Fig. 3: The categorization of hatched chicks in the Progeny Test I and II.

A Classification of chicks from Progeny Test I (recipient Chahua♂ × Chahua WT♀). Male Chahua recipients (germline chimeras) carried sperm derived from a donor Piao PGC (#P28♂) with Rp/Rp Fm/fm⁺ e⁺/e^b genotypes for three mutation traits, while the recipient Chahua males and WT Chahua hens had WT genotypes (rp⁺/rp⁺ fm⁺/fm⁺ e⁺/e⁺) for three traits. Thus, the hatched chicks in the Progeny Test I can be classified as either ♂recipient-derived chicks (Chahua♂ × Chahua♀) or ♂PGC-derived chicks (Piao♂#P28 × Chahua♀) based on the mutation phenotypes derived from the donor Piao PGC (♂#P28). In particular, the donor Piao PGCs possess homozygous genotypes for the autosomal dominant rumplessness (Rp) mutation, which results in tailbone defects. This is illustrated in Fig. 3D below. Thus, the donor Piao ♂ PGC-derived chicks (Rp/rp⁺) exhibit a tailbone defect phenotype. This can be identified based on the observation of the phenotype and palpation. In addition, some Piao ♂ PGC-derived chicks exhibit black skin (Fm/fm⁺), which results in hyperpigmentation of the skin due to the presence of the autosomal dominant fibromelanosis (Fm) mutation in a heterozygous state. This is illustrated in Fig. 3C below. Furthermore, plumage colors such as extended black (E locus, MC1R) were occasionally used for phenotyping and genotyping purposes, as the donor ♂PGCs carrying an e^b allele as heterozygous genotypes (e⁺/e^b). However, the e^b allele is almost recessive for the WT e⁺ allele. B Classification of chicks from Progeny Test II (recipient Chahua♂ × recipient Chahua♀). As mentioned above, the six recipient Chahua hens used for mating also produce oocytes from donor Piao females PGC (#P28♀) with the Rp/Rp Fm/Fm e^b/e^b genotypes for the three mutation traits, similar to the recipient Chahua males. However, the male and female Chahua recipients themselves have the three WT genotypes (rp⁺/rp⁺ fm⁺/fm⁺ e⁺/e⁺). Thus, the chicks hatched in Progeny Test II can be theoretically divided into four subgroups. These include recipient-derived chicks (Chahua♂ × Chahua♀), ♂PGC-derived chicks (Piao♂#P28 × Chahua♀), ♀PGC-derived chicks (Chahua♂ × Piao♀#P52), and ♂♀PGC-derived chicks (Piao♂#P28 × Piao♀#P52). However, since the Rp and Fm traits are autosomal dominant traits, hatched chicks can be roughly classified into either ♂♀recipient-derived chicks or donor PGC-derived chicks based on the phenotypes of the three mutant traits (Rp, Fm, and e^b). However, some chicks (Piao chicks) derived from male and female donor PGCs (Piao♂#P28 × Piao♀#P52) exhibit dark brown chick down coloration. These chicks are homozygous (e^b/e^b) for the e^b allele derived from male and female donor Piao PGCs. This coloration may also be influenced by other feather color loci, but it can be distinguished from the other three subgroups. However, subsequent classification into three donor PGC-derived subgroups (♂PGC-derived, ♀PGC-derived, and ♂♀PGC-derived chicks) was finally conducted based on genetic diagnosis of mtDNA and Rp mutations (see Supplementary Table 4, Fig. 4). C WT chicks (fm⁺/fm⁺) derived from ♂♀ Chahua recipients (left) and donor Piao PGC-derived chicks (Fm/-) with the Fm mutation (right). It was assumed that the donor PGCs were heterozygous (Fm/fm⁺) for the Fm mutation in the male donor PGC (#P28) and homozygous (Fm/Fm) in the female donor PGC (#P52). However, the recipient Chahua was WT (fm ⁺ /fm⁺ ) in both sexes. It can thus be observed that a significant proportion of the donor Piao PGC-derived chicks in both Progeny Tests exhibited black skin due to excessive melanin deposition, as indicated by the yellow dashed circles. In Progeny Test I, the donor male PGC (#P28)-derived chicks were segregated into two phenotypic groups: one with a normal skin color (fm⁺/fm⁺) and one with a black skin color (Fm/fm⁺). In contrast, all of the donor ♀ PGC (Chahua♂ × Piao♀#P52) and ♂♀PGC (Piao♂#P28 × Piao♀#P52) derived chicks in the Progeny Test II exhibited blackish or black skin coloration, indicating the presence of either the Fm/Fm or the Fm/ fm⁺ genotypes. Individual differences in melanin deposition were observed in the Fm chicks, which may be attributed to the influence of Fm genotypes. D WT chicks (rp⁺/rp⁺) derived from ♂♀Chahua recipients (left) and donor Piao PGC-derived chicks (Rp/-) with the Rp mutation (right). The male (♂#P28) and female (♀#P52) donor Piao PGCs were homozygous for the Rp mutation (Rp/Rp). In contrast, both the male and female Chahua recipients exhibited WT genotypes (rp⁺/rp⁺). All donor Piao PGC-derived chicks (Rp/Rp or Rp/rp⁺) exhibited a tailbone defect in Progeny Test I and II, as indicated by a red dashed circle.