Genomic analysis of gum disease and hypertrichosis in foxes.

Since the 1940s, a proliferative gingival disease called hereditary hyperplastic gingivitis (HHG) has been described in the farmed silver fox, Vulpes vulpes (Dyrendahl and Henricson 1960). HHG displays an autosomal recessive transmission and has a pleiotropic relationship with superior fur quality in terms of length and thickness of guard hairs. An analogous human disease, hereditary gingival fibromatosis (HGF), is characterized by a predominantly autosomal dominant transmission and a complex etiology, occurring either as an isolated condition or as a part of a syndrome. Similar to HHG, the symptom most commonly associated with syndromic HGF is hypertrichosis. Here we explore potential mechanisms involved in HHG by comparison to known genetic information about hypertrichosis co-occurring with HGF, using an Affymetrix canine genome microarray platform, quantitative PCR, and candidate gene sequencing. We conclude that the mitogen-activated protein kinase pathway is involved in HHG, however despite involvement of the mitogen-activated protein kinase kinase 6 gene in congenital hypertrichosis with gingival fibromatosis in humans, this gene did not contain any fixed mutations in exons or exon-intron boundaries in HHG-affected foxes, suggesting that it is not causative of HHG in the farmed silver fox population. Differential up-regulation of MAP2K6 gene in HHG-affected foxes does implicate this gene in the HHG phenotype.

Mitochondrial population structure and post-glacial dispersal of longnose sucker Catostomus catostomus in Labrador, Canada: evidence for multiple refugial origins and limited ongoing gene flow.

Two hundred and eighty-seven longnose sucker Catostomus catostomus were collected from 14 lakes in Labrador, 52 from three lakes in Ontario, 43 from two lakes in British Columbia and 32 from a lake in Yukon; a total of 414 in all. The resulting 34 haplotypes (20 in Labrador) contained moderate haplotypic diversity (h = 0·657) and relatively low nucleotide diversity (π = 3·730 × 10(-3) . Mean ϕST (0·453, P < 0·05) over all populations revealed distinct genetic structuring among C. catostomus populations across Canada, based on province, which was validated by the analysis and spatial analysis of molecular variance (c. 80% variation between provinces). These results probably reflect the historical imprint of recolonization from different refugia and possibly indicate limited ongoing gene flow within provinces. A haplotype network revealed one major and two minor clades within Labrador that were assigned to the Atlantic, Beringian and Mississippian refugia, respectively, with tests of neutrality and mismatch distribution indicative of a recent population expansion in Labrador, dated between c. 3500 and 8300 years ago.

Nucleotide variation in the mitochondrial genome provides evidence for dual routes of postglacial recolonization and genetic recombination in the northeastern brook trout (Salvelinus fontinalis).

Levels and patterns of mitochondrial DNA (mtDNA) variation were examined to investigate the population structure and possible routes of postglacial recolonization of the world's northernmost native populations of brook trout (Salvelinus fontinalis), which are found in Labrador, Canada. We analyzed the sequence diversity of a 1960-bp portion of the mitochondrial genome (NADH dehydrogenase 1 gene and part of cytochrome oxidase 1) of 126 fish from 32 lakes distributed throughout seven regions of northeastern Canada. These populations were found to have low levels of mtDNA diversity, a characteristic trait of populations at northern extremes, with significant structuring at the level of the watershed. Upon comparison of northeastern brook trout sequences to the publicly available brook trout whole mitochondrial genome (GenBank AF154850), we infer that the GenBank sequence is from a fish whose mtDNA has recombined with that of Arctic charr (S. alpinus). The haplotype distribution provides evidence of two different postglacial founding groups contributing to present-day brook trout populations in the northernmost part of their range; the evolution of the majority of the haplotypes coincides with the timing of glacier retreat from Labrador. Our results exemplify the strong influence that historical processes such as glaciations have had on shaping the current genetic structure of northern species such as the brook trout.

Mitogenomic and microsatellite variation in descendants of the founder population of Newfoundland: high genetic diversity in an historically isolated population.

