Genome-wide SNP and haplotype analyses reveal a rich history underlying dog domestication.

Advances in genome technology have facilitated a new understanding of the historical and genetic processes crucial to rapid phenotypic evolution under domestication. To understand the process of dog diversification better, we conducted an extensive genome-wide survey of more than 48,000 single nucleotide polymorphisms in dogs and their wild progenitor, the grey wolf. Here we show that dog breeds share a higher proportion of multi-locus haplotypes unique to grey wolves from the Middle East, indicating that they are a dominant source of genetic diversity for dogs rather than wolves from east Asia, as suggested by mitochondrial DNA sequence data. Furthermore, we find a surprising correspondence between genetic and phenotypic/functional breed groupings but there are exceptions that suggest phenotypic diversification depended in part on the repeated crossing of individuals with novel phenotypes. Our results show that Middle Eastern wolves were a critical source of genome diversity, although interbreeding with local wolf populations clearly occurred elsewhere in the early history of specific lineages. More recently, the evolution of modern dog breeds seems to have been an iterative process that drew on a limited genetic toolkit to create remarkable phenotypic diversity.

Metabolite fingerprinting of urine suggests breed-specific dietary metabolism differences in domestic dogs.

Selective breeding of dogs has culminated in a large number of modern breeds distinctive in terms of size, shape and behaviour. Inadvertently, a range of breed-specific genetic disorders have become fixed in some pure-bred populations. Several inherited conditions confer chronic metabolic defects that are influenced strongly by diet, but it is likely that many less obvious breed-specific differences in physiology exist. Using Labrador retrievers and miniature Schnauzers maintained in a simulated domestic setting on a controlled diet, an experimental design was validated in relation to husbandry, sampling and sample processing for metabolomics. Metabolite fingerprints were generated from 'spot' urine samples using flow injection electrospray MS (FIE-MS). With class based on breed, urine chemical fingerprints were modelled using Random Forest (a supervised data classification technique), and metabolite features (m/z) explanatory of breed-specific differences were putatively annotated using the ARMeC database (http://www.armec.org). GC-MS profiling to confirm FIE-MS predictions indicated major breed-specific differences centred on the metabolism of diet-related polyphenols. Metabolism of further diet components, including potentially prebiotic oligosaccharides, animal-derived fats and glycerol, appeared significantly different between the two breeds. Analysis of the urinary metabolome of young male dogs representative of a wider range of breeds from animals maintained under domestic conditions on unknown diets provided preliminary evidence that many breeds may indeed have distinctive metabolic differences, with significant differences particularly apparent in comparisons between large and smaller breeds.

Gene (mRNA) expression in canine atopic dermatitis: microarray analysis.

Genes potentially involved in the pathology of canine atopic dermatitis (AD) were identified using gene expression microarrays. Total RNA extracted from skin biopsies was hybridized to an Agilent Technologies custom-designed 22K canine array. The arrays were analysed using Genedata Analyst software. Data were corrected for multiple hypothesis testing and tested for significance using the National Institute on Aging array analysis tool. For comparison, data were divided into separate groups: lesional atopic (n = 16), nonlesional atopic (n = 17) and healthy controls (n = 9). Fifty-four genes were differentially expressed at a significance level of 0.05 in canine AD compared to healthy controls. Sixteen genes were differentially expressed in both nonlesional and lesional atopic skin, 26 genes only in nonlesional skin and 12 only in lesional skin. These genes were associated with innate immune and inflammatory responses, cell cycle, apoptosis, barrier formation and transcriptional regulation. The most dysregulated gene in lesional skin was S100A8, which showed an almost 23-fold increase in expression. This is a pro-inflammatory cytokine located in the epidermal differentiation complex. Microarray analysis is a novel technique in canine AD. Significant changes in gene expression were identified in atopic skin. These were relevant to skin barrier formation and the immune response, suggesting that they both participate in AD. Gene expression restricted to lesional skin may be involved in inflammatory changes, whereas those shared or restricted to nonlesional skin may reflect the atopic phenotype. Investigating gene polymorphisms in the targets identified in this study will help improve our understanding of the genetic basis of this disease.

Molecular recognition of metabotropic glutamate receptor type 1 (mGluR1): synergistic understanding with free energy perturbation and linear response modeling.

Metabotropic glutamate receptors (mGluRs) constitute an important family of the G-protein coupled receptors. Due to their widespread distribution in the central nervous system (CNS), these receptors are attractive candidates for understanding the molecular basis of various cognitive processes as well as for designing inhibitors for relevant psychiatric and neurological disorders. Despite many studies on drugs targeting the mGluR receptors to date, the molecular level details on the ligand binding dynamics still remain unclear. In this study, we performed in silico experiments for mGluR1 with 29 different ligands including known synthetic agonists and antagonists as well as natural amino acids. The ligand-receptor binding affinities were estimated by the use of atomistic simulations combined with the mathematically rigorous, Free Energy Perturbation (FEP) method, which successfully recognized the native agonist l-glutamate among the highly favorable binders, and also accurately distinguished antagonists from agonists. Comparative contact analysis also revealed the binding mode differences between natural and non-natural amino acid-based ligands. Several factors potentially affecting the ligand binding affinity and specificity were identified including net charges, dipole moments, and the presence of aromatic rings. On the basis of these findings, linear response models (LRMs) were built for different sets of ligands that showed high correlations (R(2) > 0.95) to the corresponding FEP binding affinities. These results identify some key factors that determine ligand-mGluR1 binding and could be used for future inhibitor designs and support a role for in silico modeling for understanding receptor ligand interactions.

