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1.  Kind granddaughters of angry grandmothers: the effect of domestication on vocalization in cross-bred silver foxes 
Behavioural processes  2009;81(3):369.
The genetic basis of the effects of domestication have previously been examined in relation to morphological, physiological and behavioural traits, but not for vocalizations. According to Belyaev (1979, Journal of Heredity 70, 301-308), directional selection for tame behaviour toward humans resulted in domestication. This hypothesis has been confirmed experimentally on the farm-bred silver fox Vulpes vulpes population that has undergone 45 years of artificial selection for tameness and 35 years of selection for aggressiveness. These foxes, with their precisely known attitudes toward people, provide a means of examining vocal indicators of tameness and aggressiveness to establish the genetic basis for vocal production in canids. We examined vocalizations toward people in foxes selected for tameness and aggressiveness compared to those of three kinds of crosses: Hybrids (Tame X Aggressive), A-Backcrosses (Aggressive X Hybrid) and T-Backcrosses (Tame X Hybrid). We report the effects of selection for tameness on usage and structure of different vocalisations and suggest that vocal indicators for tameness and aggressiveness toward people are discrete phenotypic traits in silver foxes.
doi:10.1016/j.beproc.2009.03.012
PMCID: PMC2814310  PMID: 19520236
behaviour genetic; canid-human interaction; Canidae; domestication; vocal behaviour; Vulpes vulpes
2.  Sequence comparison of prefrontal cortical brain transcriptome from a tame and an aggressive silver fox (Vulpes vulpes) 
BMC Genomics  2011;12:482.
Background
Two strains of the silver fox (Vulpes vulpes), with markedly different behavioral phenotypes, have been developed by long-term selection for behavior. Foxes from the tame strain exhibit friendly behavior towards humans, paralleling the sociability of canine puppies, whereas foxes from the aggressive strain are defensive and exhibit aggression to humans. To understand the genetic differences underlying these behavioral phenotypes fox-specific genomic resources are needed.
Results
cDNA from mRNA from pre-frontal cortex of a tame and an aggressive fox was sequenced using the Roche 454 FLX Titanium platform (> 2.5 million reads & 0.9 Gbase of tame fox sequence; >3.3 million reads & 1.2 Gbase of aggressive fox sequence). Over 80% of the fox reads were assembled into contigs. Mapping fox reads against the fox transcriptome assembly and the dog genome identified over 30,000 high confidence fox-specific SNPs. Fox transcripts for approximately 14,000 genes were identified using SwissProt and the dog RefSeq databases. An at least 2-fold expression difference between the two samples (p < 0.05) was observed for 335 genes, fewer than 3% of the total number of genes identified in the fox transcriptome.
Conclusions
Transcriptome sequencing significantly expanded genomic resources available for the fox, a species without a sequenced genome. In a very cost efficient manner this yielded a large number of fox-specific SNP markers for genetic studies and provided significant insights into the gene expression profile of the fox pre-frontal cortex; expression differences between the two fox samples; and a catalogue of potentially important gene-specific sequence variants. This result demonstrates the utility of this approach for developing genomic resources in species with limited genomic information.
doi:10.1186/1471-2164-12-482
PMCID: PMC3199282  PMID: 21967120
3.  Measurement of segregating behaviors in experimental silver fox pedigrees 
Behavior genetics  2007;38(2):185-194.
Strains of silver foxes, selectively bred at the Institute of Cytology and Genetics of the Russian Academy of Sciences, are a well established, novel model for studying the genetic basis of behavior, and the processes involved in canine domestication. Here we describe a method to measure fox behavior as quantitative phenotypes which distinguish populations and resegregate in experimental pedigrees. We defined 50 binary observations that nonredundantly and accurately distinguished behaviors in reference populations and cross-bred pedigrees. Principal-component analysis dissected out the independent elements underlying these behaviors. PC1 accounted for >44% of the total variance in measured traits. This system clearly distinguished tame foxes from aggressive and wildtype foxes. F1 foxes yield intermediate values that extend into the ranges of both the tame and aggressive foxes, while the scores of the backcross generation resegregate. These measures can thus be used for QTL mapping to explore the genetic basis of tame and aggressive behavior in foxes, which should provide new insights into the mechanisms of mammalian behavior and canine domestication.
doi:10.1007/s10519-007-9180-1
PMCID: PMC2374754  PMID: 18030612
Canidae; Vulpes vulpes; interspecies tameness; attack; domestication
4.  Selection for tameness modulates the expression of heme related genes in silver foxes 
Background
The genetic and molecular mechanisms of tameness are largely unknown. A line of silver foxes (Vulpes vulpes) selected for non-aggressive behavior has been used in Russia since the 1960's to study the effect of domestication. We have previously compared descendants of these selected (S) animals with a group of non-selected (NS) silver foxes kept under identical conditions, and showed that changes in the brain transcriptome between the two groups are small. Unexpectedly, many of the genes showing evidence of differential expression between groups were related to hemoproteins.
Results
In this study, we use quantitative RT-PCR to demonstrate that the activity of heme related genes differ between S and NS foxes in three regions of the brain. Furthermore, our analyses also indicate that changes in mRNA levels of heme related genes can be well described by an additive polygenic effect. We also show that the difference in genetic background between the two lines of foxes is limited, as estimated by mitochondrial DNA divergence.
