Genetic analysis and estimation of genetic diversity in east-European breeds of swift hounds (Canis familiaris L.) based on the data of genomic studies using RAPD markers

The method of polymerase chain reaction with a set of arbitrary primers (RAPD-PCR) was used to describe genetic variation and to estimate genetic diversity in East-European swift hounds, Russian Psovyi and Hortyi Borzois. For comparison, swift hounds of two West-European breeds (Whippet and Greyhound) and single dogs of other breed groups (shepherd, terriers, mastiffs, and bird dogs) were examined. For all dog groups, their closest related species, the wolf Canis lupus, was used as an outgroup. Variation of RAPD markers was studied at several hierarchic levels: intra- and interfamily (for individual families of Russian Psovyi and Hortyi Borzois), intra- and interbreed (for ten dog breeds), and interspecific (C. familiaris-C. lupus). In total, 57 dogs and 4 wolfs were studied. Using RAPD-PCR with three primers, 93 DNA fragments with a length of 150-1500 bp were detected in several Borzoi families with known filiation. These fragments were found to be inherited as dominant markers and to be applicable for estimation of genetic differences between parents and their offspring and for comparison of individuals and families with different level of inbreeding. A high level of intra- and interbreed variation was found in Russian Psovyi and Hortyi Borzois. In these dog groups, genetic similarity indices varied in a range of 72.2 to 93.4% (parents-offspring) and 68.0 to 94.5 (sibs). Based on the patterns of RAPD markers obtained using six primers, a dendrogram of genetic similarity between the wolf and different dog breeds was constructed, and indices of intragroup diversity were calculated. All studied breeds were found to fall into two clusters, swift hounds (Borzoi-like dogs) and other dogs. Russian Borzois represent a very heterogeneous group, in which the Russian Psovyi Borzoi is closer to Greyhound than the Russian Hortyi Borzoi. All studied wolfs constituted a separate cluster. Significant differences were found between the wolf and dogs by the number of RAPD markers (92.8 and 86.1, respectively) and by the indices of genetic diversity (54.3 and 64.8%, respectively). The reasons for the high intraspecific variation of dogs (including Russian Borzois) and the prospects of using the studied group of markers for genetic analysis and differentiation in C. familiaris are discussed.     

Genetic variation and subspecific status of the grey wolf (Canis lupus) in Saudi Arabia

This work represents the most extensive genetic study of the grey wolf (Canis lupus Linnaeus, 1758) in Arabia and the first considering genetic data from multiple locations within Saudi Arabia. Previous suggestion of the occurrence of two subspecies of wolves in Arabia is not supported by this study. The genetic evidence suggests that the wolves of Saudi Arabia are genetically variable and more closely related to the Eurasian wolf Canis lupus group (dog included) than to the Indian wolf C. l. pallipes. The genetic diversity observed for C. lupus in Saudi Arabia indicates that the subspecific status C. l. arabs should be retained for the Arabian wolf. What remains unclear is the degree to which genetic introgression from domestic dogs has influenced the composition and integrity of C. lupus in Saudi Arabia.     

Characterization of 59 canine single nucleotide polymorphisms in the Italian wolf (Canis lupus) population

We characterized 59 canine single nucleotide polymorphisms (SNPs) in the endangered Italian wolf (Canis lupus) population, which were discovered by resequencing sequence‐tagged‐site (STS) DNA sequences that are known to contain SNPs in domestic dogs. Dog SNPs were usually found also in wolves. Additional SNPs unique in dogs or wolves were discovered, which is important for detecting hybrids between dogs and wolves. We developed new primer sets and analysed 15 SNPs by Pyrosequencing. The characterized SNPs will provide an important addition to the genetic markers that are currently available for studying wild populations of canids.     

