The use of bridging systems to increase genetic variability in compound chromosome strains for genetic control of Lucilia cuprina (Wiedemann)

Summary Genetic variability in the non-compound portion of the genomes of compound-chromosome (CC) strains intended for genetic control can be increased by the use of bridging strains which can be crossed to both CC and normal strains. Two bridging systems are described for chromosome-5 CC strains of Lucilia cuprina. The first system relies on the established viability and fertility of males trisomic for chromosome 5R. Males carrying the (5L.YL)23 half-translocation, a C(5R), and a normal chromosome 5 were crossed successfully to a CC strain and a normal strain. The second system uses a pair of reciprocal whole-arm 4;5 translocations to generate gametes disomic for 5R and nullosomic for 5L, which in combination with C(5L)-bearing gametes form viable near-euploid offspring with only small duplications and deficiencies. These offspring (C(5L); (4L.5R)357; (4R.5R)194; (4L.4R)) were crossed successfully with both CC and T(4;5)357/ + individuals. The latter were in turn crossed successfully with normal strains. The T(Y;5)23 system allows replacement of the non-CC genome with wild material more rapidly than the T(4;5)357/T(4;5)194 system, but unlike the latter does not allow replacement of the Y chromosome in the CC strain. The double translocation system is currently being used in L. cuprina.     

Heritable variation in locomotion,reward sensitivity and impulsive behaviors in a genetically diverse inbred mouse panel

Drugs of abuse, including alcohol and stimulants like cocaine, produce effects that are subject to individual variability, and genetic variation accounts for at least a portion of those differences. Notably, research in both animal models and human subjects point toward reward sensitivity and impulsivity as being trait characteristics that predict relatively greater positive subjective responses to stimulant drugs. Here we describe use of the eight collaborative cross (CC) founder strains and 38 (reversal learning) or 10 (all other tests) CC strains to examine the heritability of reward sensitivity and impulsivity traits, as well as genetic correlations between these measures and existing addiction-related phenotypes. Strains were all tested for activity in an open field and reward sensitivity (intake of chocolate BOOST®). Mice were then divided into two counterbalanced groups and underwent reversal learning (impulsive action and waiting impulsivity) or delay discounting (impulsive choice). CC and founder mice show significant heritability for impulsive action, impulsive choice, waiting impulsivity, locomotor activity, and reward sensitivity, with each impulsive phenotype determined to be non-correlating, independent traits. This research was conducted within the broader, inter-laboratory effort of the Center for Systems Neurogenetics of Addiction (CSNA) to characterize CC and DO mice for multiple, cocaine abuse related traits. These data will facilitate the discovery of genetic correlations between predictive traits, which will then guide discovery of genes and genetic variants that contribute to addictive behaviors.     

Host genetic and environmental effects on mouse intestinal microbiota

The mammalian gut harbors complex and variable microbial communities, across both host phylogenetic space and conspecific individuals. A synergy of host genetic and environmental factors shape these communities and account for their variability, but their individual contributions and the selective pressures involved are still not well understood. We employed barcoded pyrosequencing of V1-2 and V4 regions of bacterial small subunit ribosomal RNA genes to characterize the effects of host genetics and environment on cecum assemblages in 10 genetically distinct, inbred mouse strains. Eight of these strains are the foundation of the Collaborative Cross (CC), a panel of mice derived from a genetically diverse set of inbred founder strains, designed specifically for complex trait analysis. Diversity of gut microbiota was characterized by complementing phylogenetic and distance-based, sequence-clustering approaches. Significant correlations were found between the mouse strains and their gut microbiota, reflected by distinct bacterial communities. Cohabitation and litter had a reduced, although detectable effect, and the microbiota response to these factors varied by strain. We identified bacterial phylotypes that appear to be discriminative and strain-specific to each mouse line used. Cohabitation of different strains of mice revealed an interaction of host genetic and environmental factors in shaping gut bacterial consortia, in which bacterial communities became more similar but retained strain specificity. This study provides a baseline analysis of intestinal bacterial communities in the eight CC progenitor strains and will be linked to integrated host genotype, phenotype and microbiota research on the resulting CC panel.     