Conclusion
Our results indicate that selection for tameness can modify the expression of heme related genes in canid brain regions known to modulate emotions and behavior. The possible involvement of heme related genes in behavior is surprising. It is possible that hemoglobin modulates the behavior of canids by interaction with CO and NO signaling. Another possibility is that hemorphins, known to be produced after enzymatic cleavage of hemoglobin, are responsible for behavioral alterations. Thus, we hypothesize that hemoglobin metabolism can be a functionally relevant aspect of the domestic phenotype in foxes selected for tameness.
doi:10.1186/1744-9081-3-18
PMCID: PMC1858698  PMID: 17439650
5.  On the origin of a domesticated species: Identifying the parent population of Russian silver foxes (Vulpes vulpes) 
The foxes at Novosibirsk, Russia, are the only population of domesticated foxes in the world. These domesticated foxes originated from farm-bred silver foxes (Vulpes vulpes), whose genetic source is unknown. In this study we examined the origin of the domesticated strain of foxes and two other farm-bred fox populations (aggressive and unselected) maintained in Novosibirsk. To identify the phylogenetic origin of these populations we sequenced two regions of mtDNA, cytochrome b and D-loop, from 24 Novosibirsk foxes (8 foxes from each population) and compared them with corresponding sequences of native red foxes from Europe, Asia, Alaska and Western Canada, Eastern Canada, and the Western Mountains of the USA. We identified seven cytochrome b - D-loop haplotypes in Novosibirsk populations, four of which were previously observed in Eastern North America. The three remaining haplotypes differed by one or two base change from the most common haplotype in Eastern Canada. ΦST analysis showed significant differentiation between Novosibirsk populations and red fox populations from all geographic regions except Eastern Canada. No haplotypes of Eurasian origin were identified in the Novosibirsk populations. These results are consistent with historical records indicating that the original breeding stock of farm-bred foxes originated from Prince Edward Island, Canada. Mitochondrial DNA data together with historical records indicate two stages in the selection of domesticated foxes: the first includes captive breeding for ~50 years with unconscious selection for behaviour; the second corresponds to over 50 further years of intensive selection for tame behaviour.
doi:10.1111/j.1095-8312.2011.01629.x
PMCID: PMC3101803  PMID: 21625363
domestication; mitochondrial DNA; phylogeography; red fox; tameness
6.  A Comparison of Brain Gene Expression Levels in Domesticated and Wild Animals 
PLoS Genetics  2012;8(9):e1002962.
Domestication has led to similar changes in morphology and behavior in several animal species, raising the question whether similarities between different domestication events also exist at the molecular level. We used mRNA sequencing to analyze genome-wide gene expression patterns in brain frontal cortex in three pairs of domesticated and wild species (dogs and wolves, pigs and wild boars, and domesticated and wild rabbits). We compared the expression differences with those between domesticated guinea pigs and a distant wild relative (Cavia aperea) as well as between two lines of rats selected for tameness or aggression towards humans. There were few gene expression differences between domesticated and wild dogs, pigs, and rabbits (30–75 genes (less than 1%) of expressed genes were differentially expressed), while guinea pigs and C. aperea differed more strongly. Almost no overlap was found between the genes with differential expression in the different domestication events. In addition, joint analyses of all domesticated and wild samples provided only suggestive evidence for the existence of a small group of genes that changed their expression in a similar fashion in different domesticated species. The most extreme of these shared expression changes include up-regulation in domesticates of SOX6 and PROM1, two modulators of brain development. There was almost no overlap between gene expression in domesticated animals and the tame and aggressive rats. However, two of the genes with the strongest expression differences between the rats (DLL3 and DHDH) were located in a genomic region associated with tameness and aggression, suggesting a role in influencing tameness. In summary, the majority of brain gene expression changes in domesticated animals are specific to the given domestication event, suggesting that the causative variants of behavioral domestication traits may likewise be different.
Author Summary
Over the millennia, humans have turned a range of wild animal species into what we today know as domesticated animals. Domestication has greatly influenced human history and evolution. The changes in the animals are even more drastic. Domesticated animals differ from their wild relatives in appearance, physiology, and behavior. Although these differences are mostly genetically encoded, little is known about which genes contribute to these domestication traits. Changes in gene expression have long been proposed to lead to phenotypic changes in evolution. In this work, we therefore compared gene expression in brains of dogs and wolves, pigs and boars, and domesticated and wild rabbits and guinea pigs. For each of the four domesticated species, we identify gene expression differences that could correlate with behavioral differences compared to wild animals. The majority of expression differences are unique to each domestication event, suggesting that domestication has proceeded through different genetic routes in different species.
doi:10.1371/journal.pgen.1002962
PMCID: PMC3459979  PMID: 23028369
7.  Vocalization toward conspecifics in silver foxes (Vulpes vulpes) selected for tame or aggressive behavior toward humans 
Behavioural processes  2010;84(2):547-554.
We examined the production of different vocalizations in three strains of silver fox (Unselected, Aggressive, and Tame) attending three kinds of behavior (aggressive, affiliative, and neutral) in response to their same-strain conspecifics. This is a follow up to previous experiments which demonstrated that in the presence of humans tame foxes produced cackles and pants but never coughed or snorted, whilst Aggressive foxes produced coughs and snorts but never cackled or panted. Thus, cackle/pant and cough/snort were indicative of the Tame and Aggressive fox strains respectively toward humans. Wild-type Unselected foxes produced cough and snort toward humans similarly to Aggressive foxes. Here we found that vocal responses to conspecifics were similar in Tame, Aggressive and Unselected fox strains. Both cackle/pant and cough/snort occurred in foxes of all strains. The difference in the use of cackle/pant and cough/snort among these strains toward humans and toward conspecifics suggest that silver foxes do not perceive humans as their conspecifics. We speculate that these vocalizations are produced in response to a triggering internal state, affiliative or aggressive, that is suppressed by default in these fox strains toward humans as a result of their strict selection for tame or aggressive behavior, whilst still remaining flexible toward conspecifics.
doi:10.1016/j.beproc.2010.01.021
PMCID: PMC2873138  PMID: 20123117
Affiliative behavior; Agonistic behavior; Canid-human interaction; Domestication; Vocalization; Vulpes vulpes
8.  Explosive vocal activity for attracting human attention is related to domestication in silver fox 
Behavioural processes  2010;86(2):216-221.
Domestication affects behavioral and vocal responses, involved in communication with humans; in particular, those that attract human attention. In this study, we found that silver foxes of Tame strain, experimentally domesticated for a few tenses generation, displayed bursts of vocal activity during the first minute after appearance of an unfamiliar human, that faded quickly during the remaining time of the test, when the experimenter stayed passively before the cage. Distinctively, foxes of Aggressive strain, artificially selected for tenses generations for aggressive behavior toward humans, and the control group of Unselected for behavior silver foxes kept steady levels of vocal activity for the duration of the tests. We found also that Aggressive foxes vocalized for a larger proportion of time than Unselected foxes for all five minutes of the test. We discuss the obtained data in relation to proposal effects of domestication on mechanisms directed to involving people into human-animal interactions and structural similarity between human laughter and vocalization of Tame foxes.
doi:10.1016/j.beproc.2010.12.001
PMCID: PMC3039033  PMID: 21145949
Vulpes vulpes; human-animal interaction; acoustic communication; vocalization; domestication; human-exposure test
9.  Targeted resequencing of a genomic region influencing tameness and aggression reveals multiple signals of positive selection 
Heredity  2011;107(3):205-214.