Bucking the Trend in Wolf-Dog Hybridization: First Evidence from Europe of Hybridization between Female Dogs and Male Wolves

Studies on hybridization have proved critical for understanding key evolutionary processes such as speciation and adaptation. However, from the perspective of conservation, hybridization poses a concern, as it can threaten the integrity and fitness of many wild species, including canids. As a result of habitat fragmentation and extensive hunting pressure, gray wolf (Canis lupus) populations have declined dramatically in Europe and elsewhere during recent centuries. Small and fragmented populations have persisted, but often only in the presence of large numbers of dogs, which increase the potential for hybridization and introgression to deleteriously affect wolf populations. Here, we demonstrate hybridization between wolf and dog populations in Estonia and Latvia, and the role of both genders in the hybridization process, using combined analysis of maternal, paternal and biparental genetic markers. Eight animals exhibiting unusual external characteristics for wolves - six from Estonia and two from Latvia - proved to be wolf-dog hybrids. However, one of the hybridization events was extraordinary. Previous field observations and genetic studies have indicated that mating between wolves and dogs is sexually asymmetrical, occurring predominantly between female wolves and male dogs. While this was also the case among the Estonian hybrids, our data revealed the existence of dog mitochondrial genomes in the Latvian hybrids and, together with Y chromosome and autosomal microsatellite data, thus provided the first evidence from Europe of mating between male wolves and female dogs. We discuss patterns of sexual asymmetry in wolf-dog hybridization.     

Genetic assessment of the Iberian wolf Canis lupus signatus captive breeding program

The main goal of ex situ conservation programs is to improve the chances of long term survival of natural populations by founding and managing captive colonies that can serve as a source of individuals for future reintroductions or to reinforce existing populations. The degree in which a captive breeding program has captured the genetic diversity existing in the source wild population has seldom been evaluated. In this study we evaluate the genetic diversity in wild and captive populations of the Iberian wolf, Canis lupus signatus, in order to assess how much genetic diversity is being preserved in the ongoing ex situ conservation program for this subspecies. A sample of domestic dogs was also included in the analysis for comparison. Seventy-four wolves and 135 dogs were genotyped at 13 unlinked microsatellite loci. The results show that genetic diversity in Iberian wolves is comparable in magnitude to that of other wild populations of gray wolf. Both the wild and the captive Iberian wolf populations have a similarly high genetic diversity indicating that no substantial loss of diversity has occurred in the captive-breeding program. The effective number of founders of the program was estimated as ∼ ∼16, suggesting that all founders in the studbook pedigree were genetically independent. Our results emphasize also the genetic divergence between wolves and domestic dogs and indicate that our set of 13 microsatellite loci provide a powerful diagnostic test to distinguish wolves, dogs and their hybrids.     

Genetic Polymorphism of Russian,European, and Asian Chicken Breeds as Revealed with DNA and Protein Markers

The variation in polymorphic DNA (RAPD and minisatellite) and protein markers was compared for nine Russian chicken breeds differing in morphological and productivity types and in origin, three European egg breeds, and three meat breeds of the Asian origin. Genetic diversity indices were calculated for each breed group and each marker type and were used to construct dendrograms of genetic similarity. In all breed groups, minisatellites and RAPD markers revealed higher genetic diversity as compared with protein markers. With any type of markers, genetic diversity of the Russian and Asian meat breeds proved to be significantly higher than that of the European egg breeds. The differentiating potentialities of molecular and genetic biochemical markers at the breed level and the origin of the Russian chicken breeds are discussed.     

Genetic polymorphism of Russian,European, and Asian chicken breeds as revealed with DNA and protein markers

The variation in polymorphic DNA (RAPD and minisatellite) and protein markers was compared for nine Russian chicken breeds differing in morphological and productivity types and in origin, three European egg breeds, and three broiler breeds of the Asian origin. Genetic diversity indices were calculated for each breed group and each marker type and were used to construct dendrograms of genetic similarity. In all breed groups, minisatellites and RAPD markers revealed higher genetic diversity as compared with protein markers. With any type of markers, genetic diversity of the Russian and Asian broiler breeds proved to be significantly higher than that of the European egg breeds. The differentiating potentialities of molecular and genetic biochemical markers at the breed level and the origin of the Russian chicken breeds are discussed.     

