Programmed DNA elimination of germline development genes in songbirds

Abstract

In some eukaryotes, germline and somatic genomes differ dramatically in their composition. Here we characterise a major germline-soma dissimilarity caused by a germline-restricted chromosome (GRC) in songbirds. We show that the zebra finch GRC contains >115 genes paralogous to single-copy genes on 18 autosomes and the Z chromosome, and is enriched in genes involved in female gonad development. Many genes are likely functional, evidenced by expression in testes and ovaries at the RNA and protein level. Using comparative genomics, we show that genes have been added to the GRC over millions of years of evolution, with embryonic development genes bicc1 and trim71 dating to the ancestor of songbirds and dozens of other genes added very recently. The somatic elimination of this evolutionarily dynamic chromosome in songbirds implies a unique mechanism to minimise genetic conflict between germline and soma, relevant to antagonistic pleiotropy, an evolutionary process underlying ageing and sexual traits.

Fig. 1

The zebra finch germline-restricted chromosome contains genes copied from many A chromosomes. a, b Cytogenetic evidence for GRC absence in muscle a and GRC presence in the testis b of the same bird (Spain_1) using fluorescence in-situ hybridisation (FISH) of our new GRC-amplified probe dph6 (selected due its high germline/soma coverage ratio; cf. e, f). Note that the single-copy A-chromosomal paralog of dph6 yields no visible FISH signal, unlike the estimated 308 dph6 copies on the GRC. The scale bar indicates 10 μm. c, d Comparison of germline/soma coverage ratios for 1 kb windows with an expected symmetrical distribution (blue bars) indicates enrichment of A-chromosomal single-copy regions in the germline (red bars), similar to lamprey, both in Seewiesen (c; linked reads) and Spain (d; average of Spain_1 and Spain_2 coverage; PCR-free short reads) samples. Y-axis is truncated for visualisation. e, f Manhattan plot of germline/soma coverage ratios in 1 kb windows across chromosomes of the somatic reference genome taeGut2. Colours indicate high-confidence GRC-linked genes and their identification (red: coverage, blue: SNVs, purple: both; Supplementary Table 5). Note that the similarities between Seewiesen e and Spain_1/Spain_2 averages f constitute independent biological replicates for GRC-amplified regions, as the data are based on different domesticated populations and different library preparation methods. Red arrows denote two FISH-verified GRC-amplified regions (cf. b). Only chromosomes >5 Mb are shown for clarity. g, h Linked-read barcode interaction heatmaps of an inter-chromosomal rearrangement on the GRC absent in Seewiesen liver g but present in Seewiesen testis h. i, j Coverage plots of two examples of GRC-linked genes that are divergent from their A-chromosomal paralog, trim71 i and napa j, and thus have very low coverage (normalised by total reads and genome size) in soma.

Fig. 2

The zebra finch germline-restricted chromosome is expressed in male and female gonads. a, b Comparison of coverage and read pileups for DNA-seq data from Spain_1 and Spain_2 testis/muscle, RNA-seq data from Spain_1 and Spain_2 testis, and available ovary RNA-seq data. Shown are 100-bp regions within trim71 a and bicc1 b. Colours indicate SNVs deviating from the reference genome taeGut2 (adenine: green; cytosine: blue; guanine: brown; thymine/uracil: red). c Example alignments of proteomics data showing a subset of peptide expression of the respective GRC-linked paralog of ugdh and napa (alternative or ‘alt’ peptide; cf. reference or ‘ref’ peptide). d Proteomic evidence for GRC peptide expression (‘alt’) in comparison to their A-chromosomal paralog (‘ref’) of 5 genes in 7 sampled testes and 2 sampled ovaries. For label-free quantification (LFQ), unique as well as razor (non-unique) peptides were used. Note that unique peptides may occur in several of the 9 samples. e Gene ontology term enrichment analysis of the 115 high-confidence GRC-linked genes (77 mapped gene symbols). Colours indicate the log₁₀ of the false discovery rate-corrected p-value (PANTHER overrepresentation test, with a Fisher exact test for significance and filtering using a false discovery rate of 0.05), circle sizes denote fold enrichment above expected values. f Expression evidence for chicken orthologs of three different sets of zebra finch GRC gene paralogs in testes, ovaries, or other tissues of chicken. Randomisation tests show a significant enrichment for germline-expressed genes among the chicken orthologs of 115 high-confidence GRC gene paralogs and all 267 GRC gene paralogs, but not the 38 GRC-amplified gene paralogs.

Fig. 3

The zebra finch germline-restricted chromosome is ancient and highly dynamic. a Phylogeny of the intergenic 27L4 locus previously sequenced by Itoh et al. suggests stable inheritance of the GRC paralog (alternative or ‘alt’ in red; cf. reference or ‘ref’) among the sampled zebra finches. b–f Phylogenies of GRC-linked genes (‘alt’, in red; most selected from expressed genes) diverging from their A-chromosomal paralogs (‘ref’) before/during early songbird evolution (b; bicc1, stratum 1; cf. Supplementary Fig. 9), during songbird evolution (c; ugdh, stratum 2), during estrildid finch evolution (d; psip1, stratum 3), in the ancestor of the zebra finch species (e; rnf17, stratum 4), and in the Australian zebra finch subspecies (f; secisbp2l; stratum 5). The maximum likelihood phylogenies in panels a–f (only bootstrap values ≥50% shown) include available somatic genome data from estrildid finches and other songbirds. g Species tree of selected songbirds showing the chronological emergence of evolutionary strata (S1–S5) on the GRC (red gene names). Molecular dates are based on previous phylogenies^,. Bird illustrations were used with permission from Lynx Edicions. h Circos plot indicating A-chromosomal origin of high-confidence GRC-linked genes from 18 autosomes and the Z chromosome. Due to the lack of chromosome-level scaffolding information for the GRC, we were unable to attribute the relative order between most of the genes in the GRC (see details in Supplementary Fig. 1c). Therefore, the represented genes are indicated in the same spot in the GRC placeholder (red box; not to scale). Note that A-chromosomal paralogs of 37 genes remain unplaced on chromosomes in the current zebra finch reference genome taeGut2.