Germline-restricted chromosome (GRC) is widespread among songbirds

Abstract

An unusual supernumerary chromosome has been reported for two related avian species, the zebra and Bengalese finches. This large, germline-restricted chromosome (GRC) is eliminated from somatic cells and spermatids and transmitted via oocytes only. Its origin, distribution among avian lineages, and function were mostly unknown so far. Using immunolocalization of key meiotic proteins, we found that GRCs of varying size and genetic content are present in all 16 songbird species investigated and absent from germline genomes of all eight examined bird species from other avian orders. Results of fluorescent in situ hybridization of microdissected GRC probes and their sequencing indicate that GRCs show little homology between songbird species and contain a variety of repetitive elements and unique sequences with paralogs in the somatic genome. Our data suggest that the GRC evolved in the common ancestor of all songbirds and underwent significant changes in the extant descendant lineages.

Keywords: Passeriformes; avian genome; chromosome evolution; meiosis; programmed DNA elimination.

Fig. 1.

Topology of the bird species examined. Our sampling covers all major passerine groups (except for the basal oscines, suboscines, and acanthisittides) as well as Galloanserae, Columbaves, Apodiformes, Charadriiformes, Falconiformes, and Psittaciformes. Black circles indicate species with a macro-GRC, and white circles indicate species with a micro-GRC. Numbers after the species’ names indicate references for SC studies; asterisks indicate new data. A consensus topology of bird orders is based on the cladogram from Reddy et al. (10). Position of the common swift is defined according to Prum et al. (11). Topology of passerine birds is shown according to Roquet et al. (12). Positions of species within the Estrildidae lineage is established according to Hooper and Price (13).

Fig. 2.

Discovery of GRCs in bird species. (A) SC spreads of four oscine species immunolabeled with antibodies against SYCP3, the main protein of the lateral element of SC (red), centromere proteins (blue) and MLH1, mismatch repair protein marking recombination sites (green). Arrowheads point to the largest chromosomes ordered according to their size ranks, ZZ (identified by its size and arm ratio), ZW (identified by heteromorphic SC and misaligned centromeres), and GRCs. Arrows in the Insets point to MLH1 foci in GRCs. Micro-GRC bivalents in female barn swallow and European pied flycatcher are indistinguishable from the microchromosomes of the somatic chromosome set. (B) DAPI-stained bone marrow cells. (C) Reverse and cross-species FISH of GRC DNA probes (green) derived from Bengalese finch (LST), zebra finch (TGU), Eurasian siskin (SSP), and pale martin (RDI) with SC spreads, immunolabeled with antibodies against SYCP3 (red). Centromeres are labeled with antibodies against centromere proteins (blue). Arrowheads point to GRCs and regions on the somatic chromosome set intensely painted with GRC probes in cross-species FISH. Insets show GRCs. The Bengalese finch GRC-specific DNA probe gives a strong signal on the Bengalese finch GRC and slightly paints some regions of the somatic chromosome set. The zebra finch GRC probe paints the distal area of the Bengalese finch GRC and a region of the short arm of SC3. The Eurasian siskin GRC probe paints a micro-GRC of European goldfinch, a region on the long arm of SC3 and some pericentromeric regions. The pale martin GRC probe gives a dispersed signal on the great tit GRC, the ZW bivalent and on SC5. Scale bar: 5 µm.

Fig. 3.

Scenario of GRC origin and evolution. A proto-GRC forms due to duplication (Ctl-D) of a microchromosome likely containing genes involved in germ cell development. Copies of unique somatic cell sequences and repetitive elements invade the GRC (Ctl-C+Ctl-V). Divergence of GRCs in different songbird lineages occurs due to amplification and deletion (Del) of their sequences.