Meiotic Mapping Research Articles

Nonredundant Role of Leishmanolysin-Like (Lmln) Zinc-Metallopeptidase in Retinal Homeostasis

To determine if Lmln, a Zn-metallopeptidase, is important for retinal homeostasis. Combining an unbiased N-ethyl-N-nitrosourea mutagenesis pipeline in mice with optical coherence tomography (OCT) screening and automated meiotic mapping, we identified an allele (nemeth) that seemed to be associated with outer nuclear layer (ONL) thinning. Since nemeth was predicted to lead to a nonsense mutation of the Lmln gene, we targeted Lmln using CRISPR/Cas-9 technology and characterized the impact on retinal anatomy and function. OCT imaging demonstrated an outer retinal degeneration in Lmln-/- mice (P = 7.3 × 10-9 for ONL at 2 m) that progressed over the first 6 months of life and then stabilized. Light microscopy showed loss of ONL nuclei (P ranged between .00033 and .0097 for posterior measurements), and a TUNEL assay revealed a small but significant increase in apoptosis (P = .034). Lmln-/- mice accumulated fundus spots (P = .0030 by 2 m of age) and activated subretinal microglia (p ranged from .0007 to 8 × 10-13 for Gal3+ cells). Scotopic electroretinography demonstrated a decrease in retinal function in Lmln-/- mice both at 6 m (only a-wave, P < .01 for all stimuli) and at 10 m of age (P < .01 for both a-wave and b-wave with all stimuli). Our work revealed a previously unknown essential requirement for Lmln in maintaining retinal anatomy and function. Further studies using this new model will be aimed at determining the cellular expression of Lmln and its mechanisms of action within the retina.

Genetic determinants of blood pressure and heart rate identified through ENU-induced mutagenesis with automated meiotic mapping.

We used N-ethyl-N-nitrosurea-induced germline mutagenesis combined with automated meiotic mapping to identify specific systolic blood pressure (SBP) and heart rate (HR) determinant loci. We analyzed 43,627 third-generation (G3) mice from 841 pedigrees to assess the effects of 45,378 variant alleles within 15,760 genes, in both heterozygous and homozygous states. We comprehensively tested 23% of all protein-encoding autosomal genes and found 87 SBP and 144 HR (with 7 affecting both) candidates exhibiting detectable hypomorphic characteristics. Unexpectedly, only 18 of the 87 SBP genes were previously known, while 26 of the 144 genes linked to HR were previously identified. Furthermore, we confirmed the influence of two genes on SBP regulation and three genes on HR control through reverse genetics. This underscores the importance of our research in uncovering genes associated with these critical cardiovascular risk factors and illustrate the effectiveness of germline mutagenesis for defining key determinants of polygenic phenotypes that must be studied in an intact organism.

Genetic influences on motor learning and performance and superperforming mutants revealed by random mutational survey of the mouse genome.

Evolution depends upon genetic variations that influence physiology. As defined in a genetic screen, phenotypic performance may be enhanced or degraded by such mutations. We set out to detect mutations that influence motor function, including motor learning. Thus, we tested the motor effects of 36,444 non-synonymous coding/splicing mutations induced in the germline of C57BL/6J mice with N-ethyl-N-nitrosourea by measuring changes in the performance of repetitive rotarod trials while blinded to genotype. Automated meiotic mapping was used to implicate individual mutations in causation. In total, 32,726 mice bearing all the variant alleles were screened. This was complemented with the simultaneous testing of 1408 normal mice for reference. In total, 16.3% of autosomal genes were thus rendered detectably hypomorphic or nullified by mutations in homozygosity and motor tested in at least three mice. This approach allowed us to identify superperformance mutations in Rif1, Tk1, Fan1 and Mn1. These genes are primarily related, among other less well-characterized functions, to nucleic acid biology. We also associated distinct motor learning patterns with groups of functionally related genes. These functional sets included, preferentially, histone H3 methyltransferase activity for mice that learnt at an accelerated rate relative to the remaining mutant mice. The results allow for an estimation of the fraction of mutations that can modify a behaviour influential for evolution such as locomotion. They may also enable, once the loci are further validated and the mechanisms elucidated, the harnessing of the activity of the newly identified genes to enhance motor ability or to counterbalance disability or disease. KEY POINTS: We studied the effect of chemically induced random mutations on mouse motor performance. An array of mutations influenced the rate of motor learning. DNA regulation genes predominated among these mutant loci. Several mutations in unsuspected genes led to superperformance. Assuming little-biased mutagenicity, the results allow for an estimation of the probability for any spontaneous mutation to influence a behaviour such as motor learning and ultimate performance.

