Mouse PRDM9 DNA-binding specificity determines sites of histone H3 lysine 4 trimethylation for initiation of meiotic recombination

Meiotic recombination generates reciprocal exchanges between homologous chromosomes (also called crossovers, COs) that are essential for proper chromosome segregation during meiosis and are a major source of genome diversity by generating new allele combinations. COs have two striking properties: they occur at specific sites, called hotspots, and these sites evolve rapidly. In mammals, the Prdm9 gene, which encodes a meiosis-specific histone H3 methyltransferase, has recently been identified as a determinant of CO hotspots. Here, using transgenic mice, we show that the sole modification of PRDM9 zinc fingers leads to changes in hotspot activity, histone H3 lysine 4 trimethylation (H3K4me3) levels, and chromosome-wide distribution of COs. We further demonstrate by an in vitro assay that the PRDM9 variant associated with hotspot activity binds specifically to DNA sequences located at the center of the three hotspots tested. Remarkably, we show that mutations in cis located at hotspot centers and associated with a decrease of hotspot activity affect PRDM9 binding. Taken together, these results provide the direct demonstration that Prdm9 is a master regulator of hotspot localization through the DNA binding specificity of its zinc finger array and that binding of PRDM9 at hotspots promotes local H3K4me3 enrichment.     

The complete mitochondrial genome of dhole <Emphasis Type="Italic">Cuon alpinus</Emphasis>: phylogenetic analysis and dating evolutionary divergence within canidae

The dhole (Cuon alpinus) is the only existent species in the genus Cuon (Carnivora: Canidae). In the present study, the complete mitochondrial genome of the dhole was sequenced. The total length is 16672 base pairs which is the shortest in Canidae. Sequence analysis revealed that most mitochondrial genomic functional regions were highly consistent among canid animals except the CSB domain of the control region. The difference in length among the Canidae mitochondrial genome sequences is mainly due to the number of short segments of tandem repeated in the CSB domain. Phylogenetic analysis was progressed based on the concatenated data set of 14 mitochondrial genes of 8 canid animals by using maximum parsimony (MP), maximum likelihood (ML) and Bayesian (BI) inference methods. The genera Vulpes and Nyctereutes formed a sister group and split first within Canidae, followed by that in the Cuon. The divergence in the genus Canis was the latest. The divarication of domestic dogs after that of the Canis lupus laniger is completely supported by all the three topologies. Pairwise sequence divergence data of different mitochondrial genes among canid animals were also determined. Except for the synonymous substitutions in protein-coding genes, the control region exhibits the highest sequence divergences. The synonymous rates are approximately two to six times higher than those of the non-synonymous sites except for a slightly higher rate in the non-synonymous substitution between Cuon alpinus and Vulpes vulpes. 16S rRNA genes have a slightly faster sequence divergence than 12S rRNA and tRNA genes. Based on nucleotide substitutions of tRNA genes and rRNA genes, the times since divergence between dhole and other canid animals, and between domestic dogs and three subspecies of wolves were evaluated. The result indicates that Vulpes and Nyctereutes have a close phylogenetic relationship and the divergence of Nyctereutes is a little earlier. The Tibetan wolf may be an archaic pedigree within wolf subspecies. The genetic distance between wolves and domestic dogs is less than that among different subspecies of wolves. The domestication of dogs was about 1.56–1.92 million years ago or even earlier.     

What are the genomic drivers of the rapid evolution of PRDM9?

Mammalian Prdm9 has been proposed to be a key determinant of the positioning of chromosome double-strand breaks during meiosis, a contributor to speciation processes, and the most rapidly evolving gene in human, and other animal, genomes. Prdm9 genes often exhibit substantial variation in their numbers of encoded zinc fingers (ZFs), not only between closely related species but also among individuals of a species. The near-identity of these ZF sequences appears to render them very unstable in copy number. The rare sequence differences, however, cluster within ZF sites that determine the DNA-binding specificity of PRDM9, and these substitutions are frequently positively selected. Here, possible drivers of the rapid evolution of Prdm9 are discussed, including selection for efficient pairing of homologous chromosomes or for recombination of deleterious linked alleles, and selection against depletion of recombination hotspots or against disease-associated genome rearrangement.     

