Analysis of genomic neighborhoods has helped

Analysis of genomic neighborhoods has helped resolve questions concerning the orthology of receptors for peptides similar to glucagon (Ng et al., 2010, Irwin and Prentice, 2011, Wang et al., 2012, Hwang et al., 2013). Using a diverse array of species with available genome sequence (see Fig. 4) the genomic neighborhoods surrounding the GCGR, GLP1R, GLP2R, GIPR and Grlr genes were examined (Fig. 5, Fig. 6, Fig. 7, Fig. 8, Fig. 9). Similar genomic neighborhoods can be seen for many of the receptor genes in the divergent species. For example, for GCGR, orthologous genes can be seen in most species shown in Fig. 5, however there are some notable differences. First, none of the genes near the presumptive lamprey Gcgr gene are orthologous to genes adjacent to GCGR in any other species. The lamprey Gcgr gene is on a shorter genomic contig with little gene content and an incomplete sequence, limiting the ability to accurately identify orthology. Based on sequence similarity and phylogenetic analysis (not shown), of the partial gene sequence, it was concluded to be a Gcgr ortholog, but the possibility remains that it is a paralogous gene duplicate, potentially one that was not retained in jawed vertebrates. In other species, such as the duck and lizard, despite sharing a large number of orthologous genes, some genes linked to Gcgr are missing or different. The Fam195b gene is missing from the duck chromosome, although whether this is due to gap in the genome assembly or a true loss of the gene cannot be resolved. In the lizard, the gene, Ddx39a is linked to Gcgr, which may reflect a change in the organization of genes on this chromosome. Indeed more divergent species typically have greater changes in gene order and composition. In teleost fish, two Gcgr-like genes are often seen, and comparison of the genomic neighborhoods of these genes suggest that they were duplicated as part of a large genomic event, likely as a consequence of the fish-specific genome CPTH2 hydrochloride (Meyer and Van de Peer, 2005). Intriguingly, in the coelacanth the Gcgr and Glp1r genes are adjacent to each other (Fig. 5, Fig. 6). This was the only instance where a pair of receptor genes was found adjacent to each other. Glp1r genes had previously been identified in tetrapod vertebrates (mammals, reptiles, birds, and amphibians) but not in bony fish. Using genomic neighborhood analysis it is clear that the Glp1r genes in tetrapods are orthologous, as similar sets of adjacent genes are found (Fig. 6). The coelacanth Glp1r gene is incomplete, but based on sequence similarity and phylogenetic analysis (not shown) it appears to be most closely related to the Glp1r genes. Whether the unexpected genomic neighborhood of this gene is due to a reorganization of the genome, incorrect assembly, or incorrect conclusions concerning its orthology requires additional analysis. No gene most closely related to GLP1R was found in the bony fish, either in representative species that diverged before (gar) or after (e.g., zebrafish) the fish-specific genome duplication (Tables S3 and S4). Searches of the fish genomes for orthologs of genes adjacent to the tetrapod Glp1r genes failed to find a conserved genomic neighborhood, as these genes were dispersed across different chromosomes in the fish genomes. The failure to find a conserved genomic neighborhood in fish may indicate either this chromosomal region was subjected to large amounts of recombination in fish, or was assembled in a contiguous fashion only in the ancestor of tetrapods. While a candidate Glp1r was identified in lamprey, this sequence was on a short genomic contig with no adjacent genes, is equally similar to the Grlr genes and yielded ambiguous phylogenetic placement, and thus, cannot be confidently classified. The GLP2R gene appears to be in single copy gene throughout vertebrates (Tables S2 and S3, Fig. 3, Fig. 7, S6, and S7). Indeed, it is the only receptor gene in the lamprey that has an adjacent gene that is also adjacent in mammals and other vertebrates (Fig. 7), thus is the most confidently classified lamprey gene for a receptor of a peptide similar to glucagon. A conserved genomic neighborhood was identified in most species examined, although some recombination has likely occurred rearranging gene order for some genes in some species (Fig. 7). Due to the fish-specific genome duplication (Meyer and Van de Peer, 2005), one might expect that there should be two Glp2r genes in teleost fish, however; only one Glp2r-like gene was found in the BLAST searches (Tables S3 and S4). As shown in Fig. 7, a pair of chromosomes were found in zebrafish that are homologous to the single copy Glp2r bearing chromosome in the gar, a species that did not experience this genome duplication. While one of the chromosomes contains a Glp2r gene, the other does not. A similar organization was found in other fish genomes (data not shown), suggesting that the second fish glp2r gene was lost prior to the radiation of teleost fish.