Supplementary Material for: Genome-Wide Association Study and Transcriptome Analysis Provide New Insights into the White/Red Earlobe Color Formation in Chicken

<b><i>Background/Aims:</i></b> Earlobe color is a typical external trait in chicken. There are some previous studies showing that the chicken white/red earlobe color is a polygenic and sex-linked trait in some breeds, but its molecular genetic and histological mechanisms still remain unclear. <b><i>Methods:</i></b> We herein utilized histological section, genome-wide association study (GWAS) and RNA-seq, further to investigate the potential histological and molecular genetic mechanisms of white/red earlobe formation in Qiangyuan Partridge chicken (QYP). <b><i>Results:</i></b> through histological section analysis, we found the dermal papillary layer of red earlobes had many more blood vessels than that of white earlobes. And we identified a total of 44 SNPs from Chromosome 1, 2, 3, 4, 9, 10, 11, 13, 19, 20, 23 and Z, that was significantly associated with the chicken white/red earlobe color from GWAS, along with 73 significantly associated genes obtained (e.g., <i>PIK3CB, B4GALT1</i> and <i>TP63</i>), supporting the fact that the white/red earlobe color was also polygenic and sex-linked in QYP. Importantly, <i>PIK3CB</i> and <i>B4GALT1</i> are both involved in the biological process of angiogenesis, which may directly give rise to the chicken white earlobe formation through regulating blood vessel density in chicken earlobe. Additionally, through contrast of RNA-seq profiles between white earlobe skins and red earlobe skins, we further identified <i>TP63</i> and <i>CDH1</i> differentially expressed. Combined with the existing knowledge of TP63 in epithelial development and tumor angiogenesis, we propose that down-regulated <i>TP63</i> in white earlobes may play roles in thickening the skin and decreasing the vessel numbers in dermal papillary layer, thereby contributing to the white earlobe formation via paling the redness of the skin in QYP, but the specific mechanism remains to be further clarified. <b><i>Conclusion:</i></b> our findings advance the existing understanding of the white earlobe formation, as well as provide new clues to understand the molecular mechanism of chicken white/red earlobe color formation