Görel Sundström

Population-scale sequencing reveals genetic differentiation due to local adaptation in Atlantic herring

The Atlantic herring (Clupea harengus), one of the most abundant marine fishes in the world, has historically been a critical food source in Northern Europe. It is one of the few marine species that can reproduce throughout the brackish salinity gradient of the Baltic Sea. Previous studies based on few genetic markers have revealed a conspicuous lack of genetic differentiation between geographic regions, consistent with huge population sizes and minute genetic drift. Here, we present a cost-effective genome-wide study in a species that lacks a genome sequence. We first assembled a muscle transcriptome and then aligned genomic reads to the transcripts, creating an “exome assembly,” capturing both exons and flanking sequences. We then resequenced pools of fish from a wide geographic range, including the Northeast Atlantic, as well as different regions in the Baltic Sea, aligned the reads to the exome assembly, and identified 440,817 SNPs. The great majority of SNPs showed no appreciable differences in allele frequency among populations; however, several thousand SNPs showed striking differences, some approaching fixation for different alleles. The contrast between low genetic differentiation at most loci and striking differences at others implies that the latter category primarily reflects natural selection. A simulation study confirmed that the distribution of the fixation index FST deviated significantly from expectation for selectively neutral loci. This study provides insights concerning the population structure of an important marine fish and establishes the Atlantic herring as a model for population genetic studies of adaptation and natural selection.

Evolution of vertebrate opioid receptors

The opioid peptides and receptors have prominent roles in pain transmission and reward mechanisms in mammals. The evolution of the opioid receptors has so far been little studied, with only a few reports on species other than tetrapods. We have investigated species representing a broader range of vertebrates and found that the four opioid receptor types (delta, kappa, mu, and NOP) are present in most of the species. The gene relationships were deduced by using both phylogenetic analyses and chromosomal location relative to 20 neighboring gene families in databases of assembled genomes. The combined results show that the vertebrate opioid receptor gene family arose by quadruplication of a large chromosomal block containing at least 14 other gene families. The quadruplication seems to coincide with, and, therefore, probably resulted from, the two proposed genome duplications in early vertebrate evolution. We conclude that the quartet of opioid receptors was already present at the origin of jawed vertebrates ≈450 million years ago. A few additional opioid receptor gene duplications have occurred in bony fishes. Interestingly, the ancestral receptor gene duplications coincide with the origin of the four opioid peptide precursor genes. Thus, the complete vertebrate opioid system was already established in the first jawed vertebrates.

An improved canine genome and a comprehensive catalogue of coding genes and non-coding transcripts.

The domestic dog, Canis familiaris, is a well-established model system for mapping trait and disease loci. While the original draft sequence was of good quality, gaps were abundant particularly in promoter regions of the genome, negatively impacting the annotation and study of candidate genes. Here, we present an improved genome build, canFam3.1, which includes 85 MB of novel sequence and now covers 99.8% of the euchromatic portion of the genome. We also present multiple RNA-Sequencing data sets from 10 different canine tissues to catalog ∼175,000 expressed loci. While about 90% of the coding genes previously annotated by EnsEMBL have measurable expression in at least one sample, the number of transcript isoforms detected by our data expands the EnsEMBL annotations by a factor of four. Syntenic comparison with the human genome revealed an additional ∼3,000 loci that are characterized as protein coding in human and were also expressed in the dog, suggesting that those were previously not annotated in the EnsEMBL canine gene set. In addition to ∼20,700 high-confidence protein coding loci, we found ∼4,600 antisense transcripts overlapping exons of protein coding genes, ∼7,200 intergenic multi-exon transcripts without coding potential, likely candidates for long intergenic non-coding RNAs (lincRNAs) and ∼11,000 transcripts were reported by two different library construction methods but did not fit any of the above categories. Of the lincRNAs, about 6,000 have no annotated orthologs in human or mouse. Functional analysis of two novel transcripts with shRNA in a mouse kidney cell line altered cell morphology and motility. All in all, we provide a much-improved annotation of the canine genome and suggest regulatory functions for several of the novel non-coding transcripts.

