New de novo assembly of the Atlantic bottlenose dolphin (Tursiops truncatus) improves genome completeness and provides haplotype phasing.

High-quality genomes are essential to resolve challenges in breeding, comparative biology, medicine, and conservation planning. New library preparation techniques along with better assembly algorithms result in continued improvements in assemblies for non-model organisms, moving them toward reference-quality genomes. We report on the latest genome assembly of the Atlantic bottlenose dolphin, leveraging Illumina sequencing data coupled with a combination of several library preparation techniques. These include Linked-Reads (Chromium, 10x Genomics), mate pairs (MP), long insert paired ends, and standard paired end. Data were assembled with the commercial DeNovoMAGIC assembly software, resulting in two assemblies, a traditional "haploid" assembly (Tur_tru_Illumina_hap_v1) that is a mosaic of the two parental haplotypes and a phased assembly (Tur_tru_Illumina_phased_v1) where each scaffold has sequence from a single homologous chromosome. We show that Tur_tru_Illumina_hap_v1 is more complete and more accurate compared to the current best reference based on the amount and composition of sequence, the consistency of the MP alignments to the assembled scaffolds, and on the analysis of conserved single-copy mammalian orthologs. The phased de novo assembly Tur_tru_Illumina_phased_v1 is the first publicly available for this species and provides the community with novel and accurate ways to explore the heterozygous nature of the dolphin genome.

Domestication and Divergence of Saccharomyces cerevisiae Beer Yeasts.

Whereas domestication of livestock, pets, and crops is well documented, it is still unclear to what extent microbes associated with the production of food have also undergone human selection and where the plethora of industrial strains originates from. Here, we present the genomes and phenomes of 157 industrial Saccharomyces cerevisiae yeasts. Our analyses reveal that today's industrial yeasts can be divided into five sublineages that are genetically and phenotypically separated from wild strains and originate from only a few ancestors through complex patterns of domestication and local divergence. Large-scale phenotyping and genome analysis further show strong industry-specific selection for stress tolerance, sugar utilization, and flavor production, while the sexual cycle and other phenotypes related to survival in nature show decay, particularly in beer yeasts. Together, these results shed light on the origins, evolutionary history, and phenotypic diversity of industrial yeasts and provide a resource for further selection of superior strains. PAPERCLIP.

A cellular genetics approach identifies gene-drug interactions and pinpoints drug toxicity pathway nodes.

New approaches to toxicity testing have incorporated high-throughput screening across a broad-range of in vitro assays to identify potential key events in response to chemical or drug treatment. To date, these approaches have primarily utilized repurposed drug discovery assays. In this study, we describe an approach that combines in vitro screening with genetic approaches for the experimental identification of genes and pathways involved in chemical or drug toxicity. Primary embryonic fibroblasts isolated from 32 genetically-characterized inbred mouse strains were treated in concentration-response format with 65 compounds, including pharmaceutical drugs, environmental chemicals, and compounds with known modes-of-action. Integrated cellular responses were measured at 24 and 72 h using high-content imaging and included cell loss, membrane permeability, mitochondrial function, and apoptosis. Genetic association analysis of cross-strain differences in the cellular responses resulted in a collection of candidate loci potentially underlying the variable strain response to each chemical. As a demonstration of the approach, one candidate gene involved in rotenone sensitivity, Cybb, was experimentally validated in vitro and in vivo. Pathway analysis on the combined list of candidate loci across all chemicals identified a number of over-connected nodes that may serve as core regulatory points in toxicity pathways.

Regenerative phenotype in mice with a point mutation in transforming growth factor beta type I receptor (TGFBR1).

