Genome-wide association analysis and genome resequencing reveal a genomic duplication possibly concerning a novel chicken comb morphology

Here, we report on a novel chicken comb phenotype, designated Antler-comb. Using a 600K Axiom® Genome-Wide Chicken Genotyping Array, we separately genotyped 12 and 24 female Hetian Wildtype-comb and Antler-comb chickens, respectively. Meanwhile, we sequenced the genomes of 10 Hetian Antler-comb and 10 Wildtype-comb chickens to interrogate the GWAS results and explore the potential genetic variants underlying this phenotype. After conducting a genome-wide association study (GWAS), a 36.5-kb candidate genomic region (chromosome 19:757,754-794,200) related to the Antler-comb phenotype was identified, which wholly and partially encompassed heat shock factor 5 (HSF5) and ring finger protein 43 (RNF43), respectively. HSF5 was ectopically expressed and RNF43 was up-regulated in Antler-comb chickens at embryo ages 7 and 9 (E7 and E9). We further genotyped the most significant single-nucleotide polymorphism (SNP) site, Chr19:794200, across 222 chickens of 16 breeds. We found that the major allele G in Antler-comb chickens remained highly significant across different breeds, and each Antler-comb chicken harbored an allele G. Whole-genome re-sequencing (WGS) involving 10 Hetian Antler-comb and 10 Wildtype-comb chickens reaffirmed the 36.5-kb candidate genomic region, and revealed a genomic duplication, which was 15.7 kb in length and pertained to the 5’-untranslated region and 5’-flanking region of HSF5 (Chr19:784,335-800,034), suggesting its possible role in inducing ectopic expression of HSF5 and altering expression of RNF43 during comb development (E7 and E9). The present study furthers our understanding of this novel chicken comb phenotype, and likely gives another example regarding interactions between genetic variation and phenotype. 24 female chickens with Antler-comb phenotype and 12 female chickens with Wildtype-comb chickens, for which all 36 chickens were from a same farm in Guangdong, were genotyped with Affymatrix 600K Axiom® Genome-Wide Chicken Genotyping Array. Based on case-control model, Plink version 1.9 was employed to perfrom genome-wide analysis, aiming for identifying candidate genomic regions concerning Antler-comb phenotype. Meanwhile, we whole-genome resequenced another 6 male Antler-comb chickens from Fujian province, 4 female Antler-comb chickens from Guangdong province, and 10 Wildtype-comb chicken from above GWAS population to interrogate the results from GWAS, and search for variations in terms of InDel or CNV inside the candidate genomic regions from GWAS. Additionally, qPCR was further performed across the genes encompassing above candidate genomic region.