Nucleotide variation in wild and inbred mice

The house mouse is a well-established model organism, particularly for studying the genetics of complex traits. However, most studies of mice use classical inbred strains, whose genomes derive from multiple species. Relatively little is known about the distribution of genetic variation among these species or how variation among strains relates to variation in the wild. We sequenced intronic regions of five X-linked loci in large samples of wild Mus domesticus and M. musculus, and we found low levels of nucleotide diversity in both species. We compared these data to published data from short portions of six X-linked and 18 autosomal loci in wild mice. We estimate that M. domesticus and M. musculus diverged <500,000 years ago. Consistent with this recent divergence, some gene genealogies were reciprocally monophyletic between these species, while others were paraphyletic or polyphyletic. In general, the X chromosome was more differentiated than the autosomes. We resequenced classical inbred strains for all 29 loci and found that inbred strains contain only a small amount of the genetic variation seen in wild mice. Notably, the X chromosome contains proportionately less variation among inbred strains than do the autosomes. Moreover, variation among inbred strains derives from differences between species as well as from differences within species, and these proportions differ in different genomic regions. Wild mice thus provide a reservoir of additional genetic variation that may be useful for mapping studies. Together these results suggest that wild mice will be a valuable complement to laboratory strains for studying the genetics of complex traits.     

The microbial community dynamics of cocaine sensitization in two behaviorally divergent strains of collaborative cross mice

The gut-brain axis is increasingly recognized as an important pathway involved in cocaine use disorder. Microbial products of the murine gut have been shown to affect striatal gene expression, and depletion of the microbiome by antibiotic treatment alters cocaine-induced behavioral sensitization in C57BL/6J male mice. Some reports suggest that cocaine-induced behavioral sensitization is correlated with drug self-administration behavior in mice. Here, we profile the composition of the naïve microbiome and its response to cocaine sensitization in two collaborative cross (CC) strains. These strains display extremely divergent behavioral responses to cocaine sensitization. A high-responding strain, CC004/TauUncJ (CC04), has a gut microbiome that contains a greater amount of Lactobacillus than the cocaine-nonresponsive strain CC041/TauUncJ (CC41). The gut microbiome of CC41 is characterized by an abundance of Eisenbergella, Robinsonella and Ruminococcus. In response to cocaine, CC04 has an increased Barnsiella population, while the gut microbiome of CC41 displays no significant changes. PICRUSt functional analysis of the functional potential of the gut microbiome in CC04 shows a significant number of potential gut-brain modules altered after exposure to cocaine, specifically those encoding for tryptophan synthesis, glutamine metabolism, and menaquinone synthesis (vitamin K2). Depletion of the microbiome by antibiotic treatment revealed an altered cocaine-sensitization response following antibiotics in female CC04 mice. Depleting the microbiome by antibiotic treatment in males revealed increased infusions for CC04 during a cocaine intravenous self-administration dose–response curve. Together these data suggest that genetic differences in cocaine-related behaviors may involve the microbiome.     

Genetic diversity of collaborative cross mice enables identification of novel rift valley fever virus encephalitis model