The identification of the causative genetic variants in quantitative trait loci (QTL) influencing phenotypic traits is challenging, especially in crosses between outbred strains. We have previously identified several QTL influencing tameness and aggression in a cross between two lines of wild-derived, outbred rats (Rattus norvegicus) selected for their behavior towards humans. Here, we use targeted sequence capture and massively parallel sequencing of all genes in the strongest QTL in the founder animals of the cross. We identify many novel sequence variants, several of which are potentially functionally relevant. The QTL contains several regions where either the tame or the aggressive founders contain no sequence variation, and two regions where alternative haplotypes are fixed between the founders. A re-analysis of the QTL signal showed that the causative site is likely to be fixed among the tame founder animals, but that several causative alleles may segregate among the aggressive founder animals. Using a formal test for the detection of positive selection, we find 10 putative positively selected regions, some of which are close to genes known to influence behavior. Together, these results show that the QTL is probably not caused by a single selected site, but may instead represent the joint effects of several sites that were targets of polygenic selection.
doi:10.1038/hdy.2011.4
PMCID: PMC3183948  PMID: 21304545
high-throughput sequencing; sequence capture; positive selection; behavior; QTL mapping
10.  Chromosomal Mapping of Canine-Derived BAC Clones to the Red Fox and American Mink Genomes 
Journal of Heredity  2009;100(Suppl 1):S42-S53.
High-quality sequencing of the dog (Canis lupus familiaris) genome has enabled enormous progress in genetic mapping of canine phenotypic variation. The red fox (Vulpes vulpes), another canid species, also exhibits a wide range of variation in coat color, morphology, and behavior. Although the fox genome has not yet been sequenced, canine genomic resources have been used to construct a meiotic linkage map of the red fox genome and begin genetic mapping in foxes. However, a more detailed gene-specific comparative map between the dog and fox genomes is required to establish gene order within homologous regions of dog and fox chromosomes and to refine breakpoints between homologous chromosomes of the 2 species. In the current study, we tested whether canine-derived gene–containing bacterial artificial chromosome (BAC) clones can be routinely used to build a gene-specific map of the red fox genome. Forty canine BAC clones were mapped to the red fox genome by fluorescence in situ hybridization (FISH). Each clone was uniquely assigned to a single fox chromosome, and the locations of 38 clones agreed with cytogenetic predictions. These results clearly demonstrate the utility of FISH mapping for construction of a whole-genome gene-specific map of the red fox. The further possibility of using canine BAC clones to map genes in the American mink (Mustela vison) genome was also explored. Much lower success was obtained for this more distantly related farm-bred species, although a few BAC clones were mapped to the predicted chromosomal locations.
doi:10.1093/jhered/esp037
PMCID: PMC3139363  PMID: 19546120
Canis lupus familiaris; comparative genomics; FISH; Mustela vison; Vulpes vulpes
11.  Effects of selection for behavior, human approach mode and sex on vocalization in silver fox 
Journal of ethology  2012;31(1):95-100.
This study presents a first direct comparison of vocal type, call rate and time spent vocalizing among Unselected, Tame and Aggressive strains of silver fox (Vulpes vulpes) in three modes of human approach (Provoking, Approach–Retreat, and Static). Also, it provides a first comparison of male and female vocal output in the Provoking test. Vocal types were found strain-specific irrespective of the fox sex or the test. Males had higher call rates and spent shorter times vocalizing than females. These results support the evidence of genetic-based emotional states, triggering vocal behavior in silver fox strains, and suggest sex dimorphism in vocal activity toward humans.
doi:10.1007/s10164-012-0353-x
PMCID: PMC3601802  PMID: 23525128
Call; Domestication; Human approach test; Gender effect; Canidae
12.  The influence of habitat structure on genetic differentiation in red fox populations in north-eastern Poland 
Acta Theriologica  2014;59:367-376.
The red fox (Vulpes vulpes) has the widest global distribution among terrestrial carnivore species, occupying most of the Northern Hemisphere in its native range. Because it carries diseases that can be transmitted to humans and domestic animals, it is important to gather information about their movements and dispersal in their natural habitat but it is difficult to do so at a broad scale with trapping and telemetry. In this study, we have described the genetic diversity and structure of red fox populations in six areas of north-eastern Poland, based on samples collected from 2002–2003. We tested 22 microsatellite loci isolated from the dog and the red fox genome to select a panel of nine polymorphic loci suitable for this study. Genetic differentiation between the six studied populations was low to moderate and analysis in Structure revealed a panmictic population in the region. Spatial autocorrelation among all individuals showed a pattern of decreasing relatedness with increasing distance and this was not significantly negative until 93 km, indicating a pattern of isolation-by-distance over a large area. However, there was no correlation between genetic distance and either Euclidean distance or least-cost path distance at the population level. There was a significant relationship between genetic distance and the proportion of large forests and water along the Euclidean distances. These types of habitats may influence dispersal paths taken by red foxes, which is useful information in terms of wildlife disease management.
Electronic supplementary material
The online version of this article (doi:10.1007/s13364-014-0180-2) contains supplementary material, which is available to authorized users.
doi:10.1007/s13364-014-0180-2
PMCID: PMC4058057  PMID: 24954926
Microsatellites; Spatial autocorrelation; Bayesian clustering; Landscape resistance; Least-cost path
13.  Molecular and histopathological detection of Hepatozoon canis in red foxes (Vulpes vulpes) from Portugal 
Parasites & Vectors  2014;7:113.