Evolution of the Insulin-Like Growth Factor Binding Protein (IGFBP) Family

The evolution of the IGF binding protein (IGFBP) gene family has been difficult to resolve. Both chromosomal and serial duplications have been suggested as mechanisms for the expansion of this gene family. We have identified and annotated IGFBP sequences from a wide selection of vertebrate species as well as Branchiostoma floridae and Ciona intestinalis. By combining detailed sequence analysis with sequence-based phylogenies and chromosome information, we arrive at the following scenario: the ancestral chordate IGFBP gene underwent a local gene duplication, resulting in a gene pair adjacent to a HOX cluster. Subsequently, the gene family expanded in the two basal vertebrate tetraploidization (2R) resulting in the six IGFBP types that are presently found in placental mammals. The teleost fish ancestor underwent a third tetraploidization (3R) that further expanded the IGFBP repertoire. The five sequenced teleost fish genomes retain 9-11 of IGFBP genes. This scenario is supported by the phylogenies of three adjacent gene families in the HOX gene regions, namely the epidermal growth factor receptors (EGFR) and the Ikaros and distal-less (DLX) transcription factors. Our sequence comparisons show that several important structural components in the IGFBPs are ancestral vertebrate features that have been maintained in all orthologs, for instance the integrin interaction motif Arg-Gly-Asp in IGFBP-2. In contrast, the Arg-Gly-Asp motif in IGFBP-1 has arisen independently in mammals. The large degree of retention of IGFBP genes after the ancient expansion of the gene family strongly suggests that each gene evolved distinct and important functions early in vertebrate evolution.

Concomitant duplications of opioid peptide and receptor genes before the origin of jawed vertebrates.

BACKGROUND The opioid system is involved in reward and pain mechanisms and consists in mammals of four receptors and several peptides. The peptides are derived from four prepropeptide genes, PENK, PDYN, PNOC and POMC, encoding enkephalins, dynorphins, orphanin/nociceptin and beta-endorphin, respectively. Previously we have described how two rounds of genome doubling (2R) before the origin of jawed vertebrates formed the receptor family. METHODOLOGY/PRINCIPAL FINDINGS Opioid peptide gene family members were investigated using a combination of sequence-based phylogeny and chromosomal locations of the peptide genes in various vertebrates. Several adjacent gene families were investigated similarly. The results show that the ancestral peptide gene gave rise to two additional copies in the genome doublings. The fourth member was generated by a local gene duplication, as the genes encoding POMC and PNOC are located on the same chromosome in the chicken genome and all three teleost genomes that we have studied. A translocation has disrupted this synteny in mammals. The PDYN gene seems to have been lost in chicken, but not in zebra finch. Duplicates of some peptide genes have arisen in the teleost fishes. Within the prepropeptide precursors, peptides have been lost or gained in different lineages. CONCLUSIONS/SIGNIFICANCE The ancestral peptide and receptor genes were located on the same chromosome and were thus duplicated concomitantly. However, subsequently genetic linkage has been lost. In conclusion, the system of opioid peptides and receptors was largely formed by the genome doublings that took place early in vertebrate evolution.

Early vertebrate chromosome duplications and the evolution of the neuropeptide Y receptor gene regions.