Linkage Disequilibrium and Demographic History of Wild and Domestic Canids

Assessing the extent of linkage disequilibrium (LD) in natural populations of a nonmodel species has been difficult due to the lack of available genomic markers. However, with advances in genotyping and genome sequencing, genomic characterization of natural populations has become feasible. Using sequence data and SNP genotypes, we measured LD and modeled the demographic history of wild canid populations and domestic dog breeds. In 11 gray wolf populations and one coyote population, we find that the extent of LD as measured by the distance at which r² = 0.2 extends <10 kb in outbred populations to >1.7 Mb in populations that have experienced significant founder events and bottlenecks. This large range in the extent of LD parallels that observed in 18 dog breeds where the r² value varies from ~20 kb to >5 Mb. Furthermore, in modeling demographic history under a composite-likelihood framework, we find that two of five wild canid populations exhibit evidence of a historical population contraction. Five domestic dog breeds display evidence for a minor population contraction during domestication and a more severe contraction during breed formation. Only a 5% reduction in nucleotide diversity was observed as a result of domestication, whereas the loss of nucleotide diversity with breed formation averaged 35%.     

Prdm9 Polymorphism Unveils Mouse Evolutionary Tracks

PR/SET domain containing 9 (Prdm9) mediates histone modifications such as H3K4me3 and marks hotspots of meiotic recombination. In many mammalian species, the Prdm9 gene is highly polymorphic. Prdm9 polymorphism is assumed to play two critical roles in evolution: to diversify the spectrum of meiotic recombination hotspots and to cause male hybrid sterility, leading to reproductive isolation and speciation. Nevertheless, information about Prdm9 sequences in natural populations is very limited. In this study, we conducted a comprehensive population survey on Prdm9 polymorphism in the house mouse, Mus musculus. Overall M. musculus Prdm9 displays an extraordinarily high level of polymorphism, particularly in regions encoding zinc finger repeats, which recognize recombination hotspots. Prdm9 alleles specific to various M. musculus subspecies dominate in subspecies territories. Moreover, introgression into other subspecies territories was found for highly divergent Prdm9 alleles associated with t-haplotype. The results of our phylogeographical analysis suggest that the requirement for hotspot diversity depends on geographical range and time span in mouse evolution, and that Prdm9 polymorphism has not been maintained by a simple balanced selection in the population of each subspecies.     

Multimer Formation Explains Allelic Suppression of PRDM9 Recombination Hotspots

Genetic recombination during meiosis functions to increase genetic diversity, promotes elimination of deleterious alleles, and helps assure proper segregation of chromatids. Mammalian recombination events are concentrated at specialized sites, termed hotspots, whose locations are determined by PRDM9, a zinc finger DNA-binding histone methyltransferase. Prdm9 is highly polymorphic with most alleles activating their own set of hotspots. In populations exhibiting high frequencies of heterozygosity, questions remain about the influences different alleles have in heterozygous individuals where the two variant forms of PRDM9 typically do not activate equivalent populations of hotspots. We now find that, in addition to activating its own hotspots, the presence of one Prdm9 allele can modify the activity of hotspots activated by the other allele. PRDM9 function is also dosage sensitive; Prdm9 ^+/- heterozygous null mice have reduced numbers and less active hotspots and increased numbers of aberrant germ cells. In mice carrying two Prdm9 alleles, there is allelic competition; the stronger Prdm9 allele can partially or entirely suppress chromatin modification and recombination at hotspots of the weaker allele. In cell cultures, PRDM9 protein variants form functional heteromeric complexes which can bind hotspots sequences. When a heteromeric complex binds at a hotspot of one PRDM9 variant, the other PRDM9 variant, which would otherwise not bind, can still methylate hotspot nucleosomes. We propose that in heterozygous individuals the underlying molecular mechanism of allelic suppression results from formation of PRDM9 heteromers, where the DNA binding activity of one protein variant dominantly directs recombination initiation towards its own hotspots, effectively titrating down recombination by the other protein variant. In natural populations with many heterozygous individuals, allelic competition will influence the recombination landscape.     

