Fig 1.
The Duplex-comb phenotypes V-shaped and Buttercup are both associated with a novel 20 Kb duplication on chicken chromosome 2.
(A) Phenotypes associated with the Duplex-comb locus V-shaped (D*V), Buttercup (D*C) and wild-type (D*N) single comb alleles. (B) A 381 Kb haplotype is IBD in D*V individuals, SNP names in the region marked in red. Yellow shading represents heterozygous genotypes with blue/orange representing reciprocal homozygous genotypes. Genotype data is from the 60K SNP chip and color coded according to genotype with missing data shown in black. A single SNP GGaluGA142157 at 38,806,246 bp (marked in yellow) is heterozygous in all D*V individuals, suggestive of a duplication. A broader view of haplotypes in more individuals is available in S1 Fig. (C) A TaqMan assay was used to investigate the genomic copy number of the putative duplicated region, showing that a duplication is present in both D*V and D*C individuals. Each bar represents a single individual. Error bars represent the minimum and maximum estimated copy number as calculated from technical replicates of each sample by Copy Caller software (ABI). (D) The Duplex-comb alleles are associated with a tandem duplication with no sequence homology at the junction point. The middle line shows the sequence at the junction point with vertical dashes indicating sequence alignment to the 5’ (bottom line) and 3’ (top line) boundaries of the duplicated region. A LTR element is shaded in gray.
Table 1.
A PCR-based diagnostic test reveals complete association of a duplication junction point with the Duplex-comb phenotypes in chickens.
Fig 2.
Characterization of the Duplex-comb locus by genetic mapping and whole genome sequencing.
(A) The genomic region to which the Duplex-comb locus was mapped to; adapted from the UCSC Genome Browser. Black bars represent regions identified from the backcross mapping population, IBD data from the 60K SNP chip, IBD data from whole genome sequencing and the 20 Kb duplication. SNPs identified from whole genome sequencing are shown in Green, Black and Red corresponding to homozygous reference allele, heterozygous, and homozygous variant allele, respectively. Although not currently annotated in the galGal3 genome build four genes are predicted in this region: EOMES (XM_426003.4), CMC1 (XM_418758.4), AZI2 (XM_418759.4) and RBMS3 (XM_004939420.1). The 20 Kb duplicated region is located ∼200 Kb upstream of EOMES. (B) Several regions within the 20 Kb duplication show elevated conservation and Genomic Evolutionary Rate Profiling (GERP) scores for 19 amniota (blue) and 3 neognath (red) species.
Fig 3.
EOMES expression is increased in D*V embryonic comb tissue.
Results of qRT-PCR analysis demonstrating increased EOMES expression in D*V embryonic comb tissue whereas CMC1 and AZI2, located nearby the duplicated region, do not show any significant change in expression. Bar graphs mean ±sem, ANOVA, *P<0.05, **P<0.001, ***P<0.0001.
Fig 4.
Ectopic expression of EOMES in the ectoderm of D*V and D*C embryonic chicken comb tissue.
A-D: Immunohistochemical labeling did not detect any expression of EOMES in either the mesenchyme or ectoderm of the D*N developing comb region. E-K: EOMES is expressed in the ectoderm of both D*V and D*C embryos as early as embryonic day 7 (E7) and continuing through E12/E15. All genotypes showed similar patterns of expression of EOMES in the brain, which was used as a positive control for the antibody specificity. By E12 and E15 a change in tissue morphology is readily apparent in both D*V and D*C embryos. ect; ectoderm, mes; dermal mesenchyme. Scale bar in K is 200 μm and applies to A-C, E-G, I and J. Bars in D and H are 100 μm and L is 1 mm.