Genomic instability within centromeres of interspecific marsupial hybrids

Abstract

Several lines of evidence suggest that, within a lineage, particular genomic regions are subject to instability that can lead to specific types of chromosome rearrangements important in species incompatibility. Within family Macropodidae (kangaroos, wallabies, bettongs, and potoroos), which exhibit recent and extensive karyotypic evolution, rearrangements involve chiefly the centromere. We propose that centromeres are the primary target for destabilization in cases of genomic instability, such as interspecific hybridization, and participate in the formation of novel chromosome rearrangements. Here we use standard cytological staining, cross-species chromosome painting, DNA probe analyses, and scanning electron microscopy to examine four interspecific macropodid hybrids (Macropus rufogriseus x Macropus agilis). The parental complements share the same centric fusions relative to the presumed macropodid ancestral karyotype, but can be differentiated on the basis of heterochromatic content, M. rufogriseus having larger centromeres with large C-banding positive regions. All hybrids exhibited the same pattern of chromosomal instability and remodeling specifically within the centromeres derived from the maternal (M. rufogriseus) complement. This instability included amplification of a satellite repeat and a transposable element, changes in chromatin structure, and de novo whole-arm rearrangements. We discuss possible reasons and mechanisms for the centromeric instability and remodeling observed in all four macropodid hybrids.

Figure 1.—

C-band karyotype of metaphase chromosomes from (A) M. rufogriseus, (B) M. agilis, and (C) M. rufogriseus × M. agilis (RA1120). Chromosome numbers are indicated below C.

Figure 2.—

Abnormal centromere structures in M. rufogriseus × M. agilis hybrids. DAPI-stained metaphase chromosomes from two cells of M. rufogriseus × M. agilis (RA1120) highlight the abnormal centromere structures of the M. rufogriseus-derived chromosomes (arrows indicate the “knob-like” structures) detected by gross karyotype analyses.

Figure 3.—

Scanning electron micrographs of M. rufogriseus × M. agilis hybrid chromosomes stained specifically for DNA with platinum blue. Some centromeres are elongated and exhibit an uneven DNA distribution similar, if narrower, to that on the chromosome arms (A). Parallel fibers and chromomeres are distributed throughout the chromosome, including the centromere (B, the SE image). DNA distribution shows distinguishable chromatids joined at a small region in the centromere (B, the BSE image); DNA distribution is uninterrupted longitudinally along the chromatid arms (B, the BSE image).

Figure 4.—

High-resolution stereo micrograph pairs of a M. rufogriseus × M. agilis hybrid chromosome centromere stained with platinum blue. (Top) SE images showing centromere topography. (Bottom) BSE images showing DNA distribution. Stereo viewing allows recognition of the spatial distribution of structural elements: chromomeres of varying sizes (top, circles) are interspersed with parallel fibers (top, arrows). Chromomeres (top, circles) correspond with local increases in DNA concentration (bottom, circles).

Figure 5.—

sat23 and KERV-1 FISH to metaphase chromosomes of M. rufogriseus × M. agilis hybrids. (A) Hybridization (from left) of sat23 (red), KERV-1 (green) to metaphase chromosomes (inverted DAPI) of RA1118 (merge). (B) Minichromosome from “new RA” containing both sat23 (red) and KERV-1 (green) DNA (merge).

Figure 6.—

De novo chromosome aberrations in M. rufogriseus × M. agilis hybrids. Examples of chromosome rearrangements identified through cross-species chromosome painting in RA1122: (A) fission, (B) translocation, and (C) isochromosome. The parental origin is labeled as M.r. (M. rufogriseus) and M.a. (M. agilis).