A method for quantifying sporulation efficiency and isolating meiotic progeny in non-GMO strains of Saccharomyces cerevisiae.

Meiotic mapping, a linkage-based method for analyzing the recombinant progeny of a cross, has long been a cornerstone of genetic research. The yeast Saccharomyces cerevisiae is a powerful system because it is possible to isolate and cultivate the four products (spores) of a single meiotic event. However, the throughput of this process has historically been limited by the process of identifying tetrads in a heterogeneous population of vegetative cells, tetrads, and dyads followed by manual separation (dissection) of the spores contained in a tetrad. To date, methods that facilitate high throughput characterization and isolation of meiotic progeny have relied on genetic engineering. Here, we characterize the ability of the fluorescent dye DiBAC4 (5) to stain yeast tetrads and dyads as well as to adhere to spores following bulk tetrad disruption. Applications include quantitative assays of sporulation rates and efficiency by flow cytometry as well as enrichment of intact tetrads, dyads, or disrupted spores by fluorescence-activated cell sorting in strains that have not been genetically modified.

Modulation of autoimmune diabetes by N-ethyl-N-nitrosourea- induced mutations in non-obese diabetic mice.

ABSTRACTGenetic association studies of type 1 diabetes (T1D) in humans, and in congenic non-obese diabetic (NOD) mice harboring DNA segments from T1D-resistant mice, face the challenge of assigning causation to specific gene variants among many within loci that affect disease risk. Here, we created random germline mutations in NOD/NckH mice and used automated meiotic mapping to identify mutations modifying T1D incidence and age of onset. In contrast with association studies in humans or congenic NOD mice, we analyzed a relatively small number of genetic changes in each pedigree, permitting implication of specific mutations as causative. Among 844 mice from 14 pedigrees bearing 594 coding/splicing changes, we identified seven mutations that accelerated T1D development, and five that delayed or suppressed T1D. Eleven mutations affected genes not previously known to influence T1D (Xpnpep1, Herc1, Srrm2, Rapgef1, Ppl, Zfp583, Aldh1l1, Col6a1, Ccdc13, Cd200r1, Atrnl1). A suppressor mutation in Coro1a validated the screen. Mutagenesis coupled with automated meiotic mapping can detect genes in which allelic variation influences T1D susceptibility in NOD mice. Variation of some of the orthologous/paralogous genes may influence T1D susceptibility in humans.

Genetic landscape of T cells identifies synthetic lethality for T-ALL

To capture the global gene network regulating the differentiation of immature T cells in an unbiased manner, large-scale forward genetic screens in zebrafish were conducted and combined with genetic interaction analysis. After ENU mutagenesis, genetic lesions associated with failure of T cell development were identified by meiotic recombination mapping, positional cloning, and whole genome sequencing. Recessive genetic variants in 33 genes were identified and confirmed as causative by additional experiments. The mutations affected T cell development but did not perturb the development of an unrelated cell type, growth hormone-expressing somatotrophs, providing an important measure of cell-type specificity of the genetic variants. The structure of the genetic network encompassing the identified components was established by a subsequent genetic interaction analysis, which identified many instances of positive (alleviating) and negative (synthetic) genetic interactions. Several examples of synthetic lethality were subsequently phenocopied using combinations of small molecule inhibitors. These drugs not only interfered with normal T cell development, but also elicited remission in a model of T cell acute lymphoblastic leukaemia. Our findings illustrate how genetic interaction data obtained in the context of entire organisms can be exploited for targeted interference with specific cell types and their malignant derivatives.