周口店变异狼(Canis lupus variabilis)在家畜狗(Canis familiaris)祖先系列中的位置

本文作者对周口店第一、第三及第十三地点的一种化石犬类——变异狼(Canis lupus uariadilis)进行了观察和测量。根据其头骨大小、形态特征以及与中国早期人类共生的情况来看,认为它有可能是从驯化的野生狼导致家畜狗出现的一种祖先类型。     

Widespread occurrence of a domestic dog mitochondrial DNA haplotype in southeastern US coyotes

Sequence analysis of the mitochondrial DNA control region from 112 southeastern US coyotes (Canis latrans) revealed 12 individuals with a haplotype closely related to those in domestic dogs. Phylogenetic analyses grouped this new haplotype in the dog/grey wolf (Canis familiaris/Canis lupus) clade with 98% bootstrap support. These results demonstrate that a male coyote hybridized with a female dog, and female hybrid offspring successfully integrated into the coyote population. The widespread distribution of this haplotype from Florida to West Virginia suggests that the hybridization event occurred long ago before the southeastern USA was colonized by coyotes. However, it could have occurred in the southeastern USA before the main front of coyotes arrived in the area between male coyotes released for sport and a local domestic dog. The introgression of domestic dog genes into the southeastern coyote population does not appear to have substantially affected the coyote's genetic, morphological, or behavioural integrity. However, our results suggest that, contrary to previous reports, hybridization can occur between domestic and wild canids, even when the latter is relatively abundant. Therefore, hybridization may be a greater threat to the persistence of wild canid populations than previously thought.     

Characterization of Prdm9 in Equids and Sterility in Mules

Prdm9 (Meisetz) is the first speciation gene discovered in vertebrates conferring reproductive isolation. This locus encodes a meiosis-specific histone H3 methyltransferase that specifies meiotic recombination hotspots during gametogenesis. Allelic differences in Prdm9, characterized for a variable number of zinc finger (ZF) domains, have been associated with hybrid sterility in male house mice via spermatogenic failure at the pachytene stage. The mule, a classic example of hybrid sterility in mammals also exhibits a similar spermatogenesis breakdown, making Prdm9 an interesting candidate to evaluate in equine hybrids. In this study, we characterized the Prdm9 gene in all species of equids by analyzing sequence variation of the ZF domains and estimating positive selection. We also evaluated the role of Prdm9 in hybrid sterility by assessing allelic differences of ZF domains in equine hybrids. We found remarkable variation in the sequence and number of ZF domains among equid species, ranging from five domains in the Tibetan kiang and Asiatic wild ass, to 14 in the Grevy’s zebra. Positive selection was detected in all species at amino acid sites known to be associated with DNA-binding specificity of ZF domains in mice and humans. Equine hybrids, in particular a quartet pedigree composed of a fertile mule showed a mosaic of sequences and number of ZF domains suggesting that Prdm9 variation does not seem by itself to contribute to equine hybrid sterility.     

Eye contact and sociability data suggests that Australian dingoes were never domesticated

Dogs were the first animal to become domesticated by humans, and they represent a classic model system for unraveling the processes of domestication. We compare Australian dingo eye contact and socialization with Basenji and German Shepherd dog (GSD) breeds. Australian dingoes arrived in Australia 5,000–8,000 BP, and there is debate whether they were domesticated before their arrival. The Basenji represents a primitive breed that diverged from the remaining breeds early in the domestication process, while GSDs are a breed dog selected from existing domestic dogs in the late 1800s. We conducted a 4-phase study with unfamiliar and familiar investigators either sitting passively or actively calling each canid. We found 75% of dingoes made eye contact in each phase. In contrast, 86% of Basenjis and 96% of GSDs made eye contact. Dingoes also exhibited shorter eye-gaze duration than breed dogs and did not respond to their name being called actively. Sociability, quantified as a canid coming within 1 m of the experimenter, was lowest for dingoes and highest for GSDs. For sociability duration, dingoes spent less time within 1 m of the experimenter than either breed dog. When compared with previous studies, these data show that the dingo is behaviorally intermediate between wild wolves and Basenji dogs and suggest that it was not domesticated before it arrived in Australia. However, it remains possible that the accumulation of mutations since colonization has obscured historical behaviors, and dingoes now exist in a feralized retamed cycle. Additional morphological and genetic data are required to resolve this conundrum.     

