BackgroundColoration is one of the most recognizable characteristics in chickens, and clarifying the coloration mechanisms will help us understand feather color formation. “Yufen I” is a commercial egg-laying chicken breed in China that was developed by a three-line cross using lines H, N and D. Columbian plumage is a typical feather character of the “Yufen I” H line. To elucidate the molecular mechanism underlying the pigmentation of Columbian plumage, this study utilizes high-throughput sequencing technology to compare the transcriptome and proteome differences in the follicular tissue of different feathers, including the dorsal neck with black and white striped feather follicles (Group A) and the ventral neck with white feather follicles (Group B) in the “Yufen I” H line.ResultsIn this study, we identified a total of 21,306 genes and 5,203 proteins in chicken feather follicles. Among these, 209 genes and 382 proteins were differentially expressed in two locations, Group A and Group B, respectively. A total of 8 differentially expressed genes (DEGs) and 9 differentially expressed proteins (DEPs) were found to be involved in the melanogenesis pathway. Additionally, a specifically expressed MED23 gene and a differentially expressed GNAQ protein were involved in melanin synthesis. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis mapped 190 DEGs and 322 DEPs to 175 and 242 pathways, respectively, and there were 166 pathways correlated with both DEGs and DEPs. 49 DEPs/DEGs overlapped and were enriched for 12 pathways. Transcriptomic and proteomic analyses revealed that the following pathways were activated: melanogenesis, cardiomyocyte adrenergic, calcium and cGMP-PKG. The expression of DEGs was validated by real-time quantitative polymerase chain reaction (qRT-PCR) that produced results similar to those from RNA-seq. In addition, we found that the expression of the MED23, FZD10, WNT7B and WNT11 genes peaked at approximately 8 weeks in the “Yufen I” H line, which is consistent with the molting cycle. As both groups showed significant differences in terms of the expression of the studied genes, this work opens up avenues for research in the future to assess their exact function in determining plumage color.ConclusionCommon DEGs and DEPs were enriched in the melanogenesis pathway. MED23 and GNAQ were also reported to play a crucial role in melanin synthesis. In addition, this study is the first to reveal gene and protein variations in in the “Yufen I” H line during Columbian feather color development and to discover principal genes and proteins that will aid in functional genomics studies in the future. The results of the present study provide a significant conceptual basis for the future breeding schemes with the “Yufen I” H line and provide a basis for research on the mechanisms of feather pigmentation.

31Background 32 Coloration is one of the most recognizable characteristics in chickens, and clarifying 33 the coloration mechanisms will help us understand feather color formation. "Yufen I" 34 is an commercial egg-laying chicken breed in China, that was developed by a 35 three-line cross using lines H, N and D. Columbian plumage is a typical feather 36 character of the "Yufen I" H line. To elucidate the molecular mechanism underlying 37 pigmentation of Columbian plumage, this study utilizes the technology of 38 high-throughput sequencing to compare the transcriptome and proteome differences 39 in different feather follicular tissue, including the dorsal neck with black and white 40 striped feather follicles (Group A) and the ventral neck with white feather follicles 41 (Group B) in the "Yufen I" H line. 42 Results 43In this study, we identified a total of 21,306 genes and 5203 proteins in chicken 44 feather follicles. Among these, 209 genes and 382 proteins were differentially 45 expressed in two different locations, Group A and Group B, respectively. A total of 8 46 differentially expressed genes (DEGs) and 9 differentially expressed proteins (DEPs) 47 were found to be involved in the melanogenesis pathway. Besides, a specifically 48 expressed MED23 gene and a differential expressed GNAQ protein were involved in 49 melanin synthesis. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis 50 mapped 190 DEGs and 322 DEPs, to 175 and 242 pathways, respectively, and there 51 were 166 pathways correlated with both DEGs and DEPs. 49 DEPs/DEGs overlapped 52 and were enriched for 12 pathways. Transcriptomic and proteomic analyses revealed 53 that the following pathways were activated: melanogenesis, cardiomycete adrenergic, 54 calcium and the cGMP-PKG. The expression of DEGs was validated by real-time 55quantitative polymerase chain reaction (qRT-PCR) that was similar to that of 56 RNA-seq. In addition, we found that MED23, FZD10, WNT7B and WNT11 genes 57 expression peaked at approximately 8 weeks in the "Yufen I" H line, which is 58 consistent with the molting cycle. As both the groups showed significant differences 59 in terms of expression of the genes studied, this study opens up avenues for study in 60 the future to assess their exact function in color of plumage. 61 Conclusion 62 These common DEGs and DEPs were enriched in the melanogenesis pathway. The 63 MED23 and GNAQ were also reported to have a crucial part synthesis of melanin. In 64 addition, this study is the first to reveal variations in gene and protein in the "Yufen I" 65 H line during Columbian feather color development, and discover principal genes and 66 proteins that would aid in the functional genomics studies in future. The results of the 67 present study provide a significant conceptual basis for the "Yufen I" H line future 68 breeding schemes and provide a basis for research on the mechanisms of feather 69 pigmentation. 70 71 Yufen I" is an egg-laying chicken breed in China, which was developed by a 106 three-line cross using line H as the ...