Reconstruction of the ancestral marsupial karyotype from comparative gene maps

Abstract

Background: The increasing number of assembled mammalian genomes makes it possible to compare genome organisation across mammalian lineages and reconstruct chromosomes of the ancestral marsupial and therian (marsupial and eutherian) mammals. However, the reconstruction of ancestral genomes requires genome assemblies to be anchored to chromosomes. The recently sequenced tammar wallaby (Macropus eugenii) genome was assembled into over 300,000 contigs. We previously devised an efficient strategy for mapping large evolutionarily conserved blocks in non-model mammals, and applied this to determine the arrangement of conserved blocks on all wallaby chromosomes, thereby permitting comparative maps to be constructed and resolve the long debated issue between a 2n = 14 and 2n = 22 ancestral marsupial karyotype.

Results: We identified large blocks of genes conserved between human and opossum, and mapped genes corresponding to the ends of these blocks by fluorescence in situ hybridization (FISH). A total of 242 genes was assigned to wallaby chromosomes in the present study, bringing the total number of genes mapped to 554 and making it the most densely cytogenetically mapped marsupial genome. We used these gene assignments to construct comparative maps between wallaby and opossum, which uncovered many intrachromosomal rearrangements, particularly for genes found on wallaby chromosomes X and 3. Expanding comparisons to include chicken and human permitted the putative ancestral marsupial (2n = 14) and therian mammal (2n = 19) karyotypes to be reconstructed.

Conclusions: Our physical mapping data for the tammar wallaby has uncovered the events shaping marsupial genomes and enabled us to predict the ancestral marsupial karyotype, supporting a 2n = 14 ancestor. Futhermore, our predicted therian ancestral karyotype has helped to understand the evolution of the ancestral eutherian genome.

Figure 1

Examples of FISH determining the orientation of adjacent BAC clones on tammar wallaby metaphase chromosomes. Orientation of (A)SERPINA1 labelled green and NUDC2 in red on chromosome 1; (B)RUNX2 in red and MRPS10 in green on chromosome 2 and (C)CORTBP2 in green and p100 in red on chromosome 3. Chromosomes have been counterstained with DAPI. Scale bar represents 10 μm.

Figure 2

Cytogenetic map of tammar wallaby chromosomes 1 and 2. The cytogenetic location of each gene mapped by FISH is indicated alongside the DAPI-banded ideograms. Gene names indicated in grey were mapped as part of previous studies. The boundaries of the conserved segments determined by chromosome painting are indicated by horizontal lines.

Figure 3

Cytogenetic map of tammar wallaby chromosomes 3 and 4. The boundaries of the conserved segments determined by chromosome painting are indicated by horizontal lines; solid lines indicate definitively determined boundaries from wallaby/opossum comparisons and dotted lines represent boundaries which could not be clearly established.

Figure 4

Cytogenetic map of tammar wallaby chromosomes 6, 7 and X.

Figure 5

Mapping of genes to the short arm of wallaby chromosome 2. FISH mapping of BET1L (green) and AIP (red) indicates homology to human 11p. Scale bar represents 1 μm.

Figure 6

Comparative maps of wallaby and opossum chromosomes. Conserved gene blocks are indicated by bars alongside chromosomes and their orientation shown by lines linking bars from the two species. The conserved segment identified from chromosome painting [6] to which each gene block belongs is indicated. Wallaby and opossum chromosomes have been colour-coded to reflect homology with human chromosomes.

Figure 7

The predicted ancestral therian chromosome containing segments C10, C11 and C12 and the derivation of opossum and wallaby chromosomes. (A) The predicted therian ancestral chromosome aligned against chicken chromosomes containing C10, C11 and C12 genes. An inversion and the addition of genes corresponding to part of human chromosomes 1 and 19 to the distal end of this chromosome and two more inversion events result in a putative marsupial ancestral chromosome consisting of all three segments in the order of C10, C12 and C11. Opossum (MDO) chromosomes 4 and 7 are derived from a fission event taking place in segment C12. (B) Wallaby (MEU) chromosomes 5 and 6 are derived from the predicted marsupial ancestor via inversions, a fission between C10 and C12 and a further inversion within C11.

Figure 8

Derivation of ancestral marsupial chromosome consisting of segments C1 to C6. The predicted therian ancestral chromosome containing segments C1-C5 essentially corresponds to four chicken chromosomes: 12, 14, Z and a large portion of chromosome 2. Inversions and addition of chromosomal segments corresponding to human chromosomes 19, 12 and 22 to the ancestral therian chromosome ultimately led to the formation of the ancestral marsupial chromosome 1.

Figure 9

Predicted ancestral marsupial and therian karyotypes. (A) The 2n = 14 ancestral marsupial karyotype, predicted based on comparative mapping data, are colour-coded to show homology to human chromosomes (same colour-code as shown in Figure 6). Segments from different human chromosomes with known associations in eutherians (light grey) indicated to the left of the chromosomes. Associations of genes in chicken are indicated in dark grey with the number of the chicken chromosome shown above. Dotted lines indicate blocks from the same chicken or ancestral eutherian chromosome. (B) The predicted 2n = 19 therian ancestral karyotype. Chromosomes have been colour-coded to reflect homology with human chromosomes (refer to key in Figure 6).

Figure 10

Derivation of (A) marsupial and (B,C) eutherian ancestral karyotypes from the predicted ancestral therian karyotype. (A) The predicted ancestral marsupial karyotype was formed by fusions of the predicted therian chromosomes. (B) Inversions, fusions and fissions led to (C) the previously predicted ancestral eutherian karyotype [40]. T – Therian, M- Marsupial. Chromosomes have been colour-coded to reflect homology with human chromosomes (refer to key in Figure 6).