Genome Sequencing Highlights the Dynamic Early History of Dogs

To identify genetic changes underlying dog domestication and reconstruct their early evolutionary history, we generated high-quality genome sequences from three gray wolves, one from each of the three putative centers of dog domestication, two basal dog lineages (Basenji and Dingo) and a golden jackal as an outgroup. Analysis of these sequences supports a demographic model in which dogs and wolves diverged through a dynamic process involving population bottlenecks in both lineages and post-divergence gene flow. In dogs, the domestication bottleneck involved at least a 16-fold reduction in population size, a much more severe bottleneck than estimated previously. A sharp bottleneck in wolves occurred soon after their divergence from dogs, implying that the pool of diversity from which dogs arose was substantially larger than represented by modern wolf populations. We narrow the plausible range for the date of initial dog domestication to an interval spanning 11–16 thousand years ago, predating the rise of agriculture. In light of this finding, we expand upon previous work regarding the increase in copy number of the amylase gene (AMY2B) in dogs, which is believed to have aided digestion of starch in agricultural refuse. We find standing variation for amylase copy number variation in wolves and little or no copy number increase in the Dingo and Husky lineages. In conjunction with the estimated timing of dog origins, these results provide additional support to archaeological finds, suggesting the earliest dogs arose alongside hunter-gathers rather than agriculturists. Regarding the geographic origin of dogs, we find that, surprisingly, none of the extant wolf lineages from putative domestication centers is more closely related to dogs, and, instead, the sampled wolves form a sister monophyletic clade. This result, in combination with dog-wolf admixture during the process of domestication, suggests that a re-evaluation of past hypotheses regarding dog origins is necessary.     

Estimation of population divergence times from non-overlapping genomic sequences: examples from dogs and wolves

Despite recent technological advances in DNA sequencing, incomplete coverage remains to be an issue in population genomics, in particular for studies that include ancient samples. Here, we describe an approach to estimate population divergence times for non-overlapping sequence data that is based on probabilities of different genealogical topologies under a structured coalescent model. We show that the approach can be adapted to accommodate common problems such as sequencing errors and postmortem nucleotide misincorporations, and we use simulations to investigate biases involved with estimating genealogical topologies from empirical data. The approach relies on three reference genomes and should be particularly useful for future analysis of genomic data that comprise of nonoverlapping sets of sequences, potentially from different points in time. We applied the method to shotgun sequence data from an ancient wolf together with extant dogs and wolves and found striking resemblance to previously described fine-scale population structure among dog breeds. When comparing modern dogs to four geographically distinct wolves, we find that the divergence time between dogs and an Indian wolf is smallest, followed by the divergence times to a Chinese wolf and a Spanish wolf, and a relatively long divergence time to an Alaskan wolf, suggesting that the origin of modern dogs is somewhere in Eurasia, potentially southern Asia. We find that less than two-thirds of all loci in the boxer and poodle genomes are more similar to each other than to a modern gray wolf and that--assuming complete isolation without gene flow--the divergence time between gray wolves and modern European dogs extends to 3,500 generations before the present, corresponding to approximately 10,000 years ago (95% confidence interval [CI]: 9,000-13,000). We explicitly study the effect of gene flow between dogs and wolves on our estimates and show that a low rate of gene flow is compatible with an even earlier domestication date ～30,000 years ago (95% CI: 15,000-90,000). This observation is in agreement with recent archaeological findings and indicates that human behavior necessary for domestication of wild animals could have appeared much earlier than the development of agriculture.     