Mitochondrial DNA restriction-fragment-length monomorphism in the Italian wolf (Canis lupus) population

Mitochondrial-DNA (mtDNA) restriction patterns were studied in 22 wolves (Canis lupus) sampled in central-northern Italy. A total of 60 restriction sites were detected, encompassing about 2 % of the mitochondrial genome of canids. All wolves showed the same restriction pattern. Therefore, a single mtDNA haplotype was detected in the Italian wolf population. Historical information on peninsular isolation and demographic decline suggest that low genetically effective population size and random drift may have strongly reduced the mtDNA variability of wolves in Italy over the last 100–200 years. A different mtDNA restriction pattern in feral dogs sampled from a wolf range in central Italy was detected. These findings suggest that the hybridization and introgression of female dog genomes into the Italian wolf population may be rare or absent.     

DLA-DRB1, DQA1, and DQB1 alleles and haplotypes in North American Gray Wolves

The canine major histocompatibility complex contains highly polymorphic genes, many of which are critical in regulating immune response. Since domestic dogs evolved from Gray Wolves (Canis lupus), common DLA class II alleles should exist. Sequencing was used to characterize 175 Gray Wolves for DLA class II alleles, and data from 1856 dogs, covering 85 different breeds of mostly European origin, were available for comparison. Within wolves, 28 new alleles were identified, all occurring in at least 2 individuals. Three DLA-DRB1, 8 DLA-DQA1, and 6 DLA-DQB1 alleles also identified in dogs were present. Twenty-eight haplotypes were identified, of which 2 three-locus haplotypes, and many DLA-DQA1/DQB1 haplotypes, are also found in dogs. The wolves studied had relatively few dog DLA alleles and may therefore represent a remnant population descended from Asian wolves. The single European wolf included carried a haplotype found in both these North American wolves and in many dog breeds. Furthermore, one wolf DQB1 allele has been found in Shih Tzu, a breed of Asian origin. These data suggest that the wolf ancestors of Asian and European dogs may have had different gene pools, currently reflected in the DLA alleles present in dog breeds.     

Detection of an East European wolf haplotype puzzles mitochondrial DNA monomorphism of the Italian wolf population

Southern European wolves suffered from reiterated population declines during glacial periods and historically due to human persecution. Differently from other European wolf populations, a single mitochondrial DNA (mtDNA) control region haplotype (W14) has been so far described in the Italian wolves, although no intensive genetic sampling has ever been conducted in historical source populations from central and southern Italy. Using non-invasive genetic techniques, we report the occurrence of an unexpected mtDNA haplotype (W16) in the wolf population of the Abruzzo, Lazio and Molise National Park (PNALM), central Italy. This haplotype, detected in three out of 90 faecal samples from the PNALM, was previously reported in wolves from the North Carpathians, Slovakia and the Balkans only. Microsatellite analysis and molecular sex determination confirmed that the W16 samples belonged to three distinct wolves. Although alternative explanations can be formulated for the origin of this mtDNA haplotype in the otherwise monomorphic Italian wolf population, assignment procedures indicated the likely admixed ancestry of one W16 sample with East European wolves. Anthropogenic introgression with dogs has been detected in the Italian wolf population using nuclear DNA microsatellites, but no population-wide genetic survey had previously reported a mtDNA control region variant in Italian wolves. Our findings strongly suggest that, in addition to wolf × dog hybridization, captive-released wolves or wolf × dog hybrids may successfully interbreed with wolves in the wild, and that human-mediated introgression may occur even in well established protected areas.     

Unequal contribution of sexes in the origin of dog breeds

Dogs (Canis familiaris) were domesticated from the gray wolf (Canis lupus) at least 14,000 years ago, and there is evidence of dogs with phenotypes similar to those in modern breeds 4000 years ago. However, recent genetic analyses have suggested that modern dog breeds have a much more recent origin, probably <200 years ago. To study the origin of contemporaneous breeds we combined the analysis of paternally inherited Y chromosome markers with maternally inherited mitochondrial DNA and biparentally inherited autosomal microsatellite markers in both domestic dogs and their wild ancestor, the gray wolf. Our results show a sex bias in the origin of breeds, with fewer males than females contributing genetically, which clearly differs from the breeding patterns in wild gray wolf populations where both sexes have similar contributions. Furthermore, a comparison of mitochondrial DNA and Y chromosome diversity in dog groups recognized by the World Canine Organization, as well as in groups defined by the breeds' genetic composition, shows that paternal lineages are more differentiated among groups than maternal lineages. This demonstrates a lower exchange of males than of females between breeds belonging to different groups, which illustrates how breed founders may have been chosen.     

