Copy number variation in intron 1 of SOX5 causes the Pea-comb phenotype in chickens

Abstract

Pea-comb is a dominant mutation in chickens that drastically reduces the size of the comb and wattles. It is an adaptive trait in cold climates as it reduces heat loss and makes the chicken less susceptible to frost lesions. Here we report that Pea-comb is caused by a massive amplification of a duplicated sequence located near evolutionary conserved non-coding sequences in intron 1 of the gene encoding the SOX5 transcription factor. This must be the causative mutation since all other polymorphisms associated with the Pea-comb allele were excluded by genetic analysis. SOX5 controls cell fate and differentiation and is essential for skeletal development, chondrocyte differentiation, and extracellular matrix production. Immunostaining in early embryos demonstrated that Pea-comb is associated with ectopic expression of SOX5 in mesenchymal cells located just beneath the surface ectoderm where the comb and wattles will subsequently develop. The results imply that the duplication expansion interferes with the regulation of SOX5 expression during the differentiation of cells crucial for the development of comb and wattles. The study provides novel insight into the nature of mutations that contribute to phenotypic evolution and is the first description of a spontaneous and fully viable mutation in this developmentally important gene.

Figure 1. Wild-type and Pea-comb chickens.

(A) Wild-type male, (B) wild-type female, (C) Pea-comb male and (D) Pea-comb female (Photo by David Gourichon).

Figure 2. Identification of the Pea-comb mutation.

(A) The region on chicken chromosome 1 harbouring SOX5. The conservation score track shows the large number of Evolutionary Conserved Non-Coding Elements at the SOX5 locus. The region upstream of SOX5 exon 2 identified through IBD mapping and showing complete association to Pea-comb is marked with a dark shaded bar. Bottom part: the Pea-comb IBD region is expanded. The position of the 3.2 kb duplicated sequence in the near vicinity of non-coding sequences conserved across vertebrate species is marked with an orange bar (adapted from the UCSC genome browser http://genome.ucsc.edu/). The GC content and the location of CpG islands are indicated. (B) Localization and composition of the duplicated sequence. CR1-F2 and CR1-Y4 are partial LINEs and a small CpG island is marked with a green bar. The region corresponding to the probe used for Southern blot analysis is indicated. (C) Southern blot analysis using genomic DNA digested with BamHI from Pea-comb and wild-type chickens; the estimated sizes of restriction fragments are given to the left. (D) Results of real-time PCR analysis of the duplicated region. Individual phenotypes were not available for the Hua-Tung breed and the real-time PCR assay indicated that one bird was homozygous wild-type which is fully possible since Pea-comb is not fixed in this breed; furthermore, this bird did not carry the Pea-comb haplotype. The results for each individual sample are compiled in Table S3. RJF, red junglefowl; BR, broiler; CGP, Czech Golden Pencilled; FF, Friesian Fowl; FL, Finnish Landrace; RV, Red Villafranquina; TNN, Transylvanian Naked Neck; WL, White Leghorn; FPC, French Pea-comb; HT, Hua-Tung; ORL, Orlov.

Figure 3. SOX5 immunostaining in Pea-comb and wild-type embryonic heads.

(A, D) Schematic drawings of sagittal and cross-sections of an E7 chick head. Green indicates SOX5 immunostainings that are identical in wild-type and Pea-comb birds, red indicates SOX5 staining unique for Pea-comb. The planes of the drawings are shown as shaded lines. Scale bar 1 mm. (B, E, I) Fluorescence micrographs of the wattle and comb regions with SOX5 immuno- and DAPI nuclear staining of E7 wild-type and (C, F, J) Pea-comb birds. (G, H) Bright-field micrographs of cRNA in situ hybridization for SOX5 mRNA in wild-type and Pea-comb. The positions of the comb and wattle regions shown in panels B, C, E–J are boxed in the schematic drawings. Scale bars 100 μm. (K–P) SOX5 immuno- and DAPI nuclear staining in the comb region of E6, E9 and E12 wild-type and Pea-comb chickens. Insets show schematic drawings of the comb-ridge shapes in wild-type and Pea-comb. The positions of corresponding fluorescence micrographs are boxed. Scale bar 100 μm. ect; ectoderm, e; eye, l; lumen of nostril, m; Meckel's cartilage, me; mesenchyme, nr; neural retina, o; optic lobe, s; interorbital septum, st; stage according to Hamburger and Hamilton , t; tongue, te; telencephalon, wt; wild-type.