Molecular genetics of the most endangered canid: the Ethiopian wolf Canis simensis

The world's most endangered canid is the Ethiopian wolf Canis simensis , which is found in six isolated areas of the Ethiopian highlands with a total population of no more than 500 individuals. Ethiopian wolf populations are declining due to habitat loss and extermination by humans. Moreover, in at least one population, Ethiopian wolves are sympatric with domestic dogs, which may hybridize with them, compete for food, and act as disease vectors. Using molecular techniques, we address four questions concerning Ethiopian wolves that have conservation implications. First, we determine the relationships of Ethiopian wolves to other wolf-like canids by phylogenetic analysis of 2001 base pairs of mitochondrial DNA (mtDNA) sequence. Our results suggest that the Ethiopian wolf is a distinct species more closely related to gray wolves and coyotes than to any African canid. The mtDNA sequence similarity with gray wolves implies that the Ethiopian wolf may hybridize with domestic dogs, a recent derivative of the gray wolf. We examine this possibility through mtDNA restriction fragment analysis and analysis of nine microsatellite loci in populations of Ethiopian wolves. The results imply that hybridization has occurred between female Ethiopian wolves and male domestic dogs in one population. Finally, we assess levels of variability within and between two Ethiopian wolf populations. Although these closely situated populations are not differentiated, the level of variability in both is low, suggesting long-term effective population sizes of less than a few hundred individuals. We recommend immediate captive breeding of Ethiopian wolves to protect their gene pool from dilution and further loss of genetic variability.     

PRDM9 Drives Evolutionary Erosion of Hotspots in Mus musculus through Haplotype-Specific Initiation of Meiotic Recombination

Meiotic recombination generates new genetic variation and assures the proper segregation of chromosomes in gametes. PRDM9, a zinc finger protein with histone methyltransferase activity, initiates meiotic recombination by binding DNA at recombination hotspots and directing the position of DNA double-strand breaks (DSB). The DSB repair mechanism suggests that hotspots should eventually self-destruct, yet genome-wide recombination levels remain constant, a conundrum known as the hotspot paradox. To test if PRDM9 drives this evolutionary erosion, we measured activity of the Prdm9 ^Cst allele in two Mus musculus subspecies, M.m. castaneus, in which Prdm9^Cst arose, and M.m. domesticus, into which Prdm9^Cst was introduced experimentally. Comparing these two strains, we find that haplotype differences at hotspots lead to qualitative and quantitative changes in PRDM9 binding and activity. Using Mus spretus as an outlier, we found most variants affecting PRDM9^Cst binding arose and were fixed in M.m. castaneus, suppressing hotspot activity. Furthermore, M.m. castaneus×M.m. domesticus F1 hybrids exhibit novel hotspots, with large haplotype biases in both PRDM9 binding and chromatin modification. These novel hotspots represent sites of historic evolutionary erosion that become activated in hybrids due to crosstalk between one parent''s Prdm9 allele and the opposite parent''s chromosome. Together these data support a model where haplotype-specific PRDM9 binding directs biased gene conversion at hotspots, ultimately leading to hotspot erosion.     

Asymmetrical intraguild interactions with coyotes,red foxes,and domestic dogs may contribute to competitive exclusion of declining gray foxes

Species coexistence is governed by availability of resources and intraguild interactions including strategies to reduce ecological overlap. Gray foxes are dietary generalist mesopredators expected to benefit from anthropogenic disturbance, but populations have declined across the midwestern USA, including severe local extirpation rates coinciding with high coyote and domestic dog occurrence and low red fox occurrence. We used data from a large‐scale camera trap survey in southern Illinois, USA to quantify intraguild spatial and temporal interactions among the canid guild including domestic dogs. We used a two‐species co‐occurrence model to make pairwise assessments of conditional occupancy and detection rates. We also estimated temporal activity overlap among species and fit a fixed‐effects hierarchical community occupancy model with the four canid species. We partitioned the posterior distributions to compare gray fox occupancy probabilities conditional on estimated state of combinations of other species to assess support for hypothesized interactions. We found no evidence of broadscale avoidance among native canids and conclude that spatial and temporal segregation were limited by ubiquitous human disturbance. Mean guild richness was two canid species at a site and gray fox occupancy was greater when any combination of sympatric canids was also present, setting the stage for competitive exclusion over time. Domestic dogs may amplify competitive interactions by increasing canid guild size to the detriment of gray foxes. Our results suggest that while human activities can benefit some mesopredators, other species such as gray foxes may serve as bellwethers for habitat degradation with trophic downgrading and continued anthropogenic homogenization.     

