A copy number variant is associated with a spectrum of pigmentation patterns in the rock pigeon (Columba livia)

Abstract

Rock pigeons (Columba livia) display an extraordinary array of pigment pattern variation. One such pattern, Almond, is characterized by a variegated patchwork of plumage colors that are distributed in an apparently random manner. Almond is a sex-linked, semi-dominant trait controlled by the classical Stipper (St) locus. Heterozygous males (ZStZ+ sex chromosomes) and hemizygous Almond females (ZStW) are favored by breeders for their attractive plumage. In contrast, homozygous Almond males (ZStZSt) develop severe eye defects and often lack plumage pigmentation, suggesting that higher dosage of the mutant allele is deleterious. To determine the molecular basis of Almond, we compared the genomes of Almond pigeons to non-Almond pigeons and identified a candidate St locus on the Z chromosome. We found a copy number variant (CNV) within the differentiated region that captures complete or partial coding sequences of four genes, including the melanosome maturation gene Mlana. We did not find fixed coding changes in genes within the CNV, but all genes are misexpressed in regenerating feather bud collar cells of Almond birds. Notably, six other alleles at the St locus are associated with depigmentation phenotypes, and all exhibit expansion of the same CNV. Structural variation at St is linked to diversity in plumage pigmentation and gene expression, and thus provides a potential mode of rapid phenotypic evolution in pigeons.

Fig 1. Phenotypes of pigeons carrying Almond alleles (St, Almond allele; +, wild type allele).

(A) Heterozygous Almond male. (B) Hemizygous Almond female. (C) Homozygous Almond male. (D) Almond females have no observable eye defects. (E) Homozygous Almond males often show severe eye defects. Defects pictured in this juvenile include bloated eyelid and anterior opacity. (F) Wing feathers from different phenotypes, left to right: non-Almond, dark Almond, light Almond, homozygous Almond.

Fig 2. Almond is associated with a CNV on a sex-linked genomic scaffold.

(A) Whole-genome pFst comparisons between Almond and non-Almond pigeons. Each dot represents a SNP position, with shades of gray indicating different genomic scaffolds. The horizontal dashed grey line indicates genome-wide significance threshold. -log10(pFst) values lower than 3 are not plotted. (B) Detail of pFst plot for candidate region on ScoHet5_227, a sex-linked scaffold. Gene models are depicted at the bottom of the plot. -log10(pFst) values lower than 1 are not plotted. (C) Detail view of the CNV region. Solid red line represents the mean normalized read depth for 10 female Almond birds in this region. The blue line is a single representative of non-Almond female coverage. Vertical dashed lines indicate positions of CNV breakpoints. Gene models are depicted below the coverage plot in grey (thick lines, exons; thin lines, introns). Asterisk indictates a drop in coverage due to a gap in the genome that was revealed to contain a CR-1 transposable element.

Fig 3. The Almond-associated CNV has a complex structure that results in duplicated, truncated, and fused genes.

(A) Coverage diagram showing different regions of the CNV normalized to a non-CNV region on the same scaffold. Two outer regions (1 and 3, above plot) have an approximately 7-fold coverage increase, while one inner region (2) has an approximately 14-fold coverage increase. Gene models are depicted below the coverage plot in grey, orange and blue (thick lines, exons; thin lines, introns). (B) Schematic of the non-Almond (top) and inferred Almond (bottom) structures of the CNV. Gene structural changes resulting from the Almond CNV include a fusion of Ermp1 and Kiaa2026 at the segment 3/1 junction (hexagon), and a truncated version of Slc16a13 at the segment 2/2 junction (star). A complete copy of Slc16a13 occurs at each 2/3 junction (diamond).

Fig 4. St-linked pigmentation phenotypes show quantitative variation in the Almond inner CNV region.

Black dots represent results of a TaqMan copy number assay targeting an intron of Mlana. Mean copy numbers for each phenotype are shown as red dots. Most individuals without St-linked phenotypes have the expected 1 or 2 copies (because St is a sex-linked locus, females have a minimum of 1 copy and males have a minimum of 2). All other St-linked phenotypes are associated with an expansion of the CNV in the Almond candidate region on scaffold ScoHet5_227, indicating an allelic series at St. Numbers above each phenotype indicate number of individuals sampled.

Fig 5. Almond and non-Almond feather buds have distinct gene expression profiles.

(A) Exons assayed within the CNV show expression differences in Almond feather buds compared to non-Almond. Boxplots show the results of qRT-PCR assays designed to assess gene expression of exons located in the CNV region. Fusion gene expression results are from qPCR primers spanning exon 7 of Ermp1 into exon 5 of Kiaa2026. (B) Exons assayed outside the CNV show no expression differences in Almond feather buds compared to non-Almond. This indicates expression differences are specific to exons inside the CNV. (C) Expression of melanocyte-related genes. qRT-PCR results indicate a decrease in expression of several genes involved in melanin production in Almond feather buds. (D) Model of interactions among genes and signaling pathways involved in different aspects of pigment synthesis. Gray boxes indicate pathways discussed in the text but not directly represented in our expression analyses. NA, feather buds from non-Almond individuals with wild type alleles at St; DA, dark Almond feather buds from hemizygous and heterozygous Almond individuals; LA, light Almond feather buds from hemizygous and heterozygous Almond individuals; HA, feather buds from a homozygous Almond individual. Bar in each box represents the median, box ends indicate upper and lower quartiles, whiskers indicate the highest and lowest value excluding outliers. Different letters indicate groups with statistically significant differences in gene expression determined by ANOVA and post-hoc Tukey test (p<0.05).