The island of Newfoundland, the first of England's overseas colonies, was settled from the 17th century onward by restricted numbers of English, Irish, and French immigrants, in small "outport" communities that have maintained geographic, religious, and linguistic isolation to the latest generations. To measure the extent of modification and loss of genetic variation through founder effect, drift, and inbreeding in this historically isolated population, we analyzed the complete mitochondrial DNA (mtDNA) genomes and 14 microsatellite loci from each of 27 individuals with matrilineal ancestries extending to the colonial period. Every individual has a unique mtDNA genome sequence. All but one of these genomes are assignable to one of five major (H,J,K,T, and U) or minor (I) European haplogroups. The possibility of homoplasy at single nucleotide polymorphism (SNP) sites that define subtypes within the H haplogroup is discussed. Observed haplogroup proportions do not differ significantly from those of western Europeans or between English and Irish Newfoundlanders. The exceptional individual is a member of haplogroup A2, who appears to be the descendant of a Mi'kmaq First Nations mother and a French father, a common marriage pattern in the early settlement of Newfoundland. Microsatellite diversity is high (HE = 0.763), unstructured with respect to mtDNA haplotype or ethnicity, and there is no evidence of linkage disequilibrium. There is a small but significant degree of inbreeding (FIS = 0.0174). Collection of whole mtDNA genome data was facilitated by the use of microarray sequencing, and we describe a simple algorithm that is 99.67% efficient for sequence recovery.

Molecular identification of prey in the stomach contents of Harp Seals (Pagophilus groenlandicus) using species-specific oligonucleotides.

All methods of diet analysis in marine mammals, including hard part analysis (HPA), have biases affecting the accuracy of prey-species identification and frequency in the estimated diet due to differential consumption, digestion and retention. Using PCR amplification of specific prey DNA with species-specific primers, we developed a DNA-based method that complements HPA and provides an alternative means to detect prey from stomach contents of Harp Seals (Pagophilus groenlandicus). The target size that could be reliably amplified was determined using a digestion time-series of Atlantic Cod (Gadus morhua) tissue in simulated seal stomachs. Various target lengths were trialed using general teleost primers; amplicons of approximately 800 bp or less were consistently obtained. Prey species-specific PCR primers for Atlantic Cod, Arctic Cod (Boreogadus saida) and Capelin (Mallotus villosus) were designed and tested with DNA from the stomach contents of 31 Harp Seals. Amplicons were obtained for all three species-specific primer sets. Amplification results compared with HPA revealed: (i) Atlantic Cod hard parts were found in five stomachs where no Atlantic Cod DNA amplified, suggesting that Atlantic Cod may be over-represented in the estimated diet, (ii) amplification of Arctic Cod DNA occurred for 17 stomachs, including all 12 stomachs with, and five stomachs without, Arctic Cod hard parts, and (iii) Capelin DNA amplified for four of five stomachs with Capelin hard parts and for one stomach without Capelin hard parts. We conclude that PCR amplification of specific prey DNA provides a viable means to complement Harp Seal diet analysis by HPA, but suggest that valuable information for quantitative diet analysis rests in a quantitative PCR approach.

The clinical and genetic epidemiology of neuronal ceroid lipofuscinosis in Newfoundland.

The neuronal ceroid lipofuscinoses (NCLs) are the commonest neurodegenerative disorders of children. The aims of this study were to determine the incidence of NCL in Newfoundland, identify the causative genes, and analyze the relationship between phenotype and genotype. Patients with NCL diagnosed between 1960 and 2005 were ascertained through the provincial genetics and pediatric neurology clinics. Fifty-two patients from 34 families were identified. DNA was obtained from 28/34 (82%) families; 18 families had mutations in the CLN2 gene, comprising five different mutations of which two were novel. One family had a CLN3 mutation, another had a novel mutation in CLN5, and five families shared the same mutation in CLN6. One family was misdiagnosed, and in two, molecular testing was inconclusive. Disease from CLN2 mutations had an earlier presentation (p = 0.003) and seizure onset (p < 0.001) compared with CLN6 mutation. There was a slower clinical course for those with CLN5 mutation compared with CLN2 mutation. NCL in Newfoundland has a high incidence, 1 in 7353 live births, and shows extensive genetic heterogeneity. The incidence of late infantile NCL, 9.0 per 100,000 (or 1 in 11,161) live births, is the highest reported in the world.

Species-specific oligonucleotides and multiplex PCR for forensic discrimination of two species of scallops, Placopecten magellanicus and Chlamys islandica.

Characterization of DNA that remains in seafood products after skin, scales, and shells are removed is widely used in forensic species identification, however, ordinary methods may be prohibitively expensive or time-consuming if large sample series need to be discriminated. Forensic discrimination of two species of bivalves commercially harvested from the North Atlantic, sea scallops (Placopecten magellanicus) and Icelandic scallops (Chlamys islandica), was made by means of species-specific oligonucleotides (SSOs) in a multiplex polymerase chain reaction (PCR). The test is a simultaneous in vitro amplification of a portion of the mitochondrial Cytochrome Oxidase I locus with a PCR anchor primer for a sequence identical in both species, and two alternative SSOs that selectively amplify either a 619-bp in Placopecten or a 459-bp DNA fragment in Chlamys. Fragment size and thus species identity are determined directly by gel electrophoresis. In the forensic application, analysis of more than 900 scallops from a series of samples seized from two fishing vessels showed significantly variable proportions of the species from the closed and open fisheries (Placopecten versus Chlamys, respectively). The multiplex SSO test provides a direct means of forensic identification of large population sample series, without the necessity of secondary DNA sequencing, RFLP mapping, or fingerprinting, and can be adapted to other loci and species.