Background
Hepatozoon canis is a protozoan tick-borne pathogen of dogs and wild canids. Hepatozoon spp. have been reported to infect foxes in different continents and recent studies have mostly used the polymerase chain reaction (PCR) for the detection and characterization of the infecting species. Surveying red foxes (Vulpes vulpes) may contribute to better understanding the epidemiology of canine vector-borne diseases, including hepatozoonosis caused by H. canis in domestic dogs. The present study investigated the prevalence of Hepatozoon spp. by means of histopathology and molecular analysis of different tissues in red foxes from different parts of Portugal.
Methods
Blood and tissues including bone marrow, heart, hind leg muscle, jejunum, kidney, liver, lung, popliteal or axillary lymph nodes, spleen and/or tongue were collected from 91 red foxes from eight districts in northern, central and southern Portugal. Tissues were formalin-fixed, paraffin-embedded, cut and stained with hematoxylin and eosin. Polymerase chain reaction (PCR) amplified a ~650 bp fragment of the 18S rRNA gene of Hepatozoon spp. and the DNA products were sequenced.
Results
Hepatozoon canis was detected in 68 out of 90 foxes (75.6%) from all the sampled areas by PCR and sequencing. Histopathology revealed H. canis meronts similar in shape to those found in dogs in the bone marrow of 11 (23.4%) and in the spleen of two (4.3%) out of 47 foxes (p = 0.007). All the 11 foxes found positive by histopathology were also positive by PCR of bone marrow and/or blood. Positivity by PCR (83.0%) was significantly higher (p < 0.001) than by histopathological examination (23.4%) in paired bone marrow samples from the same 47 foxes. Sequences of the 18S rRNA gene of H. canis were 98–99% identical to those in GenBank.
Conclusions
Hepatozoon canis was found to be highly prevalent in red fox populations from northern, central and southern Portugal. Detection of the parasite by histopathology was significantly less sensitive than by PCR. Red foxes are a presumptive reservoir of H. canis infection for domestic dogs.
doi:10.1186/1756-3305-7-113
PMCID: PMC3994325  PMID: 24655375
Canine vector-borne diseases; Hepatozoon canis; Histopathology; Polymerase chain reaction; Portugal; Red fox; Vulpes vulpes
14.  Integrative genome-scale metabolic analysis of Vibrio vulnificus for drug targeting and discovery 
Chromosome 1 of Vibrio vulnificus tends to contain larger portion of essential or housekeeping genes on the basis of the genomic analysis and gene knockout experiments performed in this study, while its chromosome 2 seems to have originated and evolved from a plasmid.The genome-scale metabolic network model of V. vulnificus was reconstructed based on databases and literature, and was used to identify 193 essential metabolites.Five essential metabolites finally selected after the filtering process are 2-amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine (AHHMP), D-glutamate (DGLU), 2,3-dihydrodipicolinate (DHDP), 1-deoxy-D-xylulose 5-phosphate (DX5P), and 4-aminobenzoate (PABA), which were predicted to be essential in V. vulnificus, absent in human, and are consumed by multiple reactions.Chemical analogs of the five essential metabolites were screened and a hit compound showing the minimal inhibitory concentration (MIC) of 2 μg/ml and the minimal bactericidal concentration (MBC) of 4 μg/ml against V. vulnificus was identified.
Discovering new antimicrobial targets and consequently new antimicrobials is important as drug resistance of pathogenic microorganisms is becoming an increasingly serious problem in human healthcare management (Fischbach and Walsh, 2009). There clearly exists a gap between genomic studies and drug discovery as the accumulation of knowledge on pathogens at genome level has not successfully transformed into the development of effective drugs (Mills, 2006; Payne et al, 2007). In this study, we dissected the genome of a microbial pathogen in detail, and subsequently developed a systems biological strategy of employing genome-scale metabolic modeling and simulation together with metabolite essentiality analysis for effective drug targeting and discovery. This strategy was used for identifying new drug targets in an opportunistic pathogen Vibrio vulnificus CMCP6 as a model.
V. vulnificus is a Gram-negative halophilic bacterium that is found in estuarine waters, brackish ponds, or coastal areas, and its Biotype 1 is an opportunistic human pathogen that can attack immune-compromised patients, and causes primary septicemia, necrotized wound infections, and gastroenteritis. We previously found that many metabolic genes were specifically induced in vivo, suggesting that specific metabolic pathways are essential for in vivo survival and virulence of this pathogen (Kim et al, 2003; Lee et al, 2007). These results motivated us to carry out systems biological analysis of the genome and the metabolic network for new drug target discovery.
V. vulnificus CMCP6 has two chromosomes. We first re-sequenced genomic regions assembled in low quality and low depth, and subsequently re-annotated the whole genome of V. vulnificus. Horizontal gene transfer was suspected to be responsible for the diversification of each chromosome of V. vulnificus, and the presence of metabolic genes was more biased to chromosome 1 than chromosome 2. Further studies on V. vulnificus genome revealed that chromosome 2 is more prone to diversification for better adaptation to the environment than its chromosome 1, while chromosome 1 tends to expand their genetic repertoire while maintaining the core genes at a constant level.
Next, a genome-scale metabolic network VvuMBEL943 was reconstructed based on literature, databases and experiments for systematic studies on the metabolism of this pathogen and prediction of drug targets. The VvuMBEL943 model is composed of 943 reactions and 765 metabolites, and covers 673 genes. The model was validated by comparing its simulated cell growth phenotype obtained by constraints-based flux analysis with the V. vulnificus-specific experimental data previously reported in the literature. In this study, constraints-based flux analysis is an optimization-based simulation method that calculates intracellular fluxes under the specific genetic and environmental condition (Kim et al, 2008). As a result, 17 growth phenotypes were correctly predicted out of 18 cases, which demonstrate the validity of VvuMBEL943.