Regeneration of peripheral differentiated tissue in mammals is rare, and regulators of this process are largely unknown. We carried out a forward genetic screen in mice using N-ethyl-N-nitrosourea mutagenesis to identify genetic mutations that affect regenerative healing in vivo. More than 400 pedigrees were screened for closure of a through-and-through punch wound in the mouse ear. This led to the identification of a single pedigree with a heritable, fast, and regenerative wound-healing phenotype. Within 5 wk after ear-punch, a threefold decrease in the diameter of the wound was observed in the mutant mice compared with the wild-type mice. At 22 wk, new cartilage, hair follicles, and sebaceous glands were observed in the newly generated tissue. This trait was mapped to a point mutation in a receptor for TGF-ß, TGFBR1. Mouse embryonic fibroblasts from the affected mice had increased expression of a subset of TGF-ß target genes, suggesting that the mutation caused partial activation of the receptor. Further, bone marrow stromal cells from the mutant mice more readily differentiated to chondrogenic precursors, providing a plausible explanation for the enhanced development of cartilage islands in the regenerated ears. This mutant mouse strain provides a unique model to further explore regeneration in mammals and, in particular, the role of TGFBR1 in chondrogenesis and regenerative wound healing.

Genetic dissection of a key reproductive barrier between nascent species of house mice.

Reproductive isolation between species is often caused by deleterious interactions among loci in hybrids. Finding the genes involved in these incompatibilities provides insight into the mechanisms of speciation. With recently diverged subspecies, house mice provide a powerful system for understanding the genetics of reproductive isolation early in the speciation process. Although previous studies have yielded important clues about the genetics of hybrid male sterility in house mice, they have been restricted to F1 sterility or incompatibilities involving the X chromosome. To provide a more complete characterization of this key reproductive barrier, we conducted an F2 intercross between wild-derived inbred strains from two subspecies of house mice, Mus musculus musculus and Mus musculus domesticus. We identified a suite of autosomal and X-linked QTL that underlie measures of hybrid male sterility, including testis weight, sperm density, and sperm morphology. In many cases, the autosomal loci were unique to a specific sterility trait and exhibited an effect only when homozygous, underscoring the importance of examining reproductive barriers beyond the F1 generation. We also found novel two-locus incompatibilities between the M. m. musculus X chromosome and M. m. domesticus autosomal alleles. Our results reveal a complex genetic architecture for hybrid male sterility and suggest a prominent role for reproductive barriers in advanced generations in maintaining subspecies integrity in house mice.

Extensive recombination rate variation in the house mouse species complex inferred from genetic linkage maps.

The rate of recombination is a key genomic parameter that displays considerable variation among taxa. Species comparisons have demonstrated that the rate of evolution in recombination rate is strongly dependent on the physical scale of measurement. Individual recombination hotspots are poorly conserved among closely related taxa, whereas genomic-scale recombination rate variation bears a strong signature of phylogenetic history. In contrast, the mode and tempo of evolution in recombination rates measured on intermediate physical scales is poorly understood. Here, we conduct a detailed statistical comparison between two whole-genome F2 genetic linkage maps constructed from experimental intercrosses between closely related house mouse subspecies (Mus musculus). Our two maps profile a common wild-derived inbred strain of M. m. domesticus crossed to distinct wild-derived inbred strains representative of two other house mouse subspecies, M. m. castaneus and M. m. musculus. We identify numerous orthologous genomic regions with significant map length differences between these two crosses. Because the genomes of these recently diverged house mice are highly collinear, observed differences in map length (centimorgans) are suggestive of variation in broadscale recombination rate (centimorgans per megabase) within M. musculus. Collectively, these divergent intervals span 19% of the house mouse genome, disproportionately aggregating on the X chromosome. In addition, we uncover strong statistical evidence for a large effect, sex-linked, site-specific modifier of recombination rate segregating within M. musculus. Our findings reveal considerable variation in the megabase-scale recombination landscape among recently diverged taxa and underscore the continued importance of genetic linkage maps in the post-genome era.

Genome wide analysis of inbred mouse lines identifies a locus containing Ppar-gamma as contributing to enhanced malaria survival.