Rift Valley fever (RVF) is an arboviral disease of humans and livestock responsible for severe economic and human health impacts. In humans, RVF spans a variety of clinical manifestations, ranging from an acute flu-like illness to severe forms of disease, including late-onset encephalitis. The large variations in human RVF disease are inadequately represented by current murine models, which overwhelmingly die of early-onset hepatitis. Existing mouse models of RVF encephalitis are either immunosuppressed, display an inconsistent phenotype, or develop encephalitis only when challenged via intranasal or aerosol exposure. In this study, the genetically defined recombinant inbred mouse resource known as the Collaborative Cross (CC) was used to identify mice with additional RVF disease phenotypes when challenged via a peripheral foot-pad route to mimic mosquito-bite exposure. Wild-type Rift Valley fever virus (RVFV) challenge of 20 CC strains revealed three distinct disease phenotypes: early-onset hepatitis, mixed phenotype, and late-onset encephalitis. Strain CC057/Unc, with the most divergent phenotype, which died of late-onset encephalitis at a median of 11 days post-infection, is the first mouse strain to develop consistent encephalitis following peripheral challenge. CC057/Unc mice were directly compared to C57BL/6 mice, which uniformly succumb to hepatitis within 2–4 days of infection. Encephalitic disease in CC057/Unc mice was characterized by high viral RNA loads in brain tissue, accompanied by clearance of viral RNA from the periphery, low ALT levels, lymphopenia, and neutrophilia. In contrast, C57BL/6 mice succumbed from hepatitis at 3 days post-infection with high viral RNA loads in the liver, viremia, high ALT levels, lymphopenia, and thrombocytopenia. The identification of a strain of CC mice as an RVFV encephalitis model will allow for future investigation into the pathogenesis and treatment of RVF encephalitic disease and indicates that genetic background makes a major contribution to RVF disease variation.     

A novel repetitive DNA sequence in the genus Oryza

Summary Repetitive DNA sequences in the genus Oryza (rice) represent a large fraction of the nuclear DNA. The isolation and characterization of major repetitive DNA sequences will lead to a better understanding of rice genome organization and evolution. Here we report the characterization of a novel repetitive sequence, CC-1, from the CC genome. This repetitive sequence is present as long tandem arrays with a repeat unit 194 bp in length in the CC-diploid genome but 172 bp in length in the BBCC and CCDD tetraploid genomes. This repetitive sequence is also present, though at lower copy numbers, in the AA and BB genomes, but is absent in the EE and FF genomes. Hybridization experiments revealed considerable differences both in copy numbers and in restriction fragment patterns of CC-1 both between and within rice species. The results support the hypothesis that the CC genome is more closely related to the AA genome than to the BB genome, and most distantly related to the EE and FF genomes.     

Genetic background influences survival of infections with Salmonella enterica serovar Typhimurium in the Collaborative Cross

Salmonella infections typically cause self-limiting gastroenteritis, but in some individuals these bacteria can spread systemically and cause disseminated disease. Salmonella Typhimurium (STm), which causes severe systemic disease in most inbred mice, has been used as a model for disseminated disease. To screen for new infection phenotypes across a range of host genetics, we orally infected 32 Collaborative Cross (CC) mouse strains with STm and monitored their disease progression for seven days by telemetry. Our data revealed a broad range of phenotypes across CC strains in many parameters including survival, bacterial colonization, tissue damage, complete blood counts (CBC), and serum cytokines. Eighteen CC strains survived to day 7, while fourteen susceptible strains succumbed to infection before day 7. Several CC strains had sex differences in survival and colonization. Surviving strains had lower pre-infection baseline temperatures and were less active during their daily active period. Core body temperature disruptions were detected earlier after STm infection than activity disruptions, making temperature a better detector of illness. All CC strains had STm in spleen and liver, but susceptible strains were more highly colonized. Tissue damage was weakly negatively correlated to survival. We identified loci associated with survival on Chromosomes (Chr) 1, 2, 4, 7. Polymorphisms in Ncf2 and Slc11a1, known to reduce survival in mice after STm infections, are located in the Chr 1 interval, and the Chr 7 association overlaps with a previously identified QTL peak called Ses2. We identified two new genetic regions on Chr 2 and 4 associated with susceptibility to STm infection. Our data reveal the diversity of responses to STm infection across a range of host genetics and identified new candidate regions for survival of STm infection.     