The main objective of constructing VvuMBEL943 in this study is to predict potential drug targets by system-wide analysis of the metabolic network for the effective treatment of V. vulnificus. To achieve this goal, a set of drug target candidates was predicted by taking a metabolite-centric approach. Metabolite essentiality analysis is a concept recently introduced for the study of cellular robustness to complement conventional reaction or gene-centric approach (Kim et al, 2007b). Metabolite essentiality analysis observes changes in flux distribution by removing each metabolite from the in silico metabolic network. Hence, metabolite essentiality predicts essential metabolites whose absence causes cell death. By selecting essential metabolites, it is possible to directly screen only their structural analogs, which substantially reduces the number of chemical compounds to screen from the chemical compound library. As a result of implementing this approach, 193 metabolites were initially identified to be essential to the cell. These essential metabolites were then further filtered based on the predetermined criteria, mainly organism specificity and multiple connectivity associated with each metabolite, in order to reduce the number of initial target candidates towards identifying the most effective ones.
Five essential metabolites finally selected are 2-amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine (AHHMP), D-glutamate (DGLU), 2,3-dihydrodipicolinate (DHDP), 1-deoxy-D-xylulose 5-phosphate (DX5P), and 4-aminobenzoate (PABA). Enzymes that consume these essential metabolites were experimentally verified to be essential, which indeed demonstrates the essentiality of these five metabolites. On the basis of the structural information of these five essential metabolites, whole-cell screening assay was performed using their analogs for possible antibacterial discovery. We screened 352 chemical analogs of the essential metabolites selected from the chemical compound library, and found a hit compound 24837, which shows the minimal inhibitory concentration (MIC) of 2 μg/ml and minimal bactericidal concentration (MBC) of 4 μg/ml, showing good antibacterial activity without further structural modification. Although this study demonstrates a proof-of-concept, the approaches and their rationale taken here should serve as a general strategy for discovering novel antibiotics and drugs based on systems-level analysis of metabolic networks.
Although the genomes of many microbial pathogens have been studied to help identify effective drug targets and novel drugs, such efforts have not yet reached full fruition. In this study, we report a systems biological approach that efficiently utilizes genomic information for drug targeting and discovery, and apply this approach to the opportunistic pathogen Vibrio vulnificus CMCP6. First, we partially re-sequenced and fully re-annotated the V. vulnificus CMCP6 genome, and accordingly reconstructed its genome-scale metabolic network, VvuMBEL943. The validated network model was employed to systematically predict drug targets using the concept of metabolite essentiality, along with additional filtering criteria. Target genes encoding enzymes that interact with the five essential metabolites finally selected were experimentally validated. These five essential metabolites are critical to the survival of the cell, and hence were used to guide the cost-effective selection of chemical analogs, which were then screened for antimicrobial activity in a whole-cell assay. This approach is expected to help fill the existing gap between genomics and drug discovery.
doi:10.1038/msb.2010.115
PMCID: PMC3049409  PMID: 21245845
drug discovery; drug targeting; genome analysis; metabolic network; Vibrio vulnificus
15.  Genome Reshuffling for Advanced Intercross Permutation (GRAIP): Simulation and Permutation for Advanced Intercross Population Analysis 
PLoS ONE  2008;3(4):e1977.
Background
Advanced intercross lines (AIL) are segregating populations created using a multi-generation breeding protocol for fine mapping complex trait loci (QTL) in mice and other organisms. Applying QTL mapping methods for intercross and backcross populations, often followed by naïve permutation of individuals and phenotypes, does not account for the effect of AIL family structure in which final generations have been expanded and leads to inappropriately low significance thresholds. The critical problem with naïve mapping approaches in AIL populations is that the individual is not an exchangeable unit.
Methodology/Principal Findings
The effect of family structure has immediate implications for the optimal AIL creation (many crosses, few animals per cross, and population expansion before the final generation) and we discuss these and the utility of AIL populations for QTL fine mapping. We also describe Genome Reshuffling for Advanced Intercross Permutation, (GRAIP) a method for analyzing AIL data that accounts for family structure. GRAIP permutes a more interchangeable unit in the final generation crosses – the parental genome – and simulating regeneration of a permuted AIL population based on exchanged parental identities. GRAIP determines appropriate genome-wide significance thresholds and locus-specific P-values for AILs and other populations with similar family structures. We contrast GRAIP with naïve permutation using a large densely genotyped mouse AIL population (1333 individuals from 32 crosses). A naïve permutation using coat color as a model phenotype demonstrates high false-positive locus identification and uncertain significance levels, which are corrected using GRAIP. GRAIP also detects an established hippocampus weight locus and a new locus, Hipp9a.
Conclusions and Significance
GRAIP determines appropriate genome-wide significance thresholds and locus-specific P-values for AILs and other populations with similar family structures. The effect of family structure has immediate implications for the optimal AIL creation and we discuss these and the utility of AIL populations.
doi:10.1371/journal.pone.0001977
PMCID: PMC2295257  PMID: 18431467
16.  Animal evolution during domestication: the domesticated fox as a model 
Summary
We review the evolution of domestic animals, emphasizing the effect of the earliest steps of domestication on its course. Using the first domesticated species, the dog (Canis familiaris) as an illustration, we describe the evolutionary specificities of the historical domestication, such as the high level and wide range of diversity. We suggest that the process of earliest domestication via unconscious and later conscious selection of human-defined behavioral traits may accelerate phenotypic variations. The review is based on the results of the long-term experiment designed to reproduce early mammalian domestication in the silver fox (Vulpes vulpes) selected for tameability, or amenability to domestication. We describe changes in behavior, morphology and physiology that appeared in the fox during its selection for tameability and that were similar to those observed in the domestic dog. Based on the experimental fox data and survey of relevant data, we discuss the developmental, genetic and possible molecular-genetic mechanisms of these changes. We assign the causative role in evolutionary transformation of domestic animals to selection for behavior and to the neurospecific regulatory genes it affects.