Genetic diversity and differentiation of Kermode bear populations.

The Kermode bear is a white phase of the North American black bear that occurs in low to moderate frequency on British Columbia's mid-coast. To investigate the genetic uniqueness of populations containing the white phase, and to ascertain levels of gene flow among populations, we surveyed 10 highly polymorphic microsatellite loci, assayed from trapped bear hairs. A total of 216 unique bear genotypes, 18 of which were white, was sampled among 12 localities. Island populations, where Kermodes are most frequent, show approximately 4% less diversity than mainland populations, and the island richest in white bears (Gribbell) exhibited substantial genetic isolation, with a mean pairwise FST of 0.14 with other localities. Among all localities, FST for the molecular variant underlying the coat-colour difference (A893G) was 0.223, which falls into the 95th percentile of the distribution of FST values among microsatellite alleles, suggestive of greater differentiation for coat colour than expected under neutrality. Control-region sequences confirm that Kermode bears are part of a coastal or western lineage of black bears whose existence predates the Wisconsin glaciation, but microsatellite variation gave no evidence of past population expansion. We conclude that Kermodism was established and is maintained in populations by a combination of genetic isolation and somewhat reduced population sizes in insular habitat, with the possible contribution of selective pressure and/or nonrandom mating.

How to tell a sea monster: molecular discrimination of large marine animals of the North Atlantic.

Remains of large marine animals that wash onshore can be difficult to identify due to decomposition and loss of external body parts, and in consequence may be dubbed "sea monsters." DNA that survives in such carcasses can provide a basis of identification. One such creature washed ashore at St. Bernard's, Fortune Bay, Newfoundland, in August 2001. DNA was extracted from the carcass and enzymatically amplified by the polymerase chain reaction (PCR): the mitochondrial NADH2 DNA sequence was identified as that of a sperm whale (Physeter catodon). Amplification and sequencing of cryptozoological DNA with "universal" PCR primers with broad specificity to vertebrate taxa and comparison with species in the GenBank taxonomic database is an effective means of discriminating otherwise unidentifiable large marine creatures.

Inheritance and population structure of the white-phased "Kermode" black bear.

We report that a single nucleotide replacement in the melanocortin 1 receptor gene [1] (mc1r) is responsible for the white coat color of the "Kermode" bear [2], a color phase of the black bear (Ursus americanus Pallus) found in the rainforests along the north coast of British Columbia. In a sample of 220 bears, of which 22 were white, there was complete association of a recessive Tyr-to-Cys replacement at codon 298 with the white phase. This variant has not been yet been reported in other mammals, and it also is the lightest-colored variant yet found at mc1r. Also, we found that heterozygotes, which act as a hidden reservoir for the allele among black bears, were infrequent outside of the three islands where Kermodes are common and that, within these three islands, heterozygotes were less frequent than expected under random mating. Immigration of black bears into Kermode populations can depress the occurrence of the white phase, and management practices should be designed to avoid facilitating higher immigration rates.

Colonization history of atlantic island common chaffinches (Fringilla coelebs) revealed by mitochondrial DNA.

Common chaffinches (Fringilla coelebs) are thought to have colonized the Atlantic island archipelagoes (the Azores, Madeira, and the Canaries) from neighboring continental populations (Iberia and north Africa) within the last million years. However, colonization may have occurred separately from north Africa to the Canaries and from Iberia to the Azores (as would be predicted geographically) or in one wave from Iberia to the Azores and then to Madeira and the Canaries. These alternatives have different implications for the evolution of morphometric and plumage differentiation in island chaffinches. To determine the most likely colonization route, we estimated the phylogenetic relationships among island and continental subspecies of common chaffinch using sequences from four mtDNA genes (cytochrome b, ATPase 6, NADH 5, and the control region). The most strongly supported mtDNA phylogeny places the continental subspecies together as the sister group to a monophyletic clade containing the island subspecies. This is consistent with a single wave of colonization, and suggests that patterns of similarity among Atlantic island common chaffinches, such as blue pigmentation, short wings, and long tarsi, are due to common colonization history rather than to convergent evolution in a common island environment. However, spectral analysis of phylogenetic splits showed that although monophyly of island haplotypes is favored, there is also substantial support for their polyphyletic origin. We attribute the latter to the confounding effect of homoplasy at multistate sites and to the relatively rapid sequence of colonization events which provided insufficient time for the accumulation of strong phylogenetic signal. These problems are likely to be significant impediments in attempts to test hypotheses of phylogenetic histories of recently evolved populations and taxa.