The genetic background of a patient determines in part if a person develops a mild form of malaria and recovers, or develops a severe form and dies. We have used a mouse model to detect genes involved in the resistance or susceptibility to Plasmodium berghei malaria infection. To this end we first characterized 32 different mouse strains infected with P. berghei and identified survival as the best trait to discriminate between the strains. We found a locus on chromosome 6 by linking the survival phenotypes of the mouse strains to their genetic variations using genome wide analyses such as haplotype associated mapping and the efficient mixed-model for association. This new locus involved in malaria resistance contains only two genes and confirms the importance of Ppar-gamma in malaria infection.

Quantitative trait locus and haplotype mapping in closely related inbred strains identifies a locus for open field behavior.

Quantitative trait locus (QTL) mapping in the mouse typically utilizes inbred strains that exhibit significant genetic and phenotypic diversity. The development of dense SNP panels in a large number of inbred strains has eliminated the need to maximize genetic diversity in QTL studies as plenty of SNP markers are now available for almost any combination of strains. We conducted a QTL mapping experiment using both a backcross (N(2)) and an intercross (F(2)) between two genetically similar inbred mouse strains: C57BL/6J (B6) and C57L/J (C57). A set of additive QTLs for activity behaviors was identified on Chrs 1, 9, 13, and 15. We also identified additive QTLs for anxiety-related behaviors on Chrs 7, 9, and 16. A QTL on Chr 11 is sex-specific, and we revealed pairwise interactions between QTLs on Chrs 1 and 13 and Chrs 10 and 18. The Chr 9 activity QTL accounts for the largest amount of phenotypic variance and was not present in our recent analysis of a B6 x C58/J (C58) intercross (Bailey et al. in Genes Brain Behav 7:761-769, 2008). To narrow this QTL interval, we used a dense SNP haplotype map with over 7 million real and imputed SNP markers across 74 inbred mouse strains (Szatkiewicz et al. in Mamm Genome 19(3):199-208, 2008). Evaluation of shared and divergent haplotype blocks among B6, C57, and C58 strains narrowed the Chr 9 QTL interval considerably and highlights the utility of QTL mapping in closely related inbred strains.

Genetic architecture of voluntary exercise in an advanced intercross line of mice.

Exercise is essential for health, yet the amount, duration, and intensity that individuals engage in are strikingly variable, even under prescription. Our focus was to identify the locations and effects of quantitative trait loci (QTL) controlling genetic predisposition for exercise-related traits, utilizing a large advanced intercross line (AIL) of mice. This AIL (G(4)) population originated from a reciprocal cross between mice with genetic propensity for increased voluntary exercise [high-runner (HR) line, selectively bred for increased wheel running] and the inbred strain C57BL/6J. After adjusting for family structure, we detected 32 significant and 13 suggestive QTL representing both daily running traits (distance, duration, average speed, and maximum speed) and the mean of these traits on days 5 and 6 (the selection criteria for HR) of a 6-day test conducted at 8 wk of age, with many co-localizing to similar genomic regions. Additionally, seven significant and five suggestive QTL were observed for the slope and intercept of a linear regression across all 6 days of running, some representing a combination of the daily traits. We also observed two significant and two suggestive QTL for body mass before exercise. These results, from a well-defined animal model, reinforce a genetic basis for the predisposition to engage in voluntary exercise, dissect this predisposition into daily segments across a continuous time period, and present unique QTL that may provide insight into the initiation, continuation, and temporal pattern of voluntary activity in mammals.

Polymorphisms of the scavenger receptor class B member 1 are associated with insulin resistance with evidence of gene by sex interaction.