Thousands of induced germline mutations affecting immune cells identified by automated meiotic mapping coupled with machine learning

Forward genetic studies use meiotic mapping to adduce evidence that a particular mutation, normally induced by a germline mutagen, is causative of a particular phenotype. Particularly in small pedigrees, cosegregation of multiple mutations, occasional unawareness of mutations, and paucity of homozygotes may lead to erroneous declarations of cause and effect. We sought to improve the identification of mutations causing immune phenotypes in mice by creating Candidate Explorer (CE), a machine-learning software program that integrates 67 features of genetic mapping data into a single numeric score, mathematically convertible to the probability of verification of any putative mutation-phenotype association. At this time, CE has evaluated putative mutation-phenotype associations arising from screening damaging mutations in ∼55% of mouse genes for effects on flow cytometry measurements of immune cells in the blood. CE has therefore identified more than half of genes within which mutations can be causative of flow cytometric phenovariation in Mus musculus The majority of these genes were not previously known to support immune function or homeostasis. Mouse geneticists will find CE data informative in identifying causative mutations within quantitative trait loci, while clinical geneticists may use CE to help connect causative variants with rare heritable diseases of immunity, even in the absence of linkage information. CE displays integrated mutation, phenotype, and linkage data, and is freely available for query online.

Saturation mutagenesis defines novel mouse models of severe spine deformity.

ABSTRACTEmbryonic formation and patterning of the vertebrate spinal column requires coordination of many molecular cues. After birth, the integrity of the spine is impacted by developmental abnormalities of the skeletal, muscular and nervous systems, which may result in deformities, such as kyphosis and scoliosis. We sought to identify novel genetic mouse models of severe spine deformity by implementing in vivo skeletal radiography as part of a high-throughput saturation mutagenesis screen. We report selected examples of genetic mouse models following radiographic screening of 54,497 mice from 1275 pedigrees. An estimated 30.44% of autosomal genes harbored predicted damaging alleles examined twice or more in the homozygous state. Of the 1275 pedigrees screened, 7.4% presented with severe spine deformity developing in multiple mice, and of these, meiotic mapping implicated N-ethyl-N-nitrosourea alleles in 21% of pedigrees. Our study provides proof of concept that saturation mutagenesis is capable of discovering novel mouse models of human disease, including conditions with skeletal, neural and neuromuscular pathologies. Furthermore, we report a mouse model of skeletal disease, including severe spine deformity, caused by recessive mutation in Scube3. By integrating results with a human clinical exome database, we identified a patient with undiagnosed skeletal disease who harbored recessive mutations in SCUBE3, and we demonstrated that disease-associated mutations are associated with reduced transactivation of Smad signaling in vitro. All radiographic results and mouse models are made publicly available through the Mutagenetix online database with the goal of advancing understanding of spine development and discovering novel mouse models of human disease.

Postembryonic screen for mutations affecting spine development in zebrafish

The spine gives structural support for the adult body, protects the spinal cord, and provides muscle attachment for moving through the environment. The development and maturation of the spine and its physiology involve the integration of multiple musculoskeletal tissues including bone, cartilage, and fibrocartilaginous joints, as well as innervation and control by the nervous system. One of the most common disorders of the spine in human is adolescent idiopathic scoliosis (AIS), which is characterized by the onset of an abnormal lateral curvature of the spine of <10° around adolescence, in otherwise healthy children. The genetic basis of AIS is largely unknown. Systematic genome-wide mutagenesis screens for embryonic phenotypes in zebrafish have been instrumental in the understanding of early patterning of embryonic tissues necessary to build and pattern the embryonic spine. However, the mechanisms required for postembryonic maturation and homeostasis of the spine remain poorly understood. Here we report the results from a small-scale forward genetic screen for adult-viable recessive and dominant zebrafish mutations, leading to overt morphological abnormalities of the adult spine. Germline mutations induced with N-ethyl N-nitrosourea (ENU) were transmitted and screened for dominant phenotypes in 1229 F1 animals, and subsequently bred to homozygosity in F3 families; from these, 314 haploid genomes were screened for adult-viable recessive phenotypes affecting general body shape. We cumulatively found 40 adult-viable (3 dominant and 37 recessive) mutations each leading to a defect in the morphogenesis of the spine. The largest phenotypic group displayed larval onset axial curvatures, leading to whole-body scoliosis without vertebral dysplasia in adult fish. Pairwise complementation testing of 16 mutant lines within this phenotypic group revealed at least 9 independent mutant loci. Using massively-parallel whole genome or whole exome sequencing and meiotic mapping we defined the molecular identity of several loci for larval onset whole-body scoliosis in zebrafish. We identified a new mutation in the skolios/kinesin family member 6 (kif6) gene, causing neurodevelopmental and ependymal cilia defects in mouse and zebrafish. We also report multiple recessive alleles of the scospondin and a disintegrin and metalloproteinase with thrombospondin motifs 9 (adamts9) genes, which all display defects in spine morphogenesis. Our results provide evidence of monogenic traits that are essential for normal spine development in zebrafish, that may help to establish new candidate risk loci for spine disorders in humans.