doi:10.1002/bies.200800070
PMCID: PMC2763232  PMID: 19260016
17.  Genetic regulation of canine skeletal traits: trade-offs between the hind limbs and forelimbs in the fox and dog 
Synopsis
Genetic variation in functionally integrated skeletal traits can be maintained over 10 million years despite bottlenecks and stringent selection. Here, we describe an analysis of the genetic architecture of the canid axial skeleton using populations of the Portuguese Water Dog Canis familiaris) and silver fox (Vulpes vulpes). Twenty-one skeletal metrics taken from radiographs of the forelimbs and hind limbs of the fox and dog were used to construct separate anatomical principal component (PC) matrices of the two species. In both species, 15 of the 21 PCs exhibited significant heritability, ranging from 25% to 70%. The second PC, in both species, represents a trade-off in which limb-bone width is inversely correlated with limb-bone length. PC2 accounts for approximately 15% of the observed skeletal variation, ~30% of the variation in shape. Many of the other significant PCs affect very small amounts of variation (e.g., 0.2–2%) along trade-off axes that partition function between the forelimbs and hind limbs. These PCs represent shape axes in which an increase in size of an element of the forelimb is associated with a decrease in size of an element of the hind limb and vice versa. In most cases, these trade-offs are heritable in both species and genetic loci have been identified in the Portuguese Water Dog for many of these. These PCs, present in both the dog and the fox, include ones that affect lengths of the forelimb versus the hind limb, length of the forefoot versus that of the hind foot, muscle moment (i.e., lever) arms of the forelimb versus hind limb, and cortical thickness of the bones of the forelimb versus hind limb. These inverse relationships suggest that genetic regulation of the axial skeleton results, in part, from the action of genes that influence suites of functionally integrated traits. Their presence in both dogs and foxes suggests that the genes controlling the regulation of these PCs of the forelimb versus hind limb may be found in other tetrapod taxa.
doi:10.1093/icb/icm023
PMCID: PMC2367254  PMID: 18458753
18.  GENETIC DISSECTION OF INTERMALE AGGRESSIVE BEHAVIOR IN BALB/cJ AND A/J MICE 
Genes, brain, and behavior  2010;10(1):57-68.
Aggressive behaviors are disabling, treatment refractory, and sometimes lethal symptoms of several neuropsychiatric disorders. However, currently available treatments for patients are inadequate, and the underlying genetics and neurobiology of aggression is only beginning to be elucidated. Inbred mouse strains are useful for identifying genomic regions, and ultimately the relevant gene variants (alleles) in these regions, that affect mammalian aggressive behaviors, which, in turn, may help to identify neurobiological pathways that mediate aggression. The BALB/cJ inbred mouse strain exhibits relatively high levels of intermale aggressive behaviors, and shows multiple brain and behavioral phenotypes relevant to neuropsychiatric syndromes associated with aggression. The A/J strain shows very low levels of aggression. We hypothesized that a cross between BALB/cJ and A/J inbred strains would reveal genomic loci that influence the tendency to initiate intermale aggressive behavior. To identify such loci, we conducted a genome-wide scan in an F2 population of 660 male mice bred from BALB/cJ and A/J inbred mouse strains. Three significant loci on chromosomes 5, 10, and 15 that influence aggression were identified. The chromosome 5 and 15 loci are completely novel, and the chromosome 10 locus overlaps an aggression locus mapped in our previous study that used NZB/B1NJ and A/J as progenitor strains. Haplotype analysis of BALB/cJ, NZB/B1NJ, and A/J strains revealed 3 positional candidate genes in the chromosome 10 locus. Future studies involving fine genetic mapping of these loci as well as additional candidate gene analysis may lead to an improved biological understanding of mammalian aggressive behaviors.
doi:10.1111/j.1601-183X.2010.00640.x
PMCID: PMC3017637  PMID: 20731721
aggression; mouse; behavior; genetics; genome scan; linkage; association
19.  mtDNA Data Indicate a Single Origin for Dogs South of Yangtze River, Less Than 16,300 Years Ago, from Numerous Wolves 
Molecular Biology and Evolution  2009;26(12):2849-2864.
There is no generally accepted picture of where, when, and how the domestic dog originated. Previous studies of mitochondrial DNA (mtDNA) have failed to establish the time and precise place of origin because of lack of phylogenetic resolution in the so far studied control region (CR), and inadequate sampling. We therefore analyzed entire mitochondrial genomes for 169 dogs to obtain maximal phylogenetic resolution and the CR for 1,543 dogs across the Old World for a comprehensive picture of geographical diversity. Hereby, a detailed picture of the origins of the dog can for the first time be suggested. We obtained evidence that the dog has a single origin in time and space and an estimation of the time of origin, number of founders, and approximate region, which also gives potential clues about the human culture involved. The analyses showed that dogs universally share a common homogenous gene pool containing 10 major haplogroups. However, the full range of genetic diversity, all 10 haplogroups, was found only in southeastern Asia south of Yangtze River, and diversity decreased following a gradient across Eurasia, through seven haplogroups in Central China and five in North China and Southwest (SW)Asia, down to only four haplogroups in Europe. The mean sequence distance to ancestral haplotypes indicates an origin 5,400–16,300 years ago (ya) from at least 51 female wolf founders. These results indicate that the domestic dog originated in southern China less than 16,300 ya, from several hundred wolves. The place and time coincide approximately with the origin of rice agriculture, suggesting that the dogs may have originated among sedentary hunter-gatherers or early farmers, and the numerous founders indicate that wolf taming was an important culture trait.
doi:10.1093/molbev/msp195
PMCID: PMC2775109  PMID: 19723671
dog; Canis familiaris; domestication; mitochondrial DNA
20.  A Sexual Ornament in Chickens Is Affected by Pleiotropic Alleles at HAO1 and BMP2, Selected during Domestication 
PLoS Genetics  2012;8(8):e1002914.
Domestication is one of the strongest forms of short-term, directional selection. Although selection is typically only exerted on one or a few target traits, domestication can lead to numerous changes in many seemingly unrelated phenotypes. It is unknown whether such correlated responses are due to pleiotropy or linkage between separate genetic architectures. Using three separate intercrosses between wild and domestic chickens, a locus affecting comb mass (a sexual ornament in the chicken) and several fitness traits (primarily medullary bone allocation and fecundity) was identified. This locus contains two tightly-linked genes, BMP2 and HAO1, which together produce the range of pleiotropic effects seen. This study demonstrates the importance of pleiotropy (or extremely close linkage) in domestication. The nature of this pleiotropy also provides insights into how this sexual ornament could be maintained in wild populations.
