Genetic Mapping and Biochemical Basis of Yellow Feather Pigmentation in Budgerigars

Abstract

Parrot feathers contain red, orange, and yellow polyene pigments called psittacofulvins. Budgerigars are parrots that have been extensively bred for plumage traits during the last century, but the underlying genes are unknown. Here we use genome-wide association mapping and gene-expression analysis to map the Mendelian blue locus, which abolishes yellow pigmentation in the budgerigar. We find that the blue trait maps to a single amino acid substitution (R644W) in an uncharacterized polyketide synthase (MuPKS). When we expressed MuPKS heterologously in yeast, yellow pigments accumulated. Mass spectrometry confirmed that these yellow pigments match those found in feathers. The R644W substitution abolished MuPKS activity. Furthermore, gene-expression data from feathers of different bird species suggest that parrots acquired their colors through regulatory changes that drive high expression of MuPKS in feather epithelia. Our data also help formulate biochemical models that may explain natural color variation in parrots. VIDEO ABSTRACT.

Keywords: Hi-C; Parrot; artificial selection; budgerigar; feather color; mendelian; pigmentation; polyene; polyketide synthase; psittacofulvin; specialized metabolism; trait mapping.

Figure 1. A Hi-C-Based Physical Map of the Budgerigar Genome

The normalized contact frequency matrix for pairs of loci was estimated from chromatin conformation capture (Hi-C) data from budgerigar erythrocytes. For a given pair of loci, the expected number of contacts (Exp) was calculated as the product of the numbers of genome-wide contacts made by each of the two loci, divided by the number of contacts between all loci. The genome was divided into 1 Mb bins, and the ratio of observed (Obs) versus expected contacts within each bin is represented by color. The LACHESIS software was used to cluster the scaffolds according to their contact frequencies by shuffling their order and orientations. The ten largest clusters were matched to the budgerigar karyotype by alignment to probes used in a previous fluores-cent in situ hybridization (FISH) experiment (Nanda et al., 2007) (see also Figure S2). The remaining clusters were assigned numbers according to size.

Figure 2. Genome-wide Association Mapping of the blue Locus

(A) Fisher’s exact test p values (3 genotype classes ×2 phenotype classes) are shown for 69,855 SNPs segregating in 249 budgerigars. Of these, 105 displayed the recessive blue phenotype caused by lack of yellow pigmentation, and 144 displayed WT pigmentation. The red line indicates the Bonferroni-corrected critical value. Unassembled scaffolds are grouped together to the right of the Z chromosome. (B) WT and blue budgerigars photographed under white light (left) or UV-A (“black light”) illumination. The crown feathers (arrowhead) of the WT bird, but not the blue, exhibit yellow fluorescence under UV-A.

Figure 3. Haplotypes Associated with theblue Phenotype

(A) Haplotypes for 63 SNPs in a 3 Mb region centered on the blue locus association peak were inferred with the software program PHASE. At a subset of these loci, outlined in gray, all blue individuals carried the same haplotype. Population-wide haplotype counts were calculated from the most likely pairs of haplotypes carried by each individual and their associated probabilities. Only haplotypes with ≥ 2 counts are shown, except haplotype 8, which was found in only one sample but shows evidence of an ancestral recombination between SNPs at 21,019,187 and 21,161,723. The ancestral alleles were determined by whole-genome sequence alignment to 14 other avian species (Green et al., 2014). (B) RefSeq gene models and descriptions for genes located within the blue-shared haplotype, with positions of SNPs shown, including the two flanking SNPs.

Figure 4. Expression of blue Locus Genes in Regenerating Budgerigar Feathers

(A) Transcript levels, measured as fragments per kilobase of transcript per million fragments mapped (FPKM), in regenerating contour feathers from WT (n = 3) or blue (n = 4) budgerigars for genes in the blue-associated haplotype (Figure 3B). LOC101880049 and TRNAN-GUU were not expressed (FPKM < 0.04). Error bars represent 95% confidence intervals calculated by cuffdiff (Trapnell et al., 2013). Violin plots to the right show the genome-wide distribution of FPKM values per gene. None of the 9 genes showed significantly different expression levels in WT versus blue after applying a Benjamini-Hochberg correction for multiple hypothesis testing. (B) Illustrated cross-sections of a regenerating feather and its follicle. The barb ridges and rachidial ridge, which give rise to the barbs and rachis, are wrapped in a cylindrical sheath (Chen et al., 2015). As the feather matures, the axial plate and marginal plate are lost by apoptosis, allowing the barbules and ramus to separate. The sheath then sloughs off, allowing the feather to open. The barb ridges closest to the rachis mature earlier than those opposite the rachis. (C) Mature stage of the same feather parts shown in (B). Yellow psittacofulvin pigment is found in the barbules and in the outer cortex of the ramus in budgerigar feathers (D’Alba et al., 2012). (D and E) In situ hybridization of regenerating contour feather follicles from a WT (D) or blue (E) budgerigar with probes against the un-characterized polyketide synthase MuPKS. Transcripts are detected in the axial-plate epithelia of more mature barb ridges. Notations: ap, axial plate; br, barb ridge; rc, rachis; rm, ramus.