Forward genetic analysis using OCT screening identifies Sfxn3 mutations leading to progressive outer retinal degeneration in mice

Retinal disease and loss of vision can result from any disruption of the complex pathways controlling retinal development and homeostasis. Forward genetics provides an excellent tool to find, in an unbiased manner, genes that are essential to these processes. Using N-ethyl-N-nitrosourea mutagenesis in mice in combination with a screening protocol using optical coherence tomography (OCT) and automated meiotic mapping, we identified 11 mutations presumably causative of retinal phenotypes in genes previously known to be essential for retinal integrity. In addition, we found multiple statistically significant gene-phenotype associations that have not been reported previously and decided to target one of these genes, Sfxn3 (encoding sideroflexin-3), using CRISPR/Cas9 technology. We demonstrate, using OCT, light microscopy, and electroretinography, that two Sfxn3-/- mouse lines developed progressive and severe outer retinal degeneration. Electron microscopy showed thinning of the retinal pigment epithelium and disruption of the external limiting membrane. Using single-cell RNA sequencing of retinal cells isolated from C57BL/6J mice, we demonstrate that Sfxn3 is expressed in several bipolar cell subtypes, retinal ganglion cells, and some amacrine cell subtypes but not significantly in Müller cells or photoreceptors. In situ hybridization confirmed these findings. Furthermore, pathway analysis suggests that Sfxn3 may be associated with synaptic homeostasis. Importantly, electron microscopy analysis showed disruption of synapses and synaptic ribbons in the outer plexiform layer of Sfxn3-/- mice. Our work describes a previously unknown requirement for Sfxn3 in retinal function.

Cold Fusion: Massive Karyotype Evolution in the Antarctic Bullhead Notothen Notothenia coriiceps.

Half of all vertebrate species share a series of chromosome fusions that preceded the teleost genome duplication (TGD), but we do not understand the causative evolutionary mechanisms. The “Robertsonian-translocation hypothesis” suggests a regular fusion of each ancestral acro- or telocentric chromosome to just one other by centromere fusions, thus halving the karyotype. An alternative “genome-stirring hypothesis” posits haphazard and repeated fusions, inversions, and reciprocal and nonreciprocal translocations. To study large-scale karyotype reduction, we investigated the decrease of chromosome numbers in Antarctic notothenioid fish. Most notothenioids have 24 haploid chromosomes, but bullhead notothen (Notothenia coriiceps) has 11. To understand mechanisms, we made a RAD-tag meiotic map with ∼10,000 polymorphic markers. Comparative genomics aligned about a thousand orthologs of platyfish and stickleback genes along bullhead chromosomes. Results revealed that 9 of 11 bullhead chromosomes arose by fusion of just two ancestral chromosomes and two others by fusion of three ancestral chromosomes. All markers from each ancestral chromosome remained contiguous, implying no inversions across fusion borders. Karyotype comparisons support a history of: (1) Robertsonian fusions of 22 ancestral chromosomes in pairs to yield 11 fused plus two small unfused chromosomes, like N. angustata; (2) fusion of one of the remaining two ancestral chromosomes to a preexisting fused pair, giving 12 chromosomes like N. rossii; and (3) fusion of the remaining ancestral chromosome to another fused pair, giving 11 chromosomes in N. coriiceps. These results raise the question of what selective forces promoted the systematic fusion of chromosomes in pairs and the suppression of pericentric inversions in this lineage, and provide a model for chromosome fusions in stem teleosts.

Validated Bayesian Differentiation of Causative and Passenger Mutations.

