agents modulating mitochondrial pH and redox potential. The presence of [2Fe-2S] cluster renders vertebrate Fech vulnerable to Atpif1-regulated mitochondrial pH and redox potential perturbations. Therefore, Atpif1 deficiency reduces the efficiency of vertebrate Fech to synthesize heme, resulting in anemia. The novel mechanism of Atpif1 as a regulator of heme synthesis advances the understanding of mitochondrial heme homeostasis and red blood cell development. A deficiency of Atpif1 may contribute to important human diseases, such as congenital sideroblastic anemias and mitochondriopathies.