BackgroundOne of the many gene families that expanded in early vertebrate evolution is the neuropeptide (NPY) receptor family of G-protein coupled receptors. Earlier work by our lab suggested that several of the NPY receptor genes found in extant vertebrates resulted from two genome duplications before the origin of jawed vertebrates (gnathostomes) and one additional genome duplication in the actinopterygian lineage, based on their location on chromosomes sharing several gene families. In this study we have investigated, in five vertebrate genomes, 45 gene families with members close to the NPY receptor genes in the compact genomes of the teleost fishes Tetraodon nigroviridis and Takifugu rubripes. These correspond to Homo sapiens chromosomes 4, 5, 8 and 10.ResultsChromosome regions with conserved synteny were identified and confirmed by phylogenetic analyses in H. sapiens, M. musculus, D. rerio, T. rubripes and T. nigroviridis. 26 gene families, including the NPY receptor genes, (plus 3 described recently by other labs) showed a tree topology consistent with duplications in early vertebrate evolution and in the actinopterygian lineage, thereby supporting expansion through block duplications. Eight gene families had complications that precluded analysis (such as short sequence length or variable number of repeated domains) and another eight families did not support block duplications (because the paralogs in these families seem to have originated in another time window than the proposed genome duplication events). RT-PCR carried out with several tissues in T. rubripes revealed that all five NPY receptors were expressed in the brain and subtypes Y2, Y4 and Y8 were also expressed in peripheral organs.ConclusionWe conclude that the phylogenetic analyses and chromosomal locations of these gene families support duplications of large blocks of genes or even entire chromosomes. Thus, these results are consistent with two early vertebrate tetraploidizations forming a paralogon comprising human chromosomes 4, 5, 8 and 10 and one teleost tetraploidization. The combination of positional and phylogenetic data further strengthens the identification of orthologs and paralogs in the NPY receptor family.

The vertebrate ancestral repertoire of visual opsins, transducin alpha subunits and oxytocin/vasopressin receptors was established by duplication of their shared genomic region in the two rounds of early vertebrate genome duplications

BackgroundVertebrate color vision is dependent on four major color opsin subtypes: RH2 (green opsin), SWS1 (ultraviolet opsin), SWS2 (blue opsin), and LWS (red opsin). Together with the dim-light receptor rhodopsin (RH1), these form the family of vertebrate visual opsins. Vertebrate genomes contain many multi-membered gene families that can largely be explained by the two rounds of whole genome duplication (WGD) in the vertebrate ancestor (2R) followed by a third round in the teleost ancestor (3R). Related chromosome regions resulting from WGD or block duplications are said to form a paralogon. We describe here a paralogon containing the genes for visual opsins, the G-protein alpha subunit families for transducin (GNAT) and adenylyl cyclase inhibition (GNAI), the oxytocin and vasopressin receptors (OT/VP-R), and the L-type voltage-gated calcium channels (CACNA1-L).ResultsSequence-based phylogenies and analyses of conserved synteny show that the above-mentioned gene families, and many neighboring gene families, expanded in the early vertebrate WGDs. This allows us to deduce the following evolutionary scenario: The vertebrate ancestor had a chromosome containing the genes for two visual opsins, one GNAT, one GNAI, two OT/VP-Rs and one CACNA1-L gene. This chromosome was quadrupled in 2R. Subsequent gene losses resulted in a set of five visual opsin genes, three GNAT and GNAI genes, six OT/VP-R genes and four CACNA1-L genes. These regions were duplicated again in 3R resulting in additional teleost genes for some of the families. Major chromosomal rearrangements have taken place in the teleost genomes. By comparison with the corresponding chromosomal regions in the spotted gar, which diverged prior to 3R, we could time these rearrangements to post-3R.ConclusionsWe present an extensive analysis of the paralogon housing the visual opsin, GNAT and GNAI, OT/VP-R, and CACNA1-L gene families. The combined data imply that the early vertebrate WGD events contributed to the evolution of vision and the other neuronal and neuroendocrine functions exerted by the proteins encoded by these gene families. In pouched lamprey all five visual opsin genes have previously been identified, suggesting that lampreys diverged from the jawed vertebrates after 2R.

Differential Evolution of Voltage-Gated Sodium Channels in Tetrapods and Teleost Fishes