In many contexts, the problem arises of determining which of many candidate mutations is the most likely to be causative for some phenotype. It is desirable to have a way to evaluate this probability that relies as little as possible on previous knowledge, to avoid bias against discovering new genes or functions. We have isolated mutants with blocked cell cycle progression in Chlamydomonas and determined mutant genome sequences. Due to the intensity of UV mutagenesis required for efficient mutant collection, the mutants contain multiple mutations altering coding sequence. To provide a quantitative estimate of probability that each individual mutation in a given mutant is the causative one, we developed a Bayesian approach. The approach employs four independent indicators: sequence conservation of the mutated coding sequence with Arabidopsis; severity of the mutation relative to Chlamydomonas wild-type based on Blosum62 scores; meiotic mapping information for location of the causative mutation relative to known molecular markers; and, for a subset of mutants, the transcriptional profile of the candidate wild-type genes through the mitotic cell cycle. These indicators are statistically independent, and so can be combined quantitatively into a single probability calculation. We validate this calculation: recently isolated mutations that were not in the training set for developing the indicators, with high calculated probability of causality, are confirmed in every case by additional genetic data to indeed be causative. Analysis of “best reciprocal BLAST” (BRB) relationships among Chlamydomonas and other eukaryotes indicate that the temperature sensitive-lethal (Ts-lethal) mutants that our procedure recovers are highly enriched for fundamental cell-essential functions conserved broadly across plants and other eukaryotes, accounting for the high information content of sequence alignment to Arabidopsis.

A Zebrafish Model for a Human Myopathy Associated with Mutation of the Unconventional Myosin MYO18B.

Myosin 18B is an unconventional myosin that has been implicated in tumor progression in humans. In addition, loss-of-function mutations of the MYO18B gene have recently been identified in several patients exhibiting symptoms of nemaline myopathy. In mouse, mutation of Myo18B results in early developmental arrest associated with cardiomyopathy, precluding analysis of its effects on skeletal muscle development. The zebrafish, frozen (fro) mutant was identified as one of a group of immotile mutants in the 1996 Tübingen genetic screen. Mutant embryos display a loss of birefringency in their skeletal muscle, indicative of disrupted sarcomeric organization. Using meiotic mapping, we localized the fro locus to the previously unannotated zebrafish myo18b gene, the product of which shares close to 50% identity with its human ortholog. Transcription of myo18b is restricted to fast-twitch myocytes in the zebrafish embryo; consistent with this, fro mutant embryos exhibit defects specifically in their fast-twitch skeletal muscles. We show that sarcomeric assembly is blocked at an early stage in fro mutants, leading to the disorganized accumulation of actin, myosin, and α-actinin and a complete loss of myofibrillar organization in fast-twitch muscles.

High-Density Genetic Mapping with Interspecific Hybrids of Two Sea Urchins, Strongylocentrotus nudus and S. intermedius, by RAD Sequencing

Sea urchins have long been used as research model organisms for developmental biology and evolutionary studies. Some of them are also important aquaculture species in East Asia. In this work, we report the construction of RAD-tag based high-density genetic maps by genotyping F1 interspecific hybrids derived from a crossing between a female sea urchin Strongylocentrotus nudus and a male Strongylocentrotus intermedius. With polymorphisms present in these two wild individuals, we constructed a female meiotic map containing 3,080 markers for S. nudus, and a male meiotic map for S. intermedius which contains 1,577 markers. Using the linkage maps, we were able to anchor a total of 1,591 scaffolds (495.9 Mb) accounting for 60.8% of the genome assembly of Strongylocentrotus purpuratus. A genome-wide scan resulted in the identification of one putative QTL for body size which spanned from 25.3 cM to 30.3 cM. This study showed the efficiency of RAD-Seq based high-density genetic map construction using F1 progenies for species with no prior genomic information. The genetic maps are essential for QTL mapping and are useful as framework to order and orientate contiguous scaffolds from sea urchin genome assembly. The integration of the genetic map with genome assembly would provide an unprecedented opportunity to conduct QTL analysis, comparative genomics, and population genetics studies.

Fast forward genetics to identify mutations causing a high light tolerant phenotype in Chlamydomonas reinhardtii by whole-genome-sequencing.