Figure 5. Candidate Causative Variant

(A) A non-synonymous SNP in budgerigar polyketide synthase segregates completely with the presence (WT) or absence (blue) of yellow feather pigment. (B) The affected arginine residue is located within the malonyl-CoA:ACP transacylase (MAT) domain and is conserved across distantly related homologs, including human and bacterial fatty-acid synthase and bacterial polyketide synthases. In the crystal structure of E. coli FabD (Oefner et al., 2006), the conserved arginine forms a salt bridge with the malonate substrate in the active site. (C) The domain structure of MuPKS is homologous to mammalian fatty-acid synthase and type I bacterial polyketide synthases. The enoyl-reductase (ER) domain is marked as inactive (Ψ) based on its lack of the canonical NADPH-binding motif. Additional notations: ketoacyl synthase, KS; dehydratase, DH; ketoreductase, KR; acyl-carrier protein, ACP; methyltransferase, ME; thioesterase, TE. (D) Proposed biochemical mechanism of yellow psittacofulvin pigment synthesis in budgerigars. The ACP is activated by attachment of phosphopantetheine. Chain initiation involves transacylation of an acetyl-primer unit from acetyl-CoA to the active-site cysteine of the ketoacyl synthase (KS) domain. Each cycle of chain elongation begins with a condensation reaction between the KS-bound growing polyketide chain and malonyl-ACP. The MAT domain is responsible for transferring malonyl extender units from malonyl-CoA to the ACP. The ketoreductase (KR) and dehydratase (DH) domains convert each β-keto-thioester intermediate to the corresponding unsaturated α,β-unsaturated thioester. The inactive ER domain of MuPKS cannot reduce this double bond, resulting in a conjugated polyene product, such as those observed in the feather pigments of the scarlet macaw (Stradi et al., 2001).

Figure 6. Reconstitution of Feather-Pigment Synthesis in Yeast

(A) Ethyl acetate extracts from yeast strain BJ5464-NpgA expressing 6 ×His-tagged MuPKS WT, blue (defined by the amino acid at position 644 but not identical at all other positions), or WT with site-directed mutagenesis to create the R644W substitution. The extracts were illuminated with white light or UV-A (“black light”). MuPKS protein levels were measured in total soluble protein extracts from the same yeast cultures by western blot with α-His antibody. (B) LC absorbance chromatograms (374 nm) for compounds extracted into methanol from yeast expressing WT MuPKS compared to budgerigar feather pigments extracted into acidified pyridine (McGraw and Nogare, 2005). (C) Comparison of mass spectrometry data from compounds produced by MuPKS in yeast versus compounds extracted from yellow feathers. The absorbance chromatograms shown in (B) are aligned to extracted ion chromatograms for m/z values identified through an untargeted search for ions enriched in the pigmented samples (see also Figures S6C–S6E). The absorbance chromatogram has been shifted forward by 2.98 s, the delay time between the diode array detector and the ion detector in our setup. The most likely chemical formula for each ion is shown in parenthesis. These formulas are consistent with the family of polyenes shown in Figure 5D.

Figure 7. Phylogeny of Metazoan Polyketide Synthases

(A) Maximum likelihood tree based on an alignment of the KS domains of metazoan polyketide synthases, fatty-acid synthases, and their homologs in fungi, eukaryotic outgroups, and bacteria. Bootstrap values (based on 1,000 replicates) are indicated at the tree nodes. The scale bar below the tree denotes substitutions per site. Species are colored according to the clades shown in (C). The tree is rooted by the outgroup mycocerosic acid synthase (MAS) from Mycobacterium. (B) Domain structures for the enzymes shown in (A). Colors denote domains commonly found in polyketide synthases, fatty-acid synthases, and non-ribosomal peptide synthases. Inactive pseudo-domains, or domains likely to be inactive based on sequence features, are denoted by “Ψ” (KS, MAT, and ACP make up the minimal set of domains for a functional polyketide synthase). Partial sequences or those containing probable artifacts from genome assembly errors are left blank. (C) Cladogram for species shown in (A).