Author Summary
The genetic analysis of phenotypes and the identification of the causative underlying genes remain central to molecular and evolutionary biology. By utilizing the domestication process, it is possible to exploit the large differences between domesticated animals and their wild counterparts to study both this and the mechanism of domestication itself. Domestication has been central to the advent of modern civilization; and yet, despite domesticated animals displaying similar adaptations in morphology, physiology, and behaviour, the genetic basis of these changes are unknown. In addition, though sexual selection theory has been the subject of a vast amount of study, very little is known about which genes are underpinning such traits. We have generated multiple intercrosses and advanced intercrosses based on wild-derived and domestic chickens to fine-map genomic regions affecting a sexual ornament. These regions have been over-laid with putative selective sweeps identified in domestic chickens and found to be significantly associated with them. By using expression QTL analysis, we show that two genes in one region, HAO1 and BMP2, are controlling multiple aspects of the domestication phenotype, from a sexual ornament to multiple life history traits. This demonstrates the importance of pleiotropy (or extremely close linkage) in controlling these genetic changes.
doi:10.1371/journal.pgen.1002914
PMCID: PMC3431302  PMID: 22956912
21.  QTL Mapping in New Arabidopsis thaliana Advanced Intercross-Recombinant Inbred Lines 
PLoS ONE  2009;4(2):e4318.
Background
Even when phenotypic differences are large between natural or domesticated strains, the underlying genetic basis is often complex, and causal genomic regions need to be identified by quantitative trait locus (QTL) mapping. Unfortunately, QTL positions typically have large confidence intervals, which can, for example, lead to one QTL being masked by another, when two closely linked loci are detected as a single QTL. One strategy to increase the power of precisely localizing small effect QTL, is the use of an intercross approach before inbreeding to produce Advanced Intercross RILs (AI-RILs).
Methodology/Principal Findings
We present two new AI-RIL populations of Arabidopsis thaliana genotyped with an average intermarker distance of 600 kb. The advanced intercrossing design led to expansion of the genetic map in the two populations, which contain recombination events corresponding to 50 kb/cM in an F2 population. We used the AI-RILs to map QTL for light response and flowering time, and to identify segregation distortion in one of the AI-RIL populations due to a negative epistatic interaction between two genomic regions.
Conclusions/Significance
The two new AI-RIL populations, EstC and KendC, derived from crosses of Columbia (Col) to Estland (Est-1) and Kendallville (Kend-L) provide an excellent resource for high precision QTL mapping. Moreover, because they have been genotyped with over 100 common markers, they are also excellent material for comparative QTL mapping.
doi:10.1371/journal.pone.0004318
PMCID: PMC2629843  PMID: 19183806
22.  Changes in the geographical distribution and abundance of the tick Ixodes ricinus during the past 30 years in Sweden 
Background
Ixodes ricinus is the main vector in Europe of human-pathogenic Lyme borreliosis (LB) spirochaetes, the tick-borne encephalitis virus (TBEV) and other pathogens of humans and domesticated mammals. The results of a previous 1994 questionnaire, directed at people living in Central and North Sweden (Svealand and Norrland) and aiming to gather information about tick exposure for humans and domestic animals, suggested that Ixodes ricinus ticks had become more widespread in Central Sweden and the southern part of North Sweden from the early 1980s to the early 1990s. To investigate whether the expansion of the tick's northern geographical range and the increasing abundance of ticks in Sweden were still occurring, in 2009 we performed a follow-up survey 16 years after the initial study.
Methods
A questionnaire similar to the one used in the 1994 study was published in Swedish magazines aimed at dog owners, home owners, and hunters. The questionnaire was published together with a popular science article about the tick's biology and role as a pathogen vector in Sweden. The magazines were selected to get information from people familiar with ticks and who spend time in areas where ticks might be present.
Results
Analyses of data from both surveys revealed that during the near 30-year period from the early 1980s to 2008, I. ricinus has expanded its distribution range northwards. In the early 1990s ticks were found in new areas along the northern coastline of the Baltic Sea, while in the 2009 study, ticks were reported for the first time from many locations in North Sweden. This included locations as far north as 66°N and places in the interior part of North Sweden. During this 16-year period the tick's range in Sweden was estimated to have increased by 9.9%. Most of the range expansion occurred in North Sweden (north of 60°N) where the tick's coverage area doubled from 12.5% in the early 1990s to 26.8% in 2008. Moreover, according to the respondents, the abundance of ticks had increased markedly in LB- and TBE-endemic areas in South (Götaland) and Central Sweden.
Conclusions
The results suggest that I. ricinus has expanded its range in North Sweden and has become distinctly more abundant in Central and South Sweden during the last three decades. However, in the northern mountain region I. ricinus is still absent. The increased abundance of the tick can be explained by two main factors: First, the high availability of large numbers of important tick maintenance hosts, i.e., cervids, particularly roe deer (Capreolus capreolus) during the last three decades. Second, a warmer climate with milder winters and a prolonged growing season that permits greater survival and proliferation over a larger geographical area of both the tick itself and deer. High reproductive potential of roe deer, high tick infestation rate and the tendency of roe deer to disperse great distances may explain the range expansion of I. ricinus and particularly the appearance of new TBEV foci far away from old TBEV-endemic localities. The geographical presence of LB in Sweden corresponds to the distribution of I. ricinus. Thus, LB is now an emerging disease risk in many parts of North Sweden. Unless countermeasures are undertaken to keep the deer populations, particularly C. capreolus and Dama dama, at the relatively low levels that prevailed before the late 1970s - especially in and around urban areas where human population density is high - by e.g. reduced hunting of red fox (Vulpes vulpes) and lynx (Lynx lynx), the incidences of human LB and TBE are expected to continue to be high or even to increase in Sweden in coming decades.
doi:10.1186/1756-3305-5-8
PMCID: PMC3311093  PMID: 22233771
23.  Replication and Explorations of High-Order Epistasis Using a Large Advanced Intercross Line Pedigree 
PLoS Genetics  2011;7(7):e1002180.