Rates and patterns of mitochondrial DNA sequence evolution in fringilline finches (fringilla spp.) and the greenfinch (Carduelis chloris).

Rates and patterns of evolution in partial sequences of five mitochondrial genes (cytochrome b, ATPase 6, NADH dehydrogenase subunit 5, tRNA(Glu), and the control region) were compared among taxa in the passerine bird genera Fringilla and Carduelis. Rates of divergence do not vary significantly among genes, even in comparisons with the control region. Rate variation among lineages is significant only for the control region and NADH dehydrogenase subunit 5, and patterns of variation are consistent with the expectations of neutral theory. Base composition is biased in all genes but is stationary among lineages, and there is evidence for directional mutation pressure only in the control region. Despite these similarities, patterns of substitution differ among genes, consistent with alternative regimes of selective constraint. Rates of nonsynonymous substitution are higher in NADH dehydrogenase subunit 5 than in other protein-coding genes, and transitions exist in elevated proportions relative to transversions. Transitions appear to accumulate linearly with time in tRNA(Glu), and despite exhibiting the highest overall rate of divergence among species, there are no transversional changes in this gene. Finally, for resolving phylogenetic relationships among Fringilla taxa, the combined protein-coding data are broadly similar to those of the control region in terms of phylogenetic informativeness and statistical support.

Enigmatic phylogeny of skuas (Aves:Stercorariidae)

Multiple sources of evidence show that the skuas (Aves:Stercorariidae) are a monophyletic group, closely related to gulls (Laridae. On morphological and behavioural evidence the Stercorariidae are divided into two widely divergent genera, Catharacta and Stercorarius, consistent with observed levels of nuclear and mitochondrial gene divergence. Catharacta skuas are large-bodied and with one exception breed in the Southern Hemisphere. Stercorarius skuas otherwise known as jaegers) are smaller bodied and breed exclusively in the Northern Hemisphere. Evidence from both mitochondrial and nuclear genomes and from ectoparasitic lice (Insecta:Phthiraptera) shows that the Pomarine skua, S. pomarinus, which has been recognized as being somewhat intermediate in certain morphological and behavioural characteristics, is much more closely related to species in the genus Catharacta, especially to the Northern Hemisphere-breeding Great skua, C. skua, than it is to the other two Stercorarius skuas, the Arctic skua, S. parasiticus and the Longtailed skua, S. longicaudus. Three possible explanations that might account for this discordant aspect of skua phylogeny are explored. These involve (i) the segregation of ancestral polymorphism, (ii) convergent evolution of morphology and behaviour or (iii) inter-generic hybridization. The available evidence from both nuclear and mitochondrial genomes does not exclude any of these hypotheses. Thus, resolution of this enigma of skua phylogeny awaits further work.

Structural conservation and variation in the mitochondrial control region of fringilline finches (Fringilla spp.) and the greenfinch (Carduelis chloris).

We sequenced the entire control region and portions of flanking genes (tRNA(Phe), tRNA(Glu), and ND6) in the common chaffinch (Fringilla coelebs), blue chaffinch (F. teydea), brambling (F. montifringilla), and greenfinch (Carduelis chloris). In these finches the control region is similar in length (1,223-1,237 bp) and has the same flanking gene order as in other birds, and contains a putative TAS element and the highly conserved CSB-1 and F, D, and C boxes recognizable in most vertebrates. Cloverleaf-like structures associated with the TAS element at the 5' end and CSB-1 at the 3' end of the control region may be involved with the stop and start of D-loop synthesis, respectively. The pattern of nucleotide and substitution bias is similar to that in other vertebrates, and consequently the finch control region can be subdivided into a central, conserved G-rich domain (domain II) flanked by hypervariable 5'-C-rich (domain I) and 3'-AT-rich (domain III) segments. In pairwise comparisons among finch species, the central domain has unusually low transition/transversion ratios, which suggests that increased G + T content is a functional constraint, possibly for DNA primase efficiency. In finches the relative rates of evolution vary among domains according to a ratio of 4.2 (domain III) to 2.2 (domain I) to 1 (domain II), and extensively among sites within domains I and II. Domain I and III sequences are extremely useful in recovering intraspecific phylogeographic splits between populations in Africa and Europe, Madeira, and a basal lineage in Nefza, Tunisia. Domain II sequences are highly conserved, and are therefore only useful in conjunction with sequences from domains I and III in phylogenetic studies of closely related species.