Martin Völker

The genome of a songbird.

The zebra finch is an important model organism in several fields with unique relevance to human neuroscience. Like other songbirds, the zebra finch communicates through learned vocalizations, an ability otherwise documented only in humans and a few other animals and lacking in the chicken—the only bird with a sequenced genome until now. Here we present a structural, functional and comparative analysis of the genome sequence of the zebra finch (Taeniopygia guttata), which is a songbird belonging to the large avian order Passeriformes. We find that the overall structures of the genomes are similar in zebra finch and chicken, but they differ in many intrachromosomal rearrangements, lineage-specific gene family expansions, the number of long-terminal-repeat-based retrotransposons, and mechanisms of sex chromosome dosage compensation. We show that song behaviour engages gene regulatory networks in the zebra finch brain, altering the expression of long non-coding RNAs, microRNAs, transcription factors and their targets. We also show evidence for rapid molecular evolution in the songbird lineage of genes that are regulated during song experience. These results indicate an active involvement of the genome in neural processes underlying vocal communication and identify potential genetic substrates for the evolution and regulation of this behaviour.

Gene duplication and fragmentation in the zebra finch major histocompatibility complex

BackgroundDue to its high polymorphism and importance for disease resistance, the major histocompatibility complex (MHC) has been an important focus of many vertebrate genome projects. Avian MHC organization is of particular interest because the chicken Gallus gallus, the avian species with the best characterized MHC, possesses a highly streamlined minimal essential MHC, which is linked to resistance against specific pathogens. It remains unclear the extent to which this organization describes the situation in other birds and whether it represents a derived or ancestral condition. The sequencing of the zebra finch Taeniopygia guttata genome, in combination with targeted bacterial artificial chromosome (BAC) sequencing, has allowed us to characterize an MHC from a highly divergent and diverse avian lineage, the passerines.ResultsThe zebra finch MHC exhibits a complex structure and history involving gene duplication and fragmentation. The zebra finch MHC includes multiple Class I and Class II genes, some of which appear to be pseudogenes, and spans a much more extensive genomic region than the chicken MHC, as evidenced by the presence of MHC genes on each of seven BACs spanning 739 kb. Cytogenetic (FISH) evidence and the genome assembly itself place core MHC genes on as many as four chromosomes with TAP and Class I genes mapping to different chromosomes. MHC Class II regions are further characterized by high endogenous retroviral content. Lastly, we find strong evidence of selection acting on sites within passerine MHC Class I and Class II genes.ConclusionThe zebra finch MHC differs markedly from that of the chicken, the only other bird species with a complete genome sequence. The apparent lack of synteny between TAP and the expressed MHC Class I locus is in fact reminiscent of a pattern seen in some mammalian lineages and may represent convergent evolution. Our analyses of the zebra finch MHC suggest a complex history involving chromosomal fission, gene duplication and translocation in the history of the MHC in birds, and highlight striking differences in MHC structure and organization among avian lineages.

Whole genome comparative studies between chicken and turkey and their implications for avian genome evolution

BackgroundComparative genomics is a powerful means of establishing inter-specific relationships between gene function/location and allows insight into genomic rearrangements, conservation and evolutionary phylogeny. The availability of the complete sequence of the chicken genome has initiated the development of detailed genomic information in other birds including turkey, an agriculturally important species where mapping has hitherto focused on linkage with limited physical information. No molecular study has yet examined conservation of avian microchromosomes, nor differences in copy number variants (CNVs) between birds.ResultsWe present a detailed comparative cytogenetic map between chicken and turkey based on reciprocal chromosome painting and mapping of 338 chicken BACs to turkey metaphases. Two inter-chromosomal changes (both involving centromeres) and three pericentric inversions have been identified between chicken and turkey; and array CGH identified 16 inter-specific CNVs.ConclusionThis is the first study to combine the modalities of zoo-FISH and array CGH between different avian species. The first insight into the conservation of microchromosomes, the first comparative cytogenetic map of any bird and the first appraisal of CNVs between birds is provided. Results suggest that avian genomes have remained relatively stable during evolution compared to mammalian equivalents.

Copy number variation, chromosome rearrangement, and their association with recombination during avian evolution

Chromosomal rearrangements and copy number variants (CNVs) play key roles in genome evolution and genetic disease; however, the molecular mechanisms underlying these types of structural genomic variation are not fully understood. The availability of complete genome sequences for two bird species, the chicken and the zebra finch, provides, for the first time, an ideal opportunity to analyze the relationship between structural genomic variation (chromosomal and CNV) and recombination on a genome-wide level. The aims of this study were therefore threefold: (1) to combine bioinformatics, physical mapping to produce comprehensive comparative maps of the genomes of chicken and zebra finch. In so doing, this allowed the identification of evolutionary chromosomal rearrangements distinguishing them. The previously reported interchromosomal conservation of synteny was confirmed, but a larger than expected number of intrachromosomal rearrangements were reported; (2) to hybridize zebra finch genomic DNA to a chicken tiling path microarray and identify CNVs in the zebra finch genome relative to chicken; 32 interspecific CNVs were identified; and (3) to test the hypothesis that there is an association between CNV, chromosomal rearrangements, and recombination by correlating data from (1) and (2) with recombination rate data from a high-resolution genetic linkage map of the zebra finch. We found a highly significant association of both chromosomal rearrangements and CNVs with elevated recombination rates. The results thus provide support for the notion of recombination-based processes playing a major role in avian genome evolution.

