Deletion of Indian hedgehog gene causes dominant semi-lethal Creeper trait in chicken

Abstract

The Creeper trait, a classical monogenic phenotype of chicken, is controlled by a dominant semi-lethal gene. This trait has been widely cited in the genetics and molecular biology textbooks for illustrating autosomal dominant semi-lethal inheritance over decades. However, the genetic basis of the Creeper trait remains unknown. Here we have utilized ultra-deep sequencing and extensive analysis for targeting causative mutation controlling the Creeper trait. Our results indicated that the deletion of Indian hedgehog (IHH) gene was only found in the whole-genome sequencing data of lethal embryos and Creeper chickens. Large scale segregation analysis demonstrated that the deletion of IHH was fully linked with early embryonic death and the Creeper trait. Expression analysis showed a much lower expression of IHH in Creeper than wild-type chickens. We therefore suggest the deletion of IHH to be the causative mutation for the Creeper trait in chicken. Our findings unravel the genetic basis of the longstanding Creeper phenotype mystery in chicken as the same gene also underlies bone dysplasia in human and mouse, and thus highlight the significance of IHH in animal development and human haploinsufficiency disorders.

Figure 1. Phenotypes of Xingyi bantam chickens.

(a,b) Female and male chickens. Left is the Creeper while right is the wild-type chickens. Shank length is shorter and body size is lower in the Creeper than in wild-type chickens. (c) Radiographic features of the lethal embryos (embryonic day 16) and day-old Creeper and wild-type chicks. Very small wings (red arrow) and legs (green arrow) were observed in the homozygous lethal embryos. (d,e) Comparison of shank (20 weeks) and wing (day-old chick) of the Creeper and wild-type chickens by digital radiography.

Figure 2. Pipelines for whole genome sequencing data analysis.

Six pairs of full-sibs were obtained for whole-genome sequencing. Several current known major mutations were detected using various algorithms/software. Once the mutations were obtained, the mutations were assessed and ranked from high to low by their potential genetic effects.

Figure 3. Identification of mutations underlying the semi-lethal Creeper trait by whole-genome sequencing.

(a) Normalized genome coverage of the deletion region on chicken chromosome 7 for lethal embryos, Creeper and wild-type chickens. The deletion region (chr7: 21,798,705-21,810,600) includes the entire IHH gene. Full deletion of IHH was observed in the lethal embryos. Each group has 6 samples (N = 6). (b) FPKM (Fragments Per Kilobase Per Million Fragments Mapped on the chromosome, FPKM) was calculated for lethal embryos, Creeper and wild-type chickens. FPKM value follows the 0:1:2 ratio for lethal embryos, Creeper and wild-type chickens, respectively. (c) Comparison of IHH gene between vertebrates and chickens. Blue and red colors represent the cDNAs of IHH in vertebrates and chickens, respectively.

Figure 4. Schematic presentation of primer design and detection of IHH deletion by a diagnostic PCR test.

(a) Diagnostic primers for wild-type and Creeper chickens. Expected size of PCR product of the wild-type chicken is 438 bp while for the Creeper chicken is 224 bp. (b) Diagnostic genotyping of three phenotypes. Lethal embryos has a single PCR product of 244 bp, Creeper chicken has two bands at 244 bp and 438 bp, and the wild-type chicken has a single band at 438 bp.

Figure 5. Relative copy number of IHH in genomic DNA from three different genotypic individuals by qPCR analysis.

Red, blue, and black colors represent lethal embryos, Creeper and wild-type chickens, respectively. Data are represented as mean ± SD (N = 16).

Figure 6. Expression analysis of IHH in tibial cartilages in three genotypic individuals.

(a) qPCR analysis of IHH at E4 (embryonic day 4), D1 (day of hatch), and D84 (day 84 post-hatch) (N = 8 for each group). (b,d) Western blot analysis of IHH from Creeper and wild-type male chicks at D1 and D84. (c,e) The quantitative analysis of western blot results at D1 and D84 using Image J software. Western blot analysis showed the expression of IHH protein in chicken tibial cartilages. β-actin was used as a loading control. Three biological replicates were conducted in this experiment. Data are expressed as mean ± SD (N = 3). *p < 0.05.