High tandem repeat content in the genome of the short-lived annual fish Nothobranchius furzeri: a new vertebrate model for aging research

Abstract

Background: The annual fish Nothobranchius furzeri is the vertebrate with the shortest known life span in captivity. Fish of the GRZ strain live only three to four months under optimal laboratory conditions, show explosive growth, early sexual maturation and age-dependent physiological and behavioral decline, and express aging related biomarkers. Treatment with resveratrol and low temperature significantly extends the maximum life span. These features make N. furzeri a promising new vertebrate model for age research.

Results: To contribute to establishing N. furzeri as a new model organism, we provide a first insight into its genome and a comparison to medaka, stickleback, tetraodon and zebrafish. The N. furzeri genome contains 19 chromosomes (2n = 38). Its genome of between 1.6 and 1.9 Gb is the largest among the analyzed fish species and has, at 45%, the highest repeat content. Remarkably, tandem repeats comprise 21%, which is 4-12 times more than in the other four fish species. In addition, G+C-rich tandem repeats preferentially localize to centromeric regions. Phylogenetic analysis based on coding sequences identifies medaka as the closest relative. Genotyping of an initial set of 27 markers and multi-locus fingerprinting of one microsatellite provides the first molecular evidence that the GRZ strain is highly inbred.

Conclusions: Our work presents a first basis for systematic genomic and genetic analyses aimed at understanding the mechanisms of life span determination in N. furzeri.

Figure 1

Cytogenetic features of N. furzeri. (a) Karyotype of DAPI-stained chromosomes of a female N. furzeri of the GRZ strain. Note the absence of bright staining at the centromeric regions. Four pairs of chromosomes (1, 6, 9, 17) are metacentric, three pairs (16, 18, 19) are acrocentric and the remaining 12 pairs are subtelo-/submetacentric. (b) C-banded karyotype of a female N. furzeri GRZ reveals centromeric heterochromatin in most chromosomes. (c) Mithramycin staining results in bright fluorescence of centromeres, which is due to G+C enriched heterochromatin.

Figure 2

Chromosomes of four African Nothobranchius species. DAPI stained chromosomes of (a) a female specimen of the N. furzeri GRZ strain, (b) a male specimen of the sympatric species N. orthonotus, and (c) a male specimen of the allopatric species N. hengstleri and (d) N. eggersi, respectively. Note the dull DAPI fluorescence at centromeric regions in N. furzeri chromosomes (indicated by arrowheads), which is indicative of the presence of G+C-rich constitutive heterochromatin and not observable in the three closely related Nothobranchius species.

Figure 3

G+C content distribution of N. furzeri compared with medaka, stickleback, tetraodon and zebrafish. (a) Histogram of the G+C content of the 5.4 Mb genomic sample of N. furzeri GRZ. The average G+C content is 44.9%. Note G+C distortions, which are seen in a second peak at approximately 62% G+C and an unusually high number of sequences with approximately 41% G+C. Green: sequences containing the most frequent G+C poor 348-nucleotide satellite repeat. Red: sequences containing the most frequent G+C-rich 77-nucleotide minisatellite repeat. (b) G+C content distribution of ten samples of random sequence sets of zebrafish (black), medaka (blue), stickleback (red) and tetraodon (green), respectively. Each data set of the four fish genomes is shown with respect to sequence length distribution and occupied genomic fraction similar to the N. furzeri GRZ 5.4 Mb sample, which, for comparison, is shown as a grey area. Average G+C content values are 36.6% for zebrafish, 40.5% for medaka, 44.6% for stickleback and 46.6% for tetraodon.

Figure 4

Sequence alignment of 189 monomers of the most abundant minisatellite of N. furzeri. The upper part shows a representative section of a ClustalW alignment of 189 monomers of the 77-nucleotide minisatellite of N. furzeri GRZ. Below, the deduced repeat consensus sequence and sequence variability are given based on all 189 monomers. Asterisks mark identical nucleotides, plus signs indicate one mismatch in 189 sequences. Numbers indicate nucleotide identities: 5 represents ≥50-60% identity for 189 sequences; 6 represents ≥60-70%; 7 represents ≥70-80%, 8 represents ≥80-90%; and 9 represents ≥90-100%.

Figure 5

FISH analysis of the most frequent N. furzeri GRZ tandem repeats. (a) The most abundant, G+C-rich minisatellite, which is comprised of 77-nucleotide monomers, is found in centromeric regions of most chromosomes. (b) The second most abundant G+C-rich minisatellite, which is comprised of 49-nucleotide monomers, also forms centromeric regions of many chromosomes. (c) A G+C-rich, 24-nucleotide minisatellite specifically stains centromeric regions of two chromosome pairs. (d) The most frequent G+C poor satellite, which is comprised of 348-nucleotide monomers, maps to centromeric regions of many chromosomes. Panels on the right side show corresponding DAPI images to better illustrate the staining of centromeric regions. Arrows highlight selected distinct DAPI dull centromeric regions.

Figure 6

Evolutionary relationships of N. furzeri to medaka, stickleback, tetraodon, zebrafish and human. The evolutionary history based on the maximum parsimony (MP) method [79] and MEGA4 [78] is shown and the most parsimonious tree given (length = 3,287). The tree topology is fully consistent with the taxonomic classification of the analyzed fish species. Accordingly, Nothobranchius is most closely related to medaka, while both zebrafish and human are distantly related to the euteleost fish division.