Figure 1.
Four comb phenotypes in chickens explained by segregation at the Rose-comb and Pea-comb loci and their interaction.
(A) Single-combed wild-type male (rr pp), (B) Rose-combed male (R- pp), (C) Pea-combed male (rr P-) and (D) walnut-combed male (R- P-). Photos by Freyja Imsland (A–C) and David Gourichon (D).
Table 1.
Two-point linkage analysis of the Rose-comb locus using 11 markers on chicken chromosome 7.
Figure 2.
Candidate structural variants identified from whole-genome resequencing.
Mate-pair information was used to plot structural variants in the region of interest (chr7:12–30 Mb) for sequenced pools of (A) single-combed White Leghorn, (B) Rose-combed Chinese Silkie (C) Rose-combed Le Mans. Structural variants were defined as 1.5 kb windows where at least 25% of the mate pairs had mapping distances exceeding ten standard deviations above the average insert size and those > = 25% that were mapped within 1 kb of each other. Y-axis indicates the size of candidate structural variants in log10 base pairs. X-axis indicates the genomic coordinates of the pairs supporting structural variants. Red colour indicates mates that map to different strands, indicative of inversion. Blue colour indicates mates that map to the same strand, indicative of a deletion or duplication. The structural variants uniquely observed in the two Rose-combed pools included an inversion candidate, stretching between approximately 16.50–23.88 Mb. In the Le Mans pool an additional inversion candidate was also observed between 16.50–23.79 Mb, supported by three read pairs. This is depicted at the magnified region at bottom of (C). Candidate structural changes shared by all genotypes may represent errors in the draft chicken assembly.
Figure 3.
Organization of wild-type and Rose-comb chromosomes and description of inversion and duplication breakpoints.
(A) Constitution of the two Rose-comb alleles, R1 and R2, in relation to the organization of the wild-type (r) chromosome 7 in chickens. Sequence orientation in relation to the wild-type chromosome is indicated by arrows. Duplicated sequence in R2 (chr7:23,790,414–23,881,384 bp) is in reverse orientation, apart from 198 bps (chr7:16,499,583–16,499,781 bp) flanking the inverted segment. Breakpoint locations are indicated by arrows (a–e). Breakpoints for the R1 inversion are at 16,499,781 and 23,881,384-23,881,392 bp in the wild-type sequence. Additional breakpoints for the R2 duplication are at 16,499,583 and 23,790,414 bp. (B) Organisation of genes in the five different chromosomal configurations associated with Rose-comb. Breakpoint locations are indicated with red arrows. mRNAs with accession numbers XM_422054.2, NM_204929.1, CR353563.1 and AJ719903.1, as well as EST sequences CD218766.1, BG713529.1 and DR426188.1 were used to define the genes illustrated. The copy of ABCB6 that occurs at the second proximal breakpoint unique to the R2 chromosome, is 5′ truncated from the duplication event, and appears 3′ truncated due to a gap in the assembly. An intact full length copy of this gene is expected to occur at its native chromosomal position (around 23.79 Mb) on R1, R2 and r chromosomes.
Table 2.
Genotyping of the Rose-comb locus in various chicken breeds.
Figure 4.
Two-colour FISH staining of metaphase chromosomes using BACs mapped to GGA7.
(A1) Staining from a heterozygous R1r bird reveals two separate localisations for CH261-95H11 and CH261-5G3 when comparing r Chr7 to R1 Chr7. (A2) The order reversal of BW27C3 and TAM32-24B23 between r Chr7 and R1 Chr7 clearly demonstrates a large inversion. Staining from a heterozygous R2r bird reveals the same localisations obtained for CH261-95H11 (B1), TAM32-24B23 and BW27C3 (B2) both r Chr7 and R2 Chr7, with CH261-5G3 showing an additional localisation on R2 Chr7 (B1), consistent with a translocated duplication of a segment from the 23.88 MB region to the 16.50 MB region. A slight spatial separation for CH261-95H11, consistent with the insertion of the translocated duplication, can be observed on R2 Chr7 (B1).
Figure 5.
Immunohistochemical labelling of MNR2 and SOX5 in various comb tissues.
Wild-type single-comb (a, b), Rose-comb (c, d), Pea-comb (e, f) and walnut-comb (g, h) sections from embryonic day (E) 6.5 (a, c, e, g) and 9 (b, d, f, h) were labelled against MNR2 and SOX5. Nuclei are visualized by DAPI. Boxed regions are shown magnified as single colour. Arrows in (g) and (h) indicate double labelled cells whereas arrowheads indicate single labelled cells. (i) E5.5 walnut comb. The two framed regions are shown magnified and arrows indicate double labelled cells. (j–l) The prospective wattle region for wild-type single-comb, Pea-comb and Rose-comb. (m) Schematic view of an E6.5 head where boxes indicate the regions of the comb depicted in (a–h) and wattles depicted in (j–l). Scale bar equals 100 µm in (h) and is valid for (a–h) and 100 µm in (l) valid for (i–l).