BACKGROUND: Genetic variation in diabetes-associated genes cumulatively explain little of the overall heritability of this trait. We sought to determine whether polymorphisms of the scavenger receptor class B, member I (SCARB1), an estrogen-regulated chromosome 12q24 positional candidate diabetes gene, were associated with type 2 diabetes or insulin resistance in a sex-specific fashion. METHODS: We evaluated 34 haplotype-tagged single-nucleotide polymorphisms (SNPs) of SCARB1 for their association with type 2 diabetes and measures of insulin resistance in two populations: a clinic-based sample of 444 Mexican-American women from Proyecto SALSA and a community-based sample of 830 white women from the Rancho Bernardo Study. RESULTS: We identified significant associations between a tagged SNP in intron 9, rs9919713, and fasting glucose in the SALSA population (P = 2.3 x 10(-4)). In the Rancho Bernardo Study, the same SNP also showed significant association with the related traits homeostasis model assessment for insulin resistance (P = 3.0 x 10(-4)), fasting glucose (P = 1.1 x 10(-3)), and type 2 diabetes (P = 9.0 x 10(-3)). In men from the Rancho Bernardo population, the opposite effect was found (genotype by sex interaction in the Rancho Bernardo population P < 10(-3) for insulin resistance). CONCLUSIONS: Our data support an association between SCARB1 variants and insulin resistance, especially in women, with evidence of significant gene by sex interaction. These findings warrant further investigation in additional populations and prompt exploration of a role for SR-BI in the development of insulin resistance.

A forward genetics screen in mice identifies recessive deafness traits and reveals that pejvakin is essential for outer hair cell function.

Deafness is the most common form of sensory impairment in the human population and is frequently caused by recessive mutations. To obtain animal models for recessive forms of deafness and to identify genes that control the development and function of the auditory sense organs, we performed a forward genetics screen in mice. We identified 13 mouse lines with defects in auditory function and six lines with auditory and vestibular defects. We mapped several of the affected genetic loci and identified point mutations in four genes. Interestingly, all identified genes are expressed in mechanosensory hair cells and required for their function. One mutation maps to the pejvakin gene, which encodes a new member of the gasdermin protein family. Previous studies have described two missense mutations in the human pejvakin gene that cause nonsyndromic recessive deafness (DFNB59) by affecting the function of auditory neurons. In contrast, the pejvakin allele described here introduces a premature stop codon, causes outer hair cell defects, and leads to progressive hearing loss. We also identified a novel allele of the human pejvakin gene in an Iranian pedigree that is afflicted with progressive hearing loss. Our findings suggest that the mechanisms of pathogenesis associated with pejvakin mutations are more diverse than previously appreciated. More generally, our findings demonstrate that recessive screens in mice are powerful tools for identifying genes that control the development and function of mechanosensory hair cells and cause deafness in humans, as well as generating animal models for disease.

Phosphoregulation of mixed-lineage kinase 1 activity by multiple phosphorylation in the activation loop.

Mixed-lineage kinase 1 (MLK1) is a mitogen-activated protein kinase kinase kinase capable of activating the c-Jun NH(2)-terminal kinase (JNK) pathway. Full-length MLK1 has 1104 amino acids and a domain structure identical to MLK2 and MLK3. Immunoblot and mass spectrometry show that MLK1 is threonine (and possibly serine) phosphorylated in or near the activation loop. A kinase-dead mutant is not, consistent with autophosphorylation. Mutation to alanine of any of the four serine or threonine residues in the activation loop reduces both the activity of the recombinant kinase domain and JNK pathway activation driven by full-length MLK1 expressed in mammalian cells. Furthermore, the gel mobility of the mutant MLK1s is closer to that of the kinase-dead than wild type, consistent with reduced phosphorylation. Thr312 is the key residue: MLK1[T312A] retains only basal activity (about 1-2% of wild type), and its gel mobility is indistinguishable from kinase-dead. Thr312 does not suffice, however; phosphorylation of multiple sites is necessary for full activation of MLK1. An activation mechanism consistent with these data involves phosphorylation of multiple sites in the activation loop, with phosphorylation of Thr312 required for full phosphorylation. This mechanism is broadly similar to that previously reported for MLK3 [Leung, I. W., and Lassam, N. (2001) J. Biol. Chem. 276, 1961-1967], but the key residue differs.