Deep Illumina-based shotgun sequencing reveals dietary effects on the structure and function of the fecal microbiome of growing kittens.

BACKGROUND: Previously, we demonstrated that dietary protein:carbohydrate ratio dramatically affects the fecal microbial taxonomic structure of kittens using targeted 16S gene sequencing. The present study, using the same fecal samples, applied deep Illumina shotgun sequencing to identify the diet-associated functional potential and analyze taxonomic changes of the feline fecal microbiome. METHODOLOGY & PRINCIPAL FINDINGS: Fecal samples from kittens fed one of two diets differing in protein and carbohydrate content (high-protein, low-carbohydrate, HPLC; and moderate-protein, moderate-carbohydrate, MPMC) were collected at 8, 12 and 16 weeks of age (n = 6 per group). A total of 345.3 gigabases of sequence were generated from 36 samples, with 99.75% of annotated sequences identified as bacterial. At the genus level, 26% and 39% of reads were annotated for HPLC- and MPMC-fed kittens, with HPLC-fed cats showing greater species richness and microbial diversity. Two phyla, ten families and fifteen genera were responsible for more than 80% of the sequences at each taxonomic level for both diet groups, consistent with the previous taxonomic study. Significantly different abundances between diet groups were observed for 324 genera (56% of all genera identified) demonstrating widespread diet-induced changes in microbial taxonomic structure. Diversity was not affected over time. Functional analysis identified 2,013 putative enzyme function groups were different (p<0.000007) between the two dietary groups and were associated to 194 pathways, which formed five discrete clusters based on average relative abundance. Of those, ten contained more (p<0.022) enzyme functions with significant diet effects than expected by chance. Six pathways were related to amino acid biosynthesis and metabolism linking changes in dietary protein with functional differences of the gut microbiome. CONCLUSIONS: These data indicate that feline feces-derived microbiomes have large structural and functional differences relating to the dietary protein:carbohydrate ratio and highlight the impact of diet early in life.

An expressed fgf4 retrogene is associated with breed-defining chondrodysplasia in domestic dogs.

Retrotransposition of processed mRNAs is a common source of novel sequence acquired during the evolution of genomes. Although the vast majority of retroposed gene copies, or retrogenes, rapidly accumulate debilitating mutations that disrupt the reading frame, a small percentage become new genes that encode functional proteins. By using a multibreed association analysis in the domestic dog, we demonstrate that expression of a recently acquired retrogene encoding fibroblast growth factor 4 (fgf4) is strongly associated with chondrodysplasia, a short-legged phenotype that defines at least 19 dog breeds including dachshund, corgi, and basset hound. These results illustrate the important role of a single evolutionary event in constraining and directing phenotypic diversity in the domestic dog.

A single IGF1 allele is a major determinant of small size in dogs.

The domestic dog exhibits greater diversity in body size than any other terrestrial vertebrate. We used a strategy that exploits the breed structure of dogs to investigate the genetic basis of size. First, through a genome-wide scan, we identified a major quantitative trait locus (QTL) on chromosome 15 influencing size variation within a single breed. Second, we examined genetic variation in the 15-megabase interval surrounding the QTL in small and giant breeds and found marked evidence for a selective sweep spanning a single gene (IGF1), encoding insulin-like growth factor 1. A single IGF1 single-nucleotide polymorphism haplotype is common to all small breeds and nearly absent from giant breeds, suggesting that the same causal sequence variant is a major contributor to body size in all small dogs.

Gene discovery and microarray analysis of cacao (Theobroma cacao L.) varieties.

The cacao bean harvest from the relatively under developed tropical tree cacao (Theobroma cacao L.) is subject to high losses in potential production due to pests and diseases. To discover and understand the stability of putative natural resistance mechanisms in this commodity crop, essential for chocolate production, we undertook a gene-discovery program and demonstrated its use in gene-expression arrays. Sequencing and assembling bean and leaf cDNA library inserts produced a unique contig set of 1,380 members. High-quality annotation of this gene set using Blast and MetaFam produced annotation for 75% of the contigs and allowed us to identify the types of gene expressed in cacao beans and leaves. Microarrays were constructed using amplified inserts of the uni-gene set and challenged with bean and leaf RNA from five cacao varieties. The microarray performed well across the five randomly chosen cacao genotypes and did not show a bias towards either leaf or bean tissues. This demonstrates that the gene sequences are useful for microarray analysis across cacao genotypes and tissue types. The array results, when compared with real-time PCR results for selected genes, showed a correlation with differential gene-expression patterns. We intend that the resultant DNA sequences and molecular microarray platform will help the cacao community to understand the basis, likely stability and pathotype resistance range of candidate cacao plants.