Increased H-2Dd expression following infection by a molecularly cloned ecotropic MuLV

The biological consequences of radiation leukemia virus (RadLV) infection include the stimulation of H-2D^d antigen expression in resistant mouse strains and thymoma induction in susceptible strains. In an effort to understand the genetic basis of these phenomena, the integrated ecotropic RadLV genome has been examined in a number of primary RadLV-induced tumors, as well as thymomas adapted to in vitro passage; considerable heterogeneity was observed. Examination of these polymorphic viral sequences should help define the viral gene(s) involved in the biological effects of RadLV infection; toward this end, integrated RadLV genomes were molecularly cloned and examined. The genomes and their flanking sequence were characterized by restriction enzyme analysis. Three unique viral genomes were obtained which represent four integration sites. The three RadLV genomes are shown to carry polymorphisms of the original tumor. Following DNA transfection, one of the three genomes replicated in and reinfected both mouse thymocytes and fibroblasts, but not mink fibroblasts in vitro. Virus encoded by the other two DNA genomes could not be recovered following transfection into any of the three cell types. One of these two apparently defective retroviruses encodes a truncated p15E molecule, while the other has elongated long terminal repeats (LTRs). The non-defective ecotropic isolate was collected from in vitro tissue culture supernatants, concentrated, and used to infect mice. Thymocytes of infected, resistant mice were shown to express elevated levels of H-2D^d antigen as early as 12 days post infection, a hallmark of RadLV infection. Offprint requests to: G. D. Brown.     

Patterns and mechanisms of sex ratio distortion in the Collaborative Cross mouse mapping population

In species with single-locus, chromosome-based mechanisms of sex determination, the laws of segregation predict an equal ratio of females to males at birth. Here, we show that departures from this Mendelian expectation are commonplace in the 8-way recombinant inbred Collaborative Cross (CC) mouse population. More than one-third of CC strains exhibit significant sex ratio distortion (SRD) at wean, with twice as many male-biased than female-biased strains. We show that these pervasive sex biases persist across multiple breeding environments, are stable over time, and are not mediated by random maternal effects. SRD exhibits a heritable component, but QTL mapping analyses fail to nominate any large effect loci. These findings, combined with the reported absence of sex ratio biases in the CC founder strains, suggest that SRD manifests from multilocus combinations of alleles only uncovered in recombined CC genomes. We explore several potential complex genetic mechanisms for SRD, including allelic interactions leading to sex-biased lethality, genetic sex reversal, chromosome drive mediated by sex-linked selfish elements, and incompatibilities between specific maternal and paternal genotypes. We show that no one mechanism offers a singular explanation for this population-wide SRD. Instead, our data present preliminary evidence for the action of distinct mechanisms of SRD at play in different strains. Taken together, our work exposes the pervasiveness of SRD in the CC population and nominates the CC as a powerful resource for investigating diverse genetic causes of biased sex chromosome transmission.     

Isozymes of NADP-dependent isocitrate dehydrogenase in normal and tumor tissues of various mouse strains and their hybrids

Normal tissues of DBA, CBA, CC57W, C3H, Balb/c, SHR mice and F1 hybrids CC57W/DBA appeared to differ in the ratios of mitochondrial and supernatant NADP-dependent isocitrate dehydrogenase (IDH). Tested inbred mice strains CC57W, C3H, SHR, Balb/c contain allelic form Idh-1a of supernatant IDH gene Idh-1, whereas allelic form Idh-1b is characteristic of mice strains DBA and CBA. In tumors IDH isozymes have the same mobility as do isozymes of homologous normal tissues; but their activity is lower. A high variability of each isozyme activity in the isozyme spectrum is revealed in various tissues of F1 hybrids CC57W/DBA. Allelic forms of gene Idh-1 were used as markers of normal and tumor cells for the experimental model: transplantation of sarcoma 37 (Idh-1a/Idh-1a) to subcutaneous tissue of the mouse strain DBA (Idh-1b/Idh-1b). It enables us to reveal isozymes of stromal cell in tumor IDH isozyme spectrum. The results indicate that the relation of normal and tumor isozymes vary in different tumors.     