BackgroundHigh light tolerance of microalgae is a desired phenotype for efficient cultivation in large scale production systems under fluctuating outdoor conditions. Outdoor cultivation requires the use of either wild-type or non-GMO derived mutant strains due to safety concerns. The identification and molecular characterization of such mutants derived from untagged forward genetics approaches was limited previously by the tedious and time-consuming methods involving techniques such as classical meiotic mapping. The combination of mapping with next generation sequencing technologies offers alternative strategies to identify genes involved in high light adaptation in untagged mutants.ResultsWe used the model alga Chlamydomonas reinhardtii in a non-GMO mutation strategy without any preceding crossing step or pooled progeny to identify genes involved in the regulatory processes of high light adaptation. To generate high light tolerant mutants, wildtype cells were mutagenized only to a low extent, followed by a stringent selection. We performed whole-genome sequencing of two independent mutants hit1 and hit2 and the parental wildtype. The availability of a reference genome sequence and the removal of shared bakground variants between the wildtype strain and each mutant, enabled us to identify two single nucleotide polymorphisms within the same gene Cre02.g085050, hereafter called LRS1 (putative Light Response Signaling protein 1). These two independent single amino acid exchanges are both located in the putative WD40 propeller domain of the corresponding protein LRS1. Both mutants exhibited an increased rate of non-photochemical-quenching (NPQ) and an improved resistance against chemically induced reactive oxygen species. In silico analyses revealed homology of LRS1 to the photoregulatory protein COP1 in plants.ConclusionsIn this work we identified the nuclear encoded gene LRS1 as an essential factor for high light adaptation in C. reinhardtii. The causative random mutation within this gene was identified by a rapid and efficient method, avoiding any preceding crossing step, meiotic mapping, or pooled progeny. Our results open up new insights into mechanisms of high light adaptation in microalgae and at the same time provide a simplified strategy for non-GMO forward genetics, a crucial precondition that could result in the identification of key factors for economically relevant biological processes within algae.

Sal-Site: research resources for the Mexican axolotl.

Sal-Site serves axolotl research efforts by providing Web access to genomic data and information, and living stocks that are reared and made available by the Ambystoma Genetic Stock Center (AGSC). In this chapter, we detail how investigators can search for genes of interest among Sal-Site resources to identify orthologous nucleotide and protein-coding sequences, determine genome positions within the Ambystoma meiotic map, and obtain estimates of gene expression. In the near future, additional genomic resources will be made available for the axolotl, including a listing of genes that are partially or wholly contained within Bacterial Artificial Chromosome (BAC) vectors, a prioritized collection of deeply sequenced BAC clones, chromosome-specific assemblies of genomic DNA, and transgenic axolotls that are engineered using TALENs and CRISPRs. Also, services provided by the AGSC will be expanded to include microinjection of user constructs into single cell embryos and distribution of axolotl tissues, DNA, and RNA. In conclusion, Sal-Site is a useful resource that generates, shares, and evolves Ambystoma associated information and databases to serve research and education.

Kinesin family member 6 (kif6) is necessary for spine development in zebrafish.

Idiopathic scoliosis is a form of spinal deformity that affects 2-3% of children and results in curvature of the spine without structural defects of the vertebral units. The pathogenesis of idiopathic scoliosis remains poorly understood, in part due to the lack of a relevant animal model. We performed a forward mutagenesis screen in zebrafish to identify new models for idiopathic scoliosis. We isolated a recessive zebrafish mutant, called skolios, which develops isolated spinal curvature that arises independent of vertebral malformations. Using meiotic mapping and whole genome sequencing, we identified a nonsense mutation in kinesin family member 6 (kif6(gw326) ) unique to skolios mutants. Three additional kif6 frameshift alleles (gw327, gw328, gw329) were generated with transcription activator-like effector nucleases (TALENs). Zebrafish homozygous or compound heterozygous for kif6 frameshift mutations developed a scoliosis phenotype indistinguishable from skolios mutants, confirming that skolios is caused by the loss of kif6. Although kif6 may play a role in cilia, no evidence for cilia dysfunction was seen in kif6(gw326) mutants. Overall, these findings demonstrate a novel role for kif6 in spinal development and identify a new candidate gene for human idiopathic scoliosis.

A RAD-tag genetic map for the platyfish (Xiphophorus maculatus) reveals mechanisms of karyotype evolution among teleost fish.