An Appraisal of Nuclear Organisation in Interphase Embryonic Fibroblasts of Chicken, Turkey and Duck

Determining the nuclear ‘addresses’ of chromosome territories is a well-documented means of assaying for nuclear organisation in many cell types and species. Data in avian species are however limited at best, despite the pivotal role played by birds (particularly chickens) in agriculture, and as model organisms in developmental biology. That is, studies have hitherto focussed mostly on mammals (especially humans) and have demonstrated the importance of chromosome territory positioning in embryology, disease and evolution. Thus a detailed study of nuclear organisation in many species, many cell types and many developmental stages in birds is warranted, however, before this is achieved, ‘baseline’ needs to be established to determine precisely the relative locations of chromosome territories in at least 1 cell type of at least 1 bird. With this in mind we hybridised FISH probes from chicken chromosomes 1–28 to embryonic fibroblast nuclei, determining nuclear addresses using a newly developed plug-in to the image analysis package ImageJ. In our experience, evenly spaced representative BAC clones yielded more consistent results than hybridisation of chromosome paints. Results suggested that chromosome territory distribution best fitted a chromosome size-based (rather than gene density-based) pattern. Identical BAC clones were then hybridised to turkey and duck in a comparative genomic strategy. Observations were consistent with those seen in chicken (although, less well-defined in duck), providing preliminary evidence of conservation throughout evolution.

Karyotype differentiation in Chromaphyosemion killifishes (Cyprinodontiformes, Nothobranchiidae). III: Extensive karyotypic variability associated with low mitochondrial haplotype differentiation in C. bivittatum

We investigated chromosomal evolution in the African killifish species Chromaphyosemion bivittatum using a combination of cytogenetic and phylogenetic methods. Specimens from five populations were examined by conventional Giemsa staining as well as sequential chromosome banding with 4′,6-diamidino-2-phenylindole (DAPI), chromomycin A3 (CMA3), AgNO3-staining and C-banding. The cytogenetic analysis revealed variability in 2n ranging from 2n = 29 to 2n = 36 and in NF ranging from NF = 38 to NF = 44. Two populations showed an extensive chromosomal polymorphism (2n = 29–34, NF = 44 and 2n = 32–34, NF = 38–42, respectively). Karyotypic variability within and among populations was mainly due to Robertsonian translocations and heterochromatin additions, and chromosome banding patterns suggested that both types of chromosomal rearrangements were related to the presence of AT-rich heterochromatin. A phylogenetic analysis of the partial mitochondrial (mt) cytochrome b gene, using specimens from eleven populations, revealed a low degree of haplotype differentiation, which suggested a relatively recent divergence of the populations examined. This finding conformed to the low degree of morphological differentiation observed among C. bivittatum populations and might indicate fast chromosomal evolution. The high karyotypic variability may be caused by an elevated chromosomal mutation rate as well as certain aspects of the mating system and population dynamics of C. bivittatum facilitating the fixation of new chromosomal variants.

Karyotype differentiation in Chromaphyosemion killifishes (Cyprinodontiformes, Nothobranchiidae). II: Cytogenetic and mitochondrial DNA analyses demonstrate karyotype differentiation and its evolutionary direction in C. riggenbachi

African killifishes of the genus Chromaphyosemion show a high degree of phenotypic and karyotypic diversity. The latter is especially pronounced in C. riggenbachi, a morphologically defined species restricted to a small distribution area in Cameroon. This study presents a detailed reconstruction of karyotype differentiation within C. riggenbachi using conventional Giemsa staining and sequential chromosome banding as well as a phylogenetic analysis based on part of the mitochondrial (mt) cytochrome b gene from eleven populations. The cytogenetic analysis revealed differences in chromosome morphology, banding patterns and/or diploid chromosome number (2n) among all populations examined. Diploid number ranged from 2n = 20 to 2n = 36 and varied mainly among populations, while C-banding patterns and NOR phenotypes showed fixed differences among populations as well as some variability within populations. The mtDNA analysis disclosed five clearly differentiated haplotype groups. Mapping the karyotype data onto the mtDNA dendrogram revealed a decrease in 2n from the most basal to the most derived groups, thus demonstrating a reduction of 2n during their evolutionary history. Our results indicate that karyotype differentiation involved Robertsonian fusions as well as non-Robertsonian processes. Causes of the high karyotypic variability may include an elevated chromosomal mutation rate as well as certain features of the ecology and mating system that could facilitate the fixation of chromosomal rearrangements. The pattern of karyotype and haplotype differentiation and the results of previous crossing experiments suggest incipient speciation in C. riggenbachi.

Karyotype differentiation in Chromaphyosemion killifishes (Cyprinodontiformes, Nothobranchiidae). I: Chromosome banding patterns of C. alpha, C. kouamense and C. lugens.

The karyotypes of three recently described species of Chromaphyosemion, namely C. lugens, C. alpha and C. kouamense, were analysed using conventional Giemsa staining, C-banding and sequential banding (fluorescence banding with 4′, 6-diamidino-2-phenylindole (DAPI) and Chromomycin A3 (CMA3), C-banding, AgNO3-staining). Diploid chromosome numbers ranged from 2n = 36 in C. lugens to 2n = 38 in C. alpha and C. kouamense. The karyotype of C. lugens consisted of one pair of metacentric (m) and 17 pairs of telocentric (t) chromosomes, that of C. alpha was composed of one pair of submetacentric (sm), six pairs of subtelocentric (st) and 12 pairs of t chromosomes, and that of C. kouamense comprised five pairs of st and 14 pairs of t chromosomes. In addition to the differences in karyotype structures and/or chromosome numbers, the karyotypes of the examined species differed with respect to NOR phenotype and distribution and base composition of heterochromatin. No heteromorphic sex chromosomes were detected in any of the species. Our findings provide cytotaxonomic evidence for the species distinctness of C. alpha, C. kouamense and C. lugens whose descriptions were based primarily on external morphology.