Figure 1.
Crested and wild-type chickens.
(A–D) Crest phenotype; (E–H) wild-type phenotype; (A and E) Silkie male; (B and F) Silkie female; (C) Chinese fatty chicken male; (D) Chinese fatty chicken female; (G) Chahua chicken male; (H) Chahua chicken female. (I) Overview of cranial feathers with gender indicated, sampled from 44 weeks old Silkie chickens. (a) Crested male; (b) Crested female; (c) Wild-type male; (d) Wild-type female. (J) There was a statistically significant difference in feather length between phenotypes. The number of feathers in each group obtained from three individuals were as follows: Crested male: 47; Crested female: 44; wild-type male: 100; wild-type female: 100 (**, P<0.01).
Table 1.
Two-point linkage analysis of the Crest locus in the CAU F2 population using eight microsatellite loci on chicken LGE22C19W28_E50C23.
Figure 2.
Manhattan plot of genome-wide association analysis for the Crest phenotype in chicken.
The x-axis shows the physical position of the SNPs by chromosome, and the y-axis shows -log10(p-values). A cutoff value of 7.63 declares a genome-wide highly significant association (P<0.001) with a Bonferroni correction. The microsatellite marker HOXC8–ssr that had the most significant p-value is located on LGE22C19W28 by HOXC8.
Table 2.
Two-point linkage analysis of the Crest locus in the INRA resource population and 15 marker loci.
Figure 3.
Expression analysis of HOXC genes in Silkie (crested) and White Leghorn (non-crested) chickens.
(A) HOXC8, HOXC12 and HOXC13 genes were detected by RT-PCR in three tissues (cranial skin, dorsal skin and heart) of Silky and White Leghorn chickens at four developmental stages (E8, E10, E12, E16). C: Cranial skin; D: Dorsal skin; H: Heart. GAPDH was used as a positive and normalised control. -RT indicates a reaction without reverse transcriptase. CK indicates a reaction with water as the template. (B)&(C) q-PCR results for HOXC8 expression level in cranial skin (B); and in dorsal skin (C) during chicken developmental stages E13 to E21 comparing Crested and non-crested chickens. No HOXC8 expression was detected in cranial skin from non-crested birds. (*, P<0.05; **, P<0.01).
Figure 4.
RT-PCR analysis of HOXC8 in different tissues in various chicken breeds.
(A) RT-PCR analysis in skin samples from the progeny of a heterozygous Cr/cr+ chicken. (B) RT-PCR of seven tissues from Crested and wild-type Silkie chickens. Samples were collected from two individuals at 12 weeks of age. -RT indicates a reaction without reverse transcriptase. CK indicates negative control. (C) RT-PCR results for the cranial skin in adult individuals of 26 Chinese local chicken breeds. Breeds are as follows: 1. Lu Yuan; 2. Gu Shi; 3. Hu Xu; 4.Chinese Fatty Chicken; 5. Xian Ju; 6. Da Gu; 7. Silkie; 8. Dou Chicken; 9. Green Egg Chicken; 10. Wen Chang; 11. You Xi Ma; 12. Ai Jiao Huang; 13. Shou Guang; 14. An Ka; 15. Bian Chicken; 16. White Ear; 17. Kuai Da Silkie; 18. Cha Hua; 19. Yin Xing Bai; 20. Chong Ren Ma; 21. Wa Hui; 22. Jin Hu Wu; 23. Tibet Chicken; 24. Shi Qi Za; 25. Black Lang Shan; 26. Qing Yuan Ma; CK: Negative control. Numbers in red indicate chicken breeds with Crest phenotype and black numbers wild-type breeds (Table S12).
Figure 5.
RT-PCR analysis of genes involved in feather development in cranial skin from Crested and wild-type chickens (*, P<0.05).
GAPDH was used as a normalising control. Relative expression levels were obtained by the 2−ΔΔCT method.
Figure 6.
Gene structure of chicken HOXC8.
The two most significant genetic markers in the GWAS of Crest are indicated.