The systematic status of the Italian wolfCanis lupus

In the past, the gray wolfCanis lupus Linnaeus, 1758, has been recognized in Italy as either the subspecieslupus oritalicus. It has also been postulated that this population has undergone introgression from the domestic dogCanis familiaris. In order to clarify these issues, multistatistical analyses were made of 10 skull measurements of 34 full grown male wolves from the Italian Peninsula, 91 other male Eurasian wolves, and 20 domestic dogs. The analyses, together with other morphological evidence and prior genetic research, support recognition of the Italian wolf as a separate subspecies,Canis lupus italicus. The same evidence indicates that the subspecies has not been affected through hybridization with the domestic dog.     

Genetic and morphometric analysis of sixteenth century <Emphasis Type="Italic">Canis</Emphasis> skull fragments: implications for historic eastern and gray wolf distribution in North America

Resolving the taxonomy and historic ranges of species are essential to recovery plans for species at risk and conservation programs that aim to restore extirpated populations. In eastern North America, planning for wolf population restoration is complicated by the disputed historic distributions of two wolf species: the Old World-evolved gray wolf (Canis lupus) and the New World-evolved eastern wolf (C. lycaon). We used genetic and morphometric data from 4- to 500-year-old Canis samples excavated in London, Ontario, Canada to help clarify the historic range of these two wolf species in the eastern temperate forests of North America. We isolated DNA and sequenced the mitochondrial control region and found that none of the samples were of gray wolf origin. Two of the DNA sequences corresponded to those found in present day coyotes (C. latrans), but morphometric comparisons show an eastern wolf, not coyote, origin. The remaining two sequences matched ancient domestic dog haplotypes. These results suggest that the New World-evolved eastern wolf, not the gray wolf, occupied this region prior to the arrival of European settlers, although eastern-gray wolf hybrids cannot be ruled out. Furthermore, our data support the idea of a shared common ancestry between eastern wolves and western coyotes, and that the distribution of gray wolves at this time probably did not include the eastern temperate forests of North America.     

Ancient DNA suggests modern wolves trace their origin to a Late Pleistocene expansion from Beringia

Grey wolves (Canis lupus) are one of the few large terrestrial carnivores that have maintained a wide geographical distribution across the Northern Hemisphere throughout the Pleistocene and Holocene. Recent genetic studies have suggested that, despite this continuous presence, major demographic changes occurred in wolf populations between the Late Pleistocene and early Holocene, and that extant wolves trace their ancestry to a single Late Pleistocene population. Both the geographical origin of this ancestral population and how it became widespread remain unknown. Here, we used a spatially and temporally explicit modelling framework to analyse a data set of 90 modern and 45 ancient mitochondrial wolf genomes from across the Northern Hemisphere, spanning the last 50,000 years. Our results suggest that contemporary wolf populations trace their ancestry to an expansion from Beringia at the end of the Last Glacial Maximum, and that this process was most likely driven by Late Pleistocene ecological fluctuations that occurred across the Northern Hemisphere. This study provides direct ancient genetic evidence that long‐range migration has played an important role in the population history of a large carnivore, and provides insight into how wolves survived the wave of megafaunal extinctions at the end of the last glaciation. Moreover, because Late Pleistocene grey wolves were the likely source from which all modern dogs trace their origins, the demographic history described in this study has fundamental implications for understanding the geographical origin of the dog.     

Combined use of maternal,paternal and bi-parental genetic markers for the identification of wolf-dog hybrids

The identification of hybrids is often a subject of primary concern for the development of conservation and management strategies, but can be difficult when the hybridizing species are closely related and do not possess diagnostic genetic markers. However, the combined use of mitochondrial DNA (mtDNA), autosomal and Y chromosome genetic markers may allow the identification of hybrids and of the direction of hybridization. We used these three types of markers to genetically characterize one possible wolf-dog hybrid in the endangered Scandinavian wolf population. We first characterized the variability of mtDNA and Y chromosome markers in Scandinavian wolves as well as in neighboring wolf populations and in dogs. While the mtDNA data suggested that the target sample could correspond to a wolf, its Y chromosome type had not been observed before in Scandinavian wolves. We compared the genotype of the target sample at 18 autosomal microsatellite markers with those expected in pure specimens and in hybrids using assignment tests. The combined results led to the conclusion that the animal was a hybrid between a Scandinavian female wolf and a male dog. This finding confirms that inter-specific hybridization between wolves and dogs can occur in natural wolf populations. A possible correlation between hybridization and wolf population density and disturbance deserves further research.     

