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All songbirds have one special accessory chromosome1–4, the so-called germline-restricted chromosome (GRC)4–7, which is only present in germline cells and absent from all somatic tissues. Earlier work on the zebra finch (Taeniopygia guttata castanotis) showed that the GRC is inherited only through the female line4,6,8,9 – like mitochondrial DNA7,9–12 – and is eliminated from the sperm during spermatogenesis5,7,9–11. Here we show that the GRC can also be paternally inherited. Confocal microscopy using GRC-specific FISH probes indicated that a considerable fraction of sperm heads (1-19%) in zebra finch ejaculates still contained the GRC. In line with these cytogenetic data, sequencing of ejaculates revealed that individual males from two families differed strongly and consistently in the number of GRCs in their ejaculates. Examining a captive-bred population of hybrids of the two zebra finch subspecies (T. g. guttata and T. g. castanotis) revealed that the descendants inherited their mitochondria from a castanotis mother but their GRC from a guttata father. Moreover, GRC haplotypes across nine different castanotis matrilines showed at best a weak tendency to be co-inherited with mtDNA haplotypes. Within castanotis, the GRC showed little variability, while the mtDNA of matrilines was highly divergent. This suggests that a single GRC haplotype has recently spread across the entire castanotis population, crossing the matriline boundaries via paternal spillover. Our findings raise the possibility that certain GRC haplotypes could selfishly spread through the population, via additional paternal transmission, thereby outcompeting other GRC haplotypes that were limited to strict maternal inheritance, even if this was partly detrimental to organismal fitness.

Summary The ability to genetically manipulate organisms has led to significant insights into functional genomics in many species. In birds, manipulation of the genome is hindered by the inaccessibility of the one-cell embryo. During embryonic development, avian primordial germ cells (PGCs) migrate through the bloodstream and reach the gonadal anlage, where they develop into mature germ cells. Here, we explored the use of PGCs to produce transgenic offspring in the zebra finch, which is a major animal model for sexual brain differentiation, vocal learning, and vocal communication. Zebra finch PGCs (zfPGCs) obtained from embryonic blood significantly proliferated when cultured in an optimized culture medium and conserved the expression of germ and stem cell markers. Transduction of cultured zfPGCs with lentiviral vectors was highly efficient, leading to strong expression of the enhanced green fluorescent protein. Transduced zfPGCs were injected into the host embryo and transgenic songbirds were successfully generated., Graphical abstract, Highlights • Blood-derived zebra finch primordial germ cells (zfPGCs) can be expanded in vitro • Lentiviral vectors can efficiently transduce zfPGCs in vitro • Genetically modified zfPGCs can be used to generate transgenic zebra finches, In this article, Gessara and colleagues used zebra finch primordial germ cells obtained from embryonic blood for the genetic modification of a songbird species using lentiviral vectors.