Dissection of the genetic architecture of complex traits persists as a major challenge in biology; despite considerable efforts, much remains unclear including the role and importance of genetic interactions. This study provides empirical evidence for a strong and persistent contribution of both second- and third-order epistatic interactions to long-term selection response for body weight in two divergently selected chicken lines. We earlier reported a network of interacting loci with large effects on body weight in an F2 intercross between these high– and low–body weight lines. Here, most pair-wise interactions in the network are replicated in an independent eight-generation advanced intercross line (AIL). The original report showed an important contribution of capacitating epistasis to growth, meaning that the genotype at a hub in the network releases the effects of one or several peripheral loci. After fine-mapping of the loci in the AIL, we show that these interactions were persistent over time. The replication of five of six originally reported epistatic loci, as well as the capacitating epistasis, provides strong empirical evidence that the originally observed epistasis is of biological importance and is a contributor in the genetic architecture of this population. The stability of genetic interaction mechanisms over time indicates a non-transient role of epistasis on phenotypic change. Third-order epistasis was for the first time examined in this study and was shown to make an important contribution to growth, which suggests that the genetic architecture of growth is more complex than can be explained by two-locus interactions only. Our results illustrate the importance of designing studies that facilitate exploration of epistasis in populations for obtaining a comprehensive understanding of the genetics underlying a complex trait.
Author Summary
This study provides evidence for a strong and persistent contribution of epistatic interactions to the selection response in two chicken lines subjected to 50 generations of divergent selection for 8-week body weight. We show that the genetic architecture of the trait involves genetic interactions of both second- and third-order and that, together, they explain a large portion of the phenotypic divergence between the lines. By replicating a radial epistatic network found in an independent intercross from the same founder individuals, we show that the type of genetic interactions affecting this complex trait is persistent over time. In addition to replicating pair-wise interactions, the size of the pedigree also facilitated evaluation of third-order interactions, which allowed us to further describe the complex genetic mechanisms underlying growth phenotype in chicken. Moreover, a new approach for measuring and detecting capacitating epistasis was proposed. By showing the importance of third-order epistasis in this system, we reinforce the importance of taking it into account when designing experiments aimed at elucidating the genetic architecture of complex traits.
doi:10.1371/journal.pgen.1002180
PMCID: PMC3140984  PMID: 21814519
24.  Fine mapping and replication of QTL in outbred chicken advanced intercross lines 
Background
Linkage mapping is used to identify genomic regions affecting the expression of complex traits. However, when experimental crosses such as F2 populations or backcrosses are used to map regions containing a Quantitative Trait Locus (QTL), the size of the regions identified remains quite large, i.e. 10 or more Mb. Thus, other experimental strategies are needed to refine the QTL locations. Advanced Intercross Lines (AIL) are produced by repeated intercrossing of F2 animals and successive generations, which decrease linkage disequilibrium in a controlled manner. Although this approach is seen as promising, both to replicate QTL analyses and fine-map QTL, only a few AIL datasets, all originating from inbred founders, have been reported in the literature.
Methods
We have produced a nine-generation AIL pedigree (n = 1529) from two outbred chicken lines divergently selected for body weight at eight weeks of age. All animals were weighed at eight weeks of age and genotyped for SNP located in nine genomic regions where significant or suggestive QTL had previously been detected in the F2 population. In parallel, we have developed a novel strategy to analyse the data that uses both genotype and pedigree information of all AIL individuals to replicate the detection of and fine-map QTL affecting juvenile body weight.
Results
Five of the nine QTL detected with the original F2 population were confirmed and fine-mapped with the AIL, while for the remaining four, only suggestive evidence of their existence was obtained. All original QTL were confirmed as a single locus, except for one, which split into two linked QTL.
Conclusions
Our results indicate that many of the QTL, which are genome-wide significant or suggestive in the analyses of large intercross populations, are true effects that can be replicated and fine-mapped using AIL. Key factors for success are the use of large populations and powerful statistical tools. Moreover, we believe that the statistical methods we have developed to efficiently study outbred AIL populations will increase the number of organisms for which in-depth complex traits can be analyzed.
doi:10.1186/1297-9686-43-3
PMCID: PMC3034666  PMID: 21241486
25.  Refined localization of the FAT1 quantitative trait locus on pig chromosome 4 by marker-assisted backcrossing 
BMC Genetics  2006;7:17.
Background
A major QTL for fatness and growth, denoted FAT1, has previously been detected on pig chromosome 4q (SSC4q) using a Large White – wild boar intercross. Progeny that carried the wild boar allele at this locus had higher fat deposition, shorter length of carcass, and reduced growth. The position and the estimated effects of the FAT1 QTL for growth and fatness have been confirmed in a previous study. In order to narrow down the QTL interval we have traced the inheritance of the wild boar allele associated with high fat deposition through six additional backcross generations.
Results
Progeny-testing was used to determine the QTL genotype for 10 backcross sires being heterozygous for different parts of the broad FAT1 region. The statistical analysis revealed that five of the sires were segregating at the QTL, two were negative while the data for three sires were inconclusive. We could confirm the QTL effects on fatness/meat content traits but not for the growth traits implying that growth and fatness are controlled by distinct QTLs on chromosome 4. Two of the segregating sires showed highly significant QTL effects that were as large as previously observed in the F2 generation. The estimates for the remaining three sires, which were all heterozygous for smaller fragments of the actual region, were markedly smaller. With the sample sizes used in the present study we cannot with great confidence determine whether these smaller effects in some sires are due to chance deviations, epistatic interactions or whether FAT1 is composed of two or more QTLs, each one with a smaller phenotypic effect. Under the assumption of a single locus, the critical region for FAT1 has been reduced to a 3.3 cM interval between the RXRG and SDHC loci.
Conclusion
We have further characterized the FAT1 QTL on pig chromosome 4 and refined its map position considerably, from a QTL interval of 70 cM to a maximum region of 20 cM and a probable region as small as 3.3 cM. The flanking markers for the small region are RXRG and SDHC and the orthologous region of FAT1 in the human genome is located on HSA1q23.3 and harbors approximately 20 genes. Our strategy to further refine the map position of this major QTL will be i) to type new markers in our pigs that are recombinant in the QTL interval and ii) to perform Identity-By-Descent (IBD) mapping across breeds that have been strongly selected for lean growth.
doi:10.1186/1471-2156-7-17
PMCID: PMC1431559  PMID: 16542463

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