Metazoans typically keep the same genetic information in all body cells over their life cycle. However, some organisms show exceptions to this rule through programmed DNA elimination from the somatic cell line during embryo development, as is the case of the germline-restricted chromosome (GRC) in songbirds. Recently, it has been demonstrated that the puzzling GRC is likely present in over half of all bird species and that the zebra finch GRC is enriched in developmental genes. However, it remains mysterious why the GRC evolved and how it may affect differential gene expression between tissues, genomic divergence between species, and the determination of cell fate between germline and soma. To address this, we first generated a high-quality reference assembly of the zebra finch GRC comprising over 70% of its expected size. We then found molecular evidence for the presence of the GRC in all the 25 Passeriformes species studied, including two Suboscines. The GRC repeat content has no traceable pattern across the phylogeny but rather seems to have expanded and contracted continuously. The comparison of the gene content of GRCs showed that elavl4 and cpeb1 are the genes more frequently present across Passeriformes GRCs and might have an important role for the GRC. Finally, we started functional analyses to elucidate the role of these genes in the early transmission in embryos with differential expression analyses of mRNA and sRNA, as well as whole-mount in situ hybridization with DNA and RNA probes to spot the GRC location and expression in embryo development. Altogether, we showed that the GRC probably arose once and early during the Passeriformes diversification. Since then, the GRC content has been changing constantly and we detected no genes in common within all the species, so function of the most important genes could be replaced by newly arrived genes.
