Dog olfactory receptor gene expression profiling using samples derived from nasal epithelium brushing.

Dogs have an exquisite sense of olfaction. In many instances this ability has been utilized by humans for a wide range of important situations including detecting explosives and illegal drugs. It is accepted that some breeds have better senses of smell than others. Dogs can detect many volatile compounds at extremely low concentrations in air. To achieve such high levels of detection, the canine olfactory system is both complex and highly developed requiring a high density of olfactory receptors capable of detecting volatiles. Consequently the dog genome encodes a large number of olfactory receptor (OR) genes. However, it remains unclear as to what extent are all of these OR genes expressed on the cell surface. To facilitate such studies, a nasal brushing method was developed to recover dog nasal epithelial cell samples from which total RNA could be extracted and used to prepare high quality cDNA libraries. After capture by hybridization with an extensive set of oligonucleotides, the level of expression of each transcript was measured following next generation sequencing (NGS). The reproducibility of this sampling approach was checked by analyzing replicate samples from the same animal (up to 6 per each naris). The quality of the hybridization capture was also checked by analyzing two DNA libraries; this offered an advantage over RNA libraries by having an equal presence for each gene. Finally, we compared this brushing method performed on living dogs to a nasal epithelium biopsy approach applied to two euthanized terminally ill dogs, following consent from their owners.Comparison the expression levels of each transcript indicate that the ratios of expression between the highest and the least expressed OR in each sample are greater than 10,000 (paralog variation). Furthermore, it was clear that a number of OR genes are not expressed.The method developed and described here will allow researchers to further address whether variations observed in the OR transcriptome relate to dog 'life experiences' and whether any differences observed between samples are dog-specific or breed-specific.

Identification and characterization of cichlid TAAR genes and comparison with other teleost TAAR repertoires.

BACKGROUND: TAARs (trace amine-associated receptors) are among the principal receptors expressed by the olfactory epithelium. We used the recent BROAD Institute release of the genome sequences of five representative fishes of the cichlid family to establish the complete TAAR repertoires of these species and to compare them with five other fish TAAR repertoires. RESULTS: The genome sequences of O. niloticus, P. nyererei, H. burtoni, N. brichardi and M. zebra were analyzed by exhaustive TBLASTN searches with a set of published TAAR gene sequences used as positive bait. A second TBLASTN analysis was then performed on the candidate genes, with a set of non-TAAR class A GPCR (G protein-coupled receptors) used as negative bait. The resulting cichlid repertoire contained 44 complete TAAR genes from O. niloticus, 18 from P. nyererei, 23 from H. burtoni, 12 from N. brichardi and 20 from M. zebra, plus a number of pseudogenes, edge genes and fragments. A large proportion of these sequences (80%) consisted of two coding exons, separated in all but two cases by an intron in the interloop 1 coding sequence. We constructed phylogenetic trees. These trees indicated that TAARs constitute a distinct clade, well separated from ORs (olfactory receptors) and other class A GPCRs. Also these repertoires consist of several families and subfamilies, a number of which are common to fugu, tetraodon, stickleback and medaka. Like all other TAARs identified to date, cichlid TAARs have a characteristic two-dimensional structure and contain a number of amino-acid motifs or amino acids, such cysteine, in particular conserved positions. CONCLUSIONS: Little is known about the functions of TAARs: in most cases their ligands have yet to be identified, partly because appropriate methods for such investigations have not been developed. Sequences analyses and comparisons of TAARs in several animal species, here fishes living in the same environment, should help reveal their roles and whether they are complementary to that of ORs.

The genomic substrate for adaptive radiation in African cichlid fish.

Cichlid fishes are famous for large, diverse and replicated adaptive radiations in the Great Lakes of East Africa. To understand the molecular mechanisms underlying cichlid phenotypic diversity, we sequenced the genomes and transcriptomes of five lineages of African cichlids: the Nile tilapia (Oreochromis niloticus), an ancestral lineage with low diversity; and four members of the East African lineage: Neolamprologus brichardi/pulcher (older radiation, Lake Tanganyika), Metriaclima zebra (recent radiation, Lake Malawi), Pundamilia nyererei (very recent radiation, Lake Victoria), and Astatotilapia burtoni (riverine species around Lake Tanganyika). We found an excess of gene duplications in the East African lineage compared to tilapia and other teleosts, an abundance of non-coding element divergence, accelerated coding sequence evolution, expression divergence associated with transposable element insertions, and regulation by novel microRNAs. In addition, we analysed sequence data from sixty individuals representing six closely related species from Lake Victoria, and show genome-wide diversifying selection on coding and regulatory variants, some of which were recruited from ancient polymorphisms. We conclude that a number of molecular mechanisms shaped East African cichlid genomes, and that amassing of standing variation during periods of relaxed purifying selection may have been important in facilitating subsequent evolutionary diversification.