A cis-regulatory mutation of PDSS2 causes silky-feather in chickens

Abstract

Silky-feather has been selected and fixed in some breeds due to its unique appearance. This phenotype is caused by a single recessive gene (hookless, h). Here we map the silky-feather locus to chromosome 3 by linkage analysis and subsequently fine-map it to an 18.9 kb interval using the identical by descent (IBD) method. Further analysis reveals that a C to G transversion located upstream of the prenyl (decaprenyl) diphosphate synthase, subunit 2 (PDSS2) gene is causing silky-feather. All silky-feather birds are homozygous for the G allele. The silky-feather mutation significantly decreases the expression of PDSS2 during feather development in vivo. Consistent with the regulatory effect, the C to G transversion is shown to remarkably reduce PDSS2 promoter activity in vitro. We report a new example of feather structure variation associated with a spontaneous mutation and provide new insight into the PDSS2 function.

Figure 1. The wild-type and silky-feather phenotype in chickens.

(A) Wild-type bird. (B) Silky-feather bird. (C) Wild-type contour feather. (D) Silky-feather contour feather. (E) Pennaceous barbules of wild-type contour feather. (F) Plumulaceous barbules of wild-type contour feather. (G) Pennaceous barbules of silky contour feather. (H) Plumulaceous barbules of silky contour feather. (E–H) Magnification: 100×; scale bar: 100 µm. Schematic magnification of the red and blue boxes in (C) and (D) are shown in (E–H). Arrows in (E) indicate the hooklet. (E) Pennaceous barbules of wild-type contour feather have hooklets, and (G) pennaceous barbules of silky contour feather don't have hooklets.

Figure 2. Fine mapping and identifying the causative mutation for silky-feather.

(A) Schematic structure of SOBP and PDSS2 gene. (B) Identical-by-descent (IBD) mapping of the recessive silky-feather (h) allele. SNP genotypes for h/h, H/h and H/H individuals are presented. Green and Red: two alternative homozygous genotypes. Yellow: heterozygous genotype. White: missing genotype. The thin black vertical lines are the boundaries defined by the homozygous genotypes in h/h birds. The thick black vertical lines are the proximal and distal boundaries of the h shared haplotype defined by the heterozygous genotypes. The minimal silky-feather shared haplotype is 18.9 kb. Asterisk indicates causative mutation position. (C) Electropherogram represents the DNA sequence across the candidate mutation (marked with an asterisk).

Figure 3. Relative mRNA expression levels of PDSS2 and SOBP in skin and liver.

The mRNA expression of (A) PDSS2 and (B) SOBP in postnatal dorsal skin, (C) PDSS2 and (D) SOBP in liver, and (E) PDSS2 and (F) SOBP in embryonic dorsal skin. The mRNA expression is compared with GAPDH. H/H, h/h and H/h represent wild-type homozygous, silky-feather homozygous and heterozygous birds respectively. P10, P60, P130 and P200 represent postnatal (P) days 10, 60, 130 and 200. E9, E11, E14 and E17 represent embryonic (E) days 9, 11, 14 and 17. * indicates P<0.05 and *** indicates P<0.001. The bar represents standard deviation.

Figure 4. Allelic expression imbalance of PDSS2 and SOBP in skin.

The allelic expression imbalance is measured using the pyrosequencing assay. (A and B) The SNPs in the PDSS2 coding sequence correspond to ss666793773 (exon 8) and ss189596174 (exon 6). (C and D) The SNPs in the SOBP coding sequence correspond to ss666793686 (exon 6) and ss666793687 (exon 6). Heterozygous (H/h) and homozygous (H/H and h/h) chickens are tested. The allele with higher expression in cDNA is regarded as the major allele in both genomic DNA (gDNA) and cDNA of the same individual. Small gray circles correspond to the allelic ratio for individual chickens. Large red circles correspond to the average mean value and bar means standard deviation. In homozygous chickens (H/H or h/h), the ratios of three different stages (P60, P130 and P200) are similar and combined into one group for fewer individuals. The P-value of the difference between gDNAs and cDNAs of the same genotype, and cDNAs of different genotypes is evaluated by a t-test. *** indicates P<0.001. ** indicates P<0.01. In (D), the allelic ratios of cDNA do not have difference among the three genotypes, although they are significantly different from the allelic ratios of gDNA. The difference between the cDNA and gDNA may be due to the SNP signal itself.

Figure 5. Immunohistochemistry of PDSS2 in wild-type and silky-feather embryonic skins.

Sections of wild-type (A–D) and silky-feather (E–H) skins at embryonic (E) days 9, 11, 14 and 17 are immunolabeled with the cPDSS2 antibody. PDSS2 staining is in green, and the nuclei are stained with DAPI (blue). (I) Schematic view of feather development and the sections corresponding to (A–H). The strong green signals that appear obviously unusual in the mesenchyme in (A and E) are suspected to originate from tissue damage caused by the experimental operation (according to bright field and not shown). Scale bar equals 100 µm.

Figure 6. Effects of the silky-feather mutation on promoter and enhancer activity using a luciferase reporter assay.

(A) Schematic description of vectors used for luciferase reporter assay. Long and short sequences for each allele are used for vector construct separately. The wild-type (H) or silky-feather (h) allele forward sequence is inserted to the empty vector pGL3-Basic. The wild-type (H) or silky-feather (h) allele reverse sequence is also inserted to the empty vector pGL3-Promoter. (B) Promoter activity and (C) Enhancer activity analyses in chicken DF1 cell are shown for each vector. The pGL3-Basic, pGL3-Promoter and pGL3-Control vectors are used as control. Three technical repeats are performed for each vector in one experiment. Firefly in relation to Renilla luciferase levels is calculated with the empty vector pGL3-Basic as reference. The average value of three technical repeats is represented as one activity value. Three separate repeats are performed and used to calculate mean and standard deviation (SD). The activity associated with the wild-type (H) and silky-feather (h) constructs are compared using a Student's t-test. ** indicates P<0.01 and *** indicates P<0.001.