Diversity of Prdm9 Zinc Finger Array in Wild Mice Unravels New Facets of the Evolutionary Turnover of this Coding Minisatellite

In humans and mice, meiotic recombination events cluster into narrow hotspots whose genomic positions are defined by the PRDM9 protein via its DNA binding domain constituted of an array of zinc fingers (ZnFs). High polymorphism and rapid divergence of the Prdm9 gene ZnF domain appear to involve positive selection at DNA-recognition amino-acid positions, but the nature of the underlying evolutionary pressures remains a puzzle. Here we explore the variability of the Prdm9 ZnF array in wild mice, and uncovered a high allelic diversity of both ZnF copy number and identity with the caracterization of 113 alleles. We analyze features of the diversity of ZnF identity which is mostly due to non-synonymous changes at codons −1, 3 and 6 of each ZnF, corresponding to amino-acids involved in DNA binding. Using methods adapted to the minisatellite structure of the ZnF array, we infer a phylogenetic tree of these alleles. We find the sister species Mus spicilegus and M. macedonicus as well as the three house mouse (Mus musculus) subspecies to be polyphyletic. However some sublineages have expanded independently in Mus musculus musculus and M. m. domesticus, the latter further showing phylogeographic substructure. Compared to random genomic regions and non-coding minisatellites, none of these patterns appears exceptional. In silico prediction of DNA binding sites for each allele, overlap of their alignments to the genome and relative coverage of the different families of interspersed repeated elements suggest a large diversity between PRDM9 variants with a potential for highly divergent distributions of recombination events in the genome with little correlation to evolutionary distance. By compiling PRDM9 ZnF protein sequences in Primates, Muridae and Equids, we find different diversity patterns among the three amino-acids most critical for the DNA-recognition function, suggesting different diversification timescales.     

Phylogenetic analysis of Porphyromonas species isolated from the oral cavity of Australian marsupials

Porphyromonas species are frequently isolated from the oral cavity and are associated with periodontal disease in both animals and humans. Black, pigmented Porphyromonas spp. isolated from the gingival margins of selected wild and captive Australian marsupials with varying degrees of periodontal disease (brushtail possums, koalas and macropods) were compared phylogenetically to Porphyromonas strains from non-marsupials (bear, wolf, coyote, cats and dogs) and Porphyromonas gingivalis strains from humans using 16S rRNA gene sequence analysis. The results of the phylogenetic analysis identified three distinct groups of strains. A monophyletic P. gingivalis group (Group 1) contained only strains isolated from humans and a Porphyromonas gulae group (Group 2) was divided into three distinct subclades, each containing both marsupial and non-marsupial strains. Group 3, which contained only marsupial strains, including all six strains isolated from captive koalas, was genetically distinct from P. gulae and may constitute a new Porphyromonas species.     

Mitochondrial DNA from prehistoric canids highlights relationships between dogs and South-East European wolves

The question of the origins of the dog has been much debated. The dog is descended from the wolf that at the end of the last glaciation (the archaeologically hypothesized period of dog domestication) was one of the most widespread among Holarctic mammals. Scenarios provided by genetic studies range from multiple dog-founding events to a single origin in East Asia. The earliest fossil dogs, dated approximately 17-12,000 radiocarbon ((14)C) years ago (YA), were found in Europe and in the Middle East. Ancient DNA (a-DNA) evidence could contribute to the identification of dog-founder wolf populations. To gain insight into the relationships between ancient European wolves and dogs we analyzed a 262-bp mitochondrial DNA control region fragment retrieved from five prehistoric Italian canids ranging in age from approximately 15,000 to approximately 3,000 (14)C YA. These canids were compared to a worldwide sample of 547 purebred dogs and 341 wolves. The ancient sequences were highly diverse and joined the three major clades of extant dog sequences. Phylogenetic investigations highlighted relationships between the ancient sequences and geographically widespread extant dog matrilines and between the ancient sequences and extant wolf matrilines of mainly East European origin. The results provide a-DNA support for the involvement of European wolves in the origins of the three major dog clades. Genetic data also suggest multiple independent domestication events. East European wolves may still reflect the genetic variation of ancient dog-founder populations.