Mammalian genomes can vary substantially in haploid chromosome number even within a small taxon (e.g., 3-40 among deer alone); in contrast, teleost fish genomes are stable (24-25 in 58% of teleosts), but we do not yet understand the mechanisms that account for differences in karyotype stability. Among perciform teleosts, platyfish (Xiphophorus maculatus) and medaka (Oryzias latipes) both have 24 chromosome pairs, but threespine stickleback (Gasterosteus aculeatus) and green pufferfish (Tetraodon nigroviridis) have just 21 pairs. To understand the evolution of teleost genomes, we made a platyfish meiotic map containing 16,114 mapped markers scored on 267 backcross fish. We tiled genomic contigs along the map to create chromosome-length genome assemblies. Genome-wide comparisons of conserved synteny showed that platyfish and medaka karyotypes remained remarkably similar with few interchromosomal translocations but with numerous intrachromosomal rearrangements (transpositions and inversions) since their lineages diverged ∼120 million years ago. Comparative genomics with platyfish shows how reduced chromosome numbers in stickleback and green pufferfish arose by fusion of pairs of ancestral chromosomes after their lineages diverged from platyfish ∼195 million years ago. Zebrafish and human genomes provide outgroups to root observed changes. These studies identify likely genome assembly errors, characterize chromosome fusion events, distinguish lineage-independent chromosome fusions, show that the teleost genome duplication does not appear to have accelerated the rate of translocations, and reveal the stability of syntenies and gene orders in teleost chromosomes over hundreds of millions of years.

Analysis of simple sequence repeats in the Gaeumannomyces graminis var. tritici genome and the development of microsatellite markers.

Understanding the genetic structure of Gaeumannomyces graminis var. tritici is essential for the establishment of efficient disease control strategies. It is becoming clear that microsatellites, or simple sequence repeats (SSRs), play an important role in genome organization and phenotypic diversity, and are a large source of genetic markers for population genetics and meiotic maps. In this study, we examined the G. graminis var. tritici genome (1) to analyze its pattern of SSRs, (2) to compare it with other plant pathogenic filamentous fungi, such as Magnaporthe oryzae and M. poae, and (3) to identify new polymorphic SSR markers for genetic diversity. The G. graminis var. tritici genome was rich in SSRs; a total 13,650 SSRs have been identified with mononucleotides being the most common motifs. In coding regions, the densities of tri- and hexanucleotides were significantly higher than in noncoding regions. The di-, tri-, tetra, penta, and hexanucleotide repeats in the G. graminis var. tritici genome were more abundant than the same repeats in M. oryzae and M. poae. From 115 devised primers, 39 SSRs are polymorphic with G. graminis var. tritici isolates, and 8 primers were randomly selected to analyze 116 isolates from China. The number of alleles varied from 2 to 7 and the expected heterozygosity (He) from 0.499 to 0.837. In conclusion, SSRs developed in this study were highly polymorphic, and our analysis indicated that G. graminis var. tritici is a species with high genetic diversity. The results provide a pioneering report for several applications, such as the assessment of population structure and genetic diversity of G. graminis var. tritici.

Fox domestication: Molecular mechanisms involved in selection for behavior

Although animal domestication has enjoyed the attention of geneticists and evolutionary biologists since Darwin’s times, the focal question about the genetic basis of this process has not been duly considered. We present some results of experimental modeling of historical domestication in silver foxes (Vulpes vulpes), an object of commercial breeding. Attention is focused on the role of artificial selection in the transformation of the behavior of these animals, which has brought about unique populations of tame and aggressive foxes. Additional resources were developed to analyze the molecular nature of differences in the behavior of these unique foxes: the meiotic map of the fox genome was constructed, informative segregating subpopulations were obtained by crosses of tame and aggressive animals, and the formerly applied method of behavioral phenotype quantification was improved. The integrated behavioral phenotypes (principal components PC1 and PC2) used in the study were obtained by analysis of ethological parameters recorded with a camcorder. The most important result is that the region most closely associated with tame behavior was identified on fox chromosome 12 by QTL interval mapping. The principle significance of this result is strengthened by the fact that the region is similar to the region on dog chromosome 5, presumed to be responsible for the early domestication of wolves and their evolutionary transformation to primitive dogs.