The voltage-gated sodium channel (SCN) alpha subunits are large proteins with central roles in the generation of action potentials. They consist of approximately 2,000 amino acids encoded by 24-27 exons. Previous evolutionary studies have been unable to reconcile the proposed gene duplication schemes with the species distribution and molecular phylogeny of the genes. We have carefully annotated the complete SCN gene sequences, correcting numerous database errors, for a broad range of vertebrate species and analyzed their phylogenetic relationships. We have also compared the chromosomal positions of the SCN genes relative to adjacent gene families. Our studies show that the ancestor of the vertebrates probably had a single sodium channel gene with two characteristic AT-AC introns, the second of which is unique to vertebrate SCN genes. This ancestral gene, located close to a HOX gene cluster, was quadrupled along with HOX in the two rounds of basal vertebrate tetraploidizations to generate the ancestors of the four channels SCN1A, SCN4A, SCN5A, and SCN8A. The third tetraploidization in the teleost fish ancestor doubled this set of genes and all eight are still present in at least three of four investigated teleost fish genomes. In tetrapods, the gene family expanded by local duplications before the radiation of amniotes, generating the cluster SCN5A, SCN10A, and SCN11A on one chromosome and the cluster SCN1A, SCN2A, SCN3A, and SCN9A on a different chromosome. In eutherian mammals, a tenth gene, SCN7A, arose in a local duplication in the SCN1A gene cluster. The SCN7A gene has undergone rapid evolution and has lost the ability to cause action potentials-instead, it functions as a sodium sensor. The three genes in the SCN5A cluster were translocated from the HOX-bearing chromosome in a mammalian ancestor along with several adjacent genes. This evolutionary scenario is supported by the adjacent TGF-β receptor superfamily (comprised of five distinct families) and the cysteine-serine-rich nuclear protein gene family as well as the HOX clusters. The independent expansions of the SCN repertoires in tetrapods and teleosts suggest that the functional diversification may differ between the two lineages.

Phylogenetic and chromosomal analyses of multiple gene families syntenic with vertebrate Hox clusters

BackgroundEver since the theory about two rounds of genome duplication (2R) in the vertebrate lineage was proposed, the Hox gene clusters have served as the prime example of quadruplicate paralogy in mammalian genomes. In teleost fishes, the observation of additional Hox clusters absent in other vertebrate lineages suggested a third tetraploidization (3R). Because the Hox clusters occupy a quite limited part of each chromosome, and are special in having position-dependent regulation within the multi-gene cluster, studies of syntenic gene families are needed to determine the extent of the duplicated chromosome segments. We have analyzed in detail 14 gene families that are syntenic with the Hox clusters to see if their phylogenies are compatible with the Hox duplications and the 2R/3R scenario. Our starting point was the gene family for the NPY family of peptides located near the Hox clusters in the pufferfish Takifugu rubripes, the zebrafish Danio rerio, and human.ResultsSeven of the gene families have members on at least three of the human Hox chromosomes and two families are present on all four. Using both neighbor-joining and quartet-puzzling maximum likelihood methods we found that 13 families have a phylogeny that supports duplications coinciding with the Hox cluster duplications. One additional family also has a topology consistent with 2R but due to lack of urochordate or cephalocordate sequences the time window when these duplications could have occurred is wider. All but two gene families also show teleost-specific duplicates.ConclusionBased on this analysis we conclude that the Hox cluster duplications involved a large number of adjacent gene families, supporting expansion of these families in the 2R, as well as in the teleost 3R tetraploidization. The gene duplicates presumably provided raw material in early vertebrate evolution for neofunctionalization and subfunctionalization.

Neuropeptide Y-family peptides and receptors in the elephant shark, Callorhinchus milii confirm gene duplications before the gnathostome radiation

We describe here the repertoire of neuropeptide Y (NPY) peptides and receptors in the elephant shark Callorhinchus milii, belonging to the chondrichthyans that diverged from the rest of the gnathostome (jawed vertebrate) lineage about 450 million years ago and the first chondrichthyan with a genome project. We have identified two peptide genes that are orthologous to NPY and PYY (peptide YY) in other vertebrates, and seven receptor genes orthologous to the Y1, Y2, Y4, Y5, Y6, Y7 and Y8 subtypes found in tetrapods and teleost fishes. The repertoire of peptides and receptors seems to reflect the ancestral configuration in the predecessor of all gnathostomes, whereas other lineages such as mammals and teleosts have lost one or more receptor genes or have acquired 1-2 additional peptide genes. Both the peptides and receptors showed broad and overlapping mRNA expression which may explain why some receptor gene losses could take place in some lineages, but leaves open the question why all the known ancestral receptors have been retained in the elephant shark.