Ni Uptake and Limitation in Marine Synechococcus Strains

Ni accumulation and utilization were studied in two strains of marine Synechococcus, isolated from both coastal (CC9311; clade I) and open-ocean (WH8102; clade III) environments, for which complete genome sequences are available. Both strains have genes encoding an Ni-containing urease and when grown on urea without Ni become Ni-N colimited. The Ni requirements of these strains also depend upon the genomic complement of genes encoding superoxide dismutase (SOD). WH8102, with a gene encoding only an Ni-SOD, has a novel obligate requirement for Ni, regardless of the N source. Reduced SOD activity in Ni-depleted cultures of WH8102 supports the link of this strain's Ni requirement to Ni-SOD. The genome of CC9311 contains a gene for a Cu/Zn-SOD in addition to a predicted pair of Ni-SODs, yet this strain cannot grow without Ni on NO₃⁻ and can grow only slowly on NH₄⁺ without Ni, implying that the Cu/Zn-SOD cannot completely replace Ni-SOD in marine cyanobacteria. CC9311 does have a greater tolerance for Ni starvation. Both strains increase their Ni uptake capabilities and actively bioconcentrate Ni in response to decreasing extracellular and intracellular Ni. The changes in Ni uptake rates were more pronounced in WH8102 than in CC9311 and for growth on urea or nitrate than for growth on ammonia. These results, combined with an analysis of fully sequenced marine cyanobacterial genomes, suggest that the growth of many marine Synechococcus and all Prochlorococcus strains is dependent upon Ni.     

The genome architecture of the Collaborative Cross mouse genetic reference population

The Collaborative Cross Consortium reports here on the development of a unique genetic resource population. The Collaborative Cross (CC) is a multiparental recombinant inbred panel derived from eight laboratory mouse inbred strains. Breeding of the CC lines was initiated at multiple international sites using mice from The Jackson Laboratory. Currently, this innovative project is breeding independent CC lines at the University of North Carolina (UNC), at Tel Aviv University (TAU), and at Geniad in Western Australia (GND). These institutions aim to make publicly available the completed CC lines and their genotypes and sequence information. We genotyped, and report here, results from 458 extant lines from UNC, TAU, and GND using a custom genotyping array with 7500 SNPs designed to be maximally informative in the CC and used a novel algorithm to infer inherited haplotypes directly from hybridization intensity patterns. We identified lines with breeding errors and cousin lines generated by splitting incipient lines into two or more cousin lines at early generations of inbreeding. We then characterized the genome architecture of 350 genetically independent CC lines. Results showed that founder haplotypes are inherited at the expected frequency, although we also consistently observed highly significant transmission ratio distortion at specific loci across all three populations. On chromosome 2, there is significant overrepresentation of WSB/EiJ alleles, and on chromosome X, there is a large deficit of CC lines with CAST/EiJ alleles. Linkage disequilibrium decays as expected and we saw no evidence of gametic disequilibrium in the CC population as a whole or in random subsets of the population. Gametic equilibrium in the CC population is in marked contrast to the gametic disequilibrium present in a large panel of classical inbred strains. Finally, we discuss access to the CC population and to the associated raw data describing the genetic structure of individual lines. Integration of rich phenotypic and genomic data over time and across a wide variety of fields will be vital to delivering on one of the key attributes of the CC, a common genetic reference platform for identifying causative variants and genetic networks determining traits in mammals.     

Restriction fragment length polymorphism analysis of CCDD genome species of the genus Oryza L.