Sympatric wolf and coyote populations of the western Great Lakes region are reproductively isolated

Interpretation of the genetic composition and taxonomic history of wolves in the western Great Lakes region (WGLR) of the United States has long been debated and has become more important to their conservation given the recent changes in their status under the Endangered Species Act. Currently, the two competing hypotheses on WGLR wolves are that they resulted from hybridization between (i) grey wolves (Canis lupus) and western coyotes (C. latrans) or (ii) between grey wolves and eastern wolves (C. lycaon). We performed a genetic analysis of sympatric wolves and coyotes from the region to assess the degree of reproductive isolation between them and to clarify the taxonomic status of WGLR wolves. Based on data from maternal, paternal and bi‐parental genetic markers, we demonstrate a clear genetic distinction between sympatric wolves and coyotes and conclude that they are reproductively isolated and that wolf–coyote hybridization in the WGLR is uncommon. The data reject the hypothesis that wolves in the WGLR derive from hybridization between grey wolves and western coyotes, and we conclude that the extant WGLR wolf population is derived from hybridization between grey wolves and eastern wolves. Grey‐eastern wolf hybrids (C. lupus × lycaon) comprise a substantial population that extends across Michigan, Wisconsin, Minnesota and western Ontario. These findings have important implications for the conservation and management of wolves in North America, specifically concerning the overestimation of grey wolf numbers in the United States and the need to address policies for hybrids.     

MHC variability supports dog domestication from a large number of wolves: high diversity in Asia

The process of dog domestication is still somewhat unresolved. Earlier studies indicate that domestic dogs from all over the world have a common origin in Asia. So far, major histocompatibility complex (MHC) diversity has not been studied in detail in Asian dogs, although high levels of genetic diversity are expected at the domestication locality. We sequenced the second exon of the canine MHC gene DLA–DRB1 from 128 Asian dogs and compared our data with a previously published large data set of MHC alleles, mostly from European dogs. Our results show that Asian dogs have a higher MHC diversity than European dogs. We also estimated that there is only a small probability that new alleles have arisen by mutation since domestication. Based on the assumption that all of the currently known 102 DLA–DRB1 alleles come from the founding wolf population, we simulated the number of founding wolf individuals. Our simulations indicate an effective population size of at least 500 founding wolves, suggesting that the founding wolf population was large or that backcrossing has taken place.     

Evolutionary genomics of dog domestication

We review the underlying principles and tools used in genomic studies of domestic dogs aimed at understanding the genetic changes that have occurred during domestication. We show that there are two principle modes of evolution within dogs. One primary mode that accounts for much of the remarkable diversity of dog breeds is the fixation of discrete mutations of large effect in individual lineages that are then crossed to various breed groupings. This transfer of mutations across the dog evolutionary tree leads to the appearance of high phenotypic diversity that in actuality reflects a small number of major genes. A second mechanism causing diversification involves the selective breeding of dogs within distinct phenotypic or functional groups, which enhances specific group attributes such as heading or tracking. Such progressive selection leads to a distinct genetic structure in evolutionary trees such that functional and phenotypic groups cluster genetically. We trace the origin of the nuclear genome in dogs based on haplotype-sharing analyses between dogs and gray wolves and show that contrary to previous mtDNA analyses, the nuclear genome of dogs derives primarily from Middle Eastern or European wolves, a result more consistent with the archeological record. Sequencing analysis of the IGF1 gene, which has been the target of size selection in small breeds, further supports this conclusion. Finally, we discuss how a black coat color mutation that evolved in dogs has transformed North American gray wolf populations, providing a first example of a mutation that appeared under domestication and selectively swept through a wild relative.