Restriction fragment length polymorphisms (RFLPs) were studied in fourteen accessions of CCDD genome allotetraploid wild rice species (Oryza latifolia, O. alta and O. grandiglumis). Fourteen nuclear RFLP markers previously mapped in AA genome-cultivated rice were used as probes. A phylogenetic tree, constructed by parsimony analysis based on RFLPs, grouped the accessions according to their geographic origin from Central or South America. Oryza alta, O. grandiglumis and one accession of O. latifolia grouped together as a subgroup, and our results suggested that the three taxa should be considered as populations of a single complex species. Duplicate loci, representing the two constituent genomes of the allotetraploid, were observed for most RFLP markers. By comparing RFLPs from the allotetraploids with those from a CC genome diploid wild species (O. officinalis), it was possible to detect RFLPs specific for both the CC and DD genomes of the allotetraploid. In inter-accession F2 populations, independent segregation of RFLP markers for CC and DD genomes was observed.     

PCR-analyzed microsatellites of the mouse genome—additional polymorphisms among ten inbred mouse strains

Eighty sequences from the mouse genome database containing microsatellites (simple sequence repeats) have been analyzed for size variation among ten different inbred strains of mice; 62/80 (77.5%) showed polymorphism of at least three alleles. We have been able to detect all the polymorphims by agarose gel electrophoresis, often running the gels for up to 3 h. Between individual pairs of mouse strains to be used in chromosomal mapping studies in our laboratory, 35–60% polymorphism occurred. There are potentially enough microsatellites within the mouse and human genome to have a marker at every 1-cM distance. This simple approach will, therefore, continue to be useful in genome mapping studies, leading eventually to high-resolution maps of both the mouse and human genomes; this should allow for physical mapping and cloning of specific genes.     

A General Modeling Framework for Genome Ancestral Origins in Multiparental Populations

The next generation of QTL (quantitative trait loci) mapping populations have been designed with multiple founders, where one to a number of generations of intercrossing are introduced prior to the inbreeding phase to increase accumulated recombinations and thus mapping resolution. Examples of such populations are Collaborative Cross (CC) in mice and Multiparent Advanced Generation Inter-Cross (MAGIC) lines in Arabidopsis. The genomes of the produced inbred lines are fine-grained random mosaics of the founder genomes. In this article, we present a novel framework for modeling ancestral origin processes along two homologous autosomal chromosomes from mapping populations, which is a major component in the reconstruction of the ancestral origins of each line for QTL mapping. We construct a general continuous time Markov model for ancestral origin processes, where the rate matrix is deduced from the expected densities of various types of junctions (recombination breakpoints). The model can be applied to monoecious populations with or without self-fertilizations and to dioecious populations with two separate sexes. The analytic expressions for map expansions and expected junction densities are obtained for mapping populations that have stage-wise constant mating schemes, such as CC and MAGIC. Our studies on the breeding design of MAGIC populations show that the intercross mating schemes do not matter much for large population size and that the overall expected junction density, and thus map resolution, are approximately proportional to the inverse of the number of founders.     

A second pilus type in Streptococcus pneumoniae is prevalent in emerging serotypes and mediates adhesion to host cells

Analysis of publicly available genomes of Streptococcus pneumoniae has led to the identification of a new genomic element containing genes typical of gram-positive pilus islets (PIs). Here, we demonstrate that this genomic region, herein referred to as PI-2 (consisting of pitA, sipA, pitB, srtG1, and srtG2) codes for a second functional pilus in pneumococcus. Polymerization of the PI-2 pilus requires the backbone protein PitB as well as the sortase SrtG1 and the signal peptidase-like protein SipA. Presence of PI-2 correlates with the genotype as defined by multilocus sequence typing and clonal complex (CC). The PI-2-positive CCs are associated with serotypes 1, 2, 7F, 19A, and 19F, considered to be emerging serotypes in both industrialized and developing countries. Interestingly, strains belonging to CC271 (where sequence type 271 is the predicted founder of the CC) contain both PI-1 and PI-2, as revealed by genome analyses. In these strains both pili are surface exposed and independently assembled. Furthermore, in vitro experiments provide evidence that the pilus encoded by PI-2 of S. pneumoniae is involved in adherence. Thus, pneumococci encode at least two types of pili that play a role in the initial host cell contact to the respiratory tract and are potential antigens for inclusion